U.S. Department of the Interior

U.S. Geological Survey

Assessing Biological Effects from

Highway-Runoff Constituents

Open-File Report 99-240

A Contribution to the 

National Highway Runoff Data and Methodology Synthesis
U.S. Department of the Interior

U.S. Geological Survey

Assessing Biological Effects from

Highway-Runoff Constituents

By DENNY R. BUCKLER and GREGORY E. GRANATO

A Contribution to the 

National Highway Runoff Data and Methodology Synthesis

Northborough, Massachusetts

1999
U.S. DEPARTMENT OF THE INTERIOR

BRUCE BABBITT, Secretary 

U.S. GEOLOGICAL SURVEY

Charles G. Groat, Director 

The use of trade or product names in this report is for identification 

purposes only and does not constitute endorsement by the 

U.S. Geological Survey.

For additional information write to: Copies of this report can be purchased 

District Chief U.S. Geological Survey

Massachussetts–Rhode Island District Information Services

U.S. Geological Survey Box 25286, Denver Federal Center

10 Bearfoot Rd. Denver, CO 80225-0286

Northborough, MA 01532
PREFACE

Preface 

Knowledge of the characteristics of highway runoff (concentrations and loads of 

constituents and the physical and chemical processes that produce this runoff) is 

important for decision makers, planners, and highway engineers to assess and 

mitigate possible adverse impacts of highway runoff on the Nation’s receiving 

waters. In October, 1996, the Federal Highway Administration and the U.S. 

Geological Survey began the National Highway Runoff Data and Methodology 

Synthesis to provide a catalog of the pertinent information available; to define 

the necessary documentation to determine if data are valid (useful for intended 

purposes), current, and technically supportable; and to evaluate available 

sources in terms of current and foreseeable information needs. This paper is 

one contribution to the National Highway Runoff Data and Methodology 

Synthesis and is being made available as a U.S. Geological Survey Open-File 

Report pending its inclusion in a volume or series to be published by the Federal 

Highway Administration. More information about this project is available on the 

World Wide Web at http://ma.water.usgs.gov/fhwa/runwater.htm

Fred G. Bank

Team Leader

Water and Ecosystems Team

Office of Natural Environment

Federal Highway Administration

Patricia A. Cazenas

Highway Engineer

Water and Ecosystems Team

Office of Natural Environment

Federal Highway Administration

Gregory E. Granato

Hydrologist 

U.S. Geological Survey

Con

CONTENTS

Abstract .............................................................................................................................................................................

Introduction .......................................................................................................................................................................

Background .............................................................................................................................................................

Purpose and Scope ..................................................................................................................................................

Factors for Assessing Biological Effects...........................................................................................................................

Contaminants of Interest .........................................................................................................................................

Other Factors ...........................................................................................................................................................

Biological Assessment Techniques ...................................................................................................................................

Biochemical, Physiological, and Histological Techniques......................................................................................

Metal Sequestration and Regulation..............................................................................................................

Oxidative Metabolism ...................................................................................................................................

Reproductive Parameters...............................................................................................................................

Histopathology ..............................................................................................................................................

Tissue Analysis..............................................................................................................................................

Semipermeable Membrane Devices..............................................................................................................

Whole-Organism and Single-Species Techniques...................................................................................................

Microbial Assays ...........................................................................................................................................

Algal Assays..................................................................................................................................................

Aquatic Invertebrate Assays ..........................................................................................................................

Early Life-Stage Toxicity Studies with Fish .................................................................................................

In-Situ Toxicity Assessment..........................................................................................................................

Population and Community Techniques..................................................................................................................

Algal Population and Community Assessments............................................................................................

Aquatic Invertebrate Population and Community Assessments....................................................................

Fish Population and Community Assessments..............................................................................................

Data-Quality Considerations .............................................................................................................................................

Documentation of Methods.....................................................................................................................................

Quality Assurance and Quality Control ..................................................................................................................

Description of Environmental Setting.....................................................................................................................

Comparability Issues for a National Synthesis .......................................................................................................

Biological Effects of Highway-Runoff Quality: A Literature Review..............................................................................

Factors for Assessing Biological Effects.................................................................................................................

Biological Assessment Techniques .........................................................................................................................

Data-Quality Considerations ...................................................................................................................................

Summary ...........................................................................................................................................................................

References .........................................................................................................................................................................

Tables

1. Comparative strengths of categories of biological assessment techniques.........................................................

2. Documented metadata for reports examining the effects of highway runoff on biota in receiving water..........
VI Contents SI* (MODERN METRIC) CONVERSION FACTORS

* SI is the symbol for the International System of Units. Appropriaterounding should be made to comply with Section 4 of ASTM E380.
Ab

Assessing Biological Effects from

Highway-Runoff Constituents

By Denny R. Buckler and Gregory E. Granato

Abstract

Increased emphasis on evaluation of 

nonpoint-source pollution has intensified the need 

for techniques that can be used to discern the toxicological effects of complex chemical mixtures. In 

response, the use of biological assessment techniques is receiving increased regulatory emphasis. 

When applied with documented habitat assessment and chemical analysis, these techniques can 

increase our understanding of the influence of 

environmental contaminants on the biological 

integrity and ecological function of aquatic communities. 

The contaminants of greatest potential concern in highway runoff are those that arise from 

highway construction, maintenance, and use. The 

major contaminants of interest are deicers; nutrients; metals; petroleum-related organic compounds, such as polycyclic aromatic hydrocarbons 

(PAHs), benzene, toluene, ethylbenzene, and 

xylene (BTEX), and methyl tert-butyl ether 

(MTBE); sediment washed off the road surface; 

and agricultural chemicals used in highway 

maintenance.

Hundreds, if not thousands, of biological 

endpoints (measurable responses of living organisms) may be either directly or associatively 

affected by contaminant exposure. Measurable 

effects can occur throughout ecosystem processes 

across the wide range of biological complex

ranging from responses at the biochemical l

the community level. 

The challenge to the environmental sc

is to develop an understanding of the relatio

of effects at various levels of biological org

tion in order to determine whether a causal 

tionship exists between chemical exposure 

substantial ecological impairment. This rep

vides a brief history of the evolution of biol

assessment techniques, a description of the 

classes of contaminants that are of particula

est in highway runoff, an overview of repre

tive biological assessment techniques, and a 

discussion of data-quality considerations.

Published reports with a focus on the 

of highway runoff on the local ecosystem w

reviewed to provide information on (1) the 

ability of the existing data for a quantitative 

national synthesis, (2) the methods availabl

study the effects of highway runoff on local 

systems, and (3) the potential for adverse ef

on the roadside environment and receiving w

Although many biological studies have been 

the use of different methods and a general l

sufficient documentation precludes a quanti

national synthesis on the basis of the existin

The Federal Highway Administration, the U

Environmental Protection Agency, the U.S. 

Geological Survey, the Intergovernmental T

Force on Monitoring Water Quality, and the 
2 Assessing Biological Effects from Highway-Runoff Constituents

National Resources Conservation Service all have 

developed and documented methods for assessing 

the effects of contaminants on ecosystems in 

receiving waters. These published methods can be 

used to formulate a set of protocols to provide 

consistent information from highway-runoff 

studies. 

Review of the literature indicates (qualitatively) that highway runoff (even from highways 

with high traffic volume) may not usually be 

acutely toxic. Tissue analysis and community 

assessments, however, indicate effects from highway-runoff sediments near discharge points (even 

from sites near highways with relatively low traffic 

volumes). At many sites, elevated concentrations 

of highway-runoff constituents were measured in 

tissues of species associated with aquatic sediments. Community assessments also indicate 

decreases in the diversity and productivity of 

aquatic ecosystems at some sites receiving highway runoff. These results are not definitive, however, and depend on many site-specific criteria that 

were not sufficiently documented in most of the 

studies reviewed.

INTRODUCTION

Biological effects are an important component 

of studies of the effects of highway runoff on water 

quality. The sensitivity of biota to changes in water 

quality, coupled with a high degree of public interest 

in their welfare, as well as legal requirements, make 

consideration of biological effects a high priority. The 

Clean Water Act (Federal Water Pollution Control Act 

of 1972, 33 USC §§ 1251 et seq.) and its amendments 

and revisions represent the primary Federal legislative 

actions directed toward the protection and restoration 

of the physical, chemical, and biological integrity 

of the Nation’s waters. More recently, the States 

have been integrating biological criteria into the development and enforcement of their water-quality 

standards (Davis and others, 1996). The intended 

use of biological criteria is to evaluate the effects of 

anthropogenic activities on the biological integrity and 

ecological function of aquatic communities. Because 

of these factors, biological assessment of the ef

of nonpoint-source pollution has gained increas

regulatory and scientific attention in recent year

Background

The complexity of mixtures of chemical 

inants has, in part, lead to an increased regulato

emphasis on biological endpoints (using the res

of living organisms as indicators of contaminan

ence and effects). Chemical measurements com

do not allow prediction of the complex interactio

can occur in biological organisms. Scientific ad

in the development and application of biologica

ation procedures also have lead to increased use 

logical endpoints. These advances are a result o

technological improvements in the area of envir

tal toxicology and chemistry. The early history 

aquatic toxicology was a period in which the bio

responses of organisms in the field were used to 

the extent of aquatic pollution. During this peri

analytical chemistry methods for measuring env

mentally relevant concentrations of contaminan

were available for relatively few compounds. A

stage in the development of the science, the org

provided the most sensitive measure of environ

contaminants. 

Over the past 15 to 20 years, major advan

analytical chemistry have made it possible to m

very low concentrations of many contaminants 

ronmental samples. These technological advanc

stimulated a period in which the regulation and 

toring of environmental contaminants were driv

largely by the measurement of chemical concen

in water, soil, or organisms. Water-quality criter

developed for a wide range of contaminants by 

information from laboratory toxicity studies to e

acceptable environmental concentrations, but en

ment of water-pollution regulations was based l

upon the measurement of chemicals. During thi

period, efforts were made to develop models to 

biological effects from highway-runoff chemist

and regulatory drinking water and aquatic life s

dards (Horner and Mar, 1985). It was recognize

ever, that these planning-level estimates were sp

to the area for which they were developed. Mon
Factors for Assessing Biological E

and assessment programs also were based on measuring chemical residues in water, sediments, and biota. 

Although these individual chemical-based approaches 

have served as the foundation for advancements in 

point-source pollution control, they also have drawn 

attention to the difficulties associated with collecting, 

analyzing, and interpreting the significance of complex 

mixtures of contaminants that can now be measured.

The most recent trend in environmental pollution 

assessment has been to return to the use of biological 

responses as measures of pollutant effects. However, 

current approaches usually are coupled tightly with 

analytical chemistry for measuring pollutant exposure. 

This integration of the biological and physical sciences 

is particularly important in the arena of nonpointsource pollution, where the goal is to understand the 

environmental significance of complex mixtures of 

diverse arrays of contaminants. In this manner, the scientist or regulator can take advantage of current capabilities to measure low levels of myriad environmental 

contaminants, while relying upon data from the biota to 

integrate the cumulative biological effects of such 

exposure. 

Purpose and Scope

The purpose of this report is to survey tools and 

techniques commonly used for the assessment of the 

biological effects from contaminants in stormwater 

runoff that are available for the study of highway runoff, and other sources of nonpoint-source pollution. 

This report describes the types of contaminants that are 

of particular interest to the study of highway runoff, 

discusses an array of biological assessment techniques 

that are appropriate for use in evaluating the effects of 

these contaminants, and evaluates data-quality issues 

for biological assessment techniques. A review of 

reports that focus on the effects of highway runoff on 

the local ecosystems provides information on (1) the 

suitability of the existing data for a quantitative 

national synthesis, (2) available methods to study the 

effects of highway runoff on local ecosystems, and 

(3) information about the potential for adverse effects 

on the roadside environment and receiving waters. 

FACTORS FOR ASSESSING

BIOLOGICAL EFFECTS

Factors of particular interest in the study 

way runoff are logically those that arise from co

tion, maintenance, and use of the highway syste

Asphalt, concrete, rock, and steel are the prima

highway-construction materials. Road oils, deic

abrasives, solvents, paints, and vegetation-contr

agents are the primary highway-maintenance ma

Highway use results in the introduction of meta

fuels, combustion products, and toxic chemical 

as potential environmental contaminants. Finall

ical habitat disturbance resulting from construc

maintenance, and use of the highway system m

affect biota. General descriptions of the major c

of the factors associated with these activities ar

vided below.

Contaminants of Interest

Many of the contaminants normally assoc

with runoff from the Nation's highways have th

tial for biological effects. Different contaminan

have varying biological effects depending on th

cal and chemical properties of each constituent

concentrations found in an environmental syste

sensitivities of organisms to adverse physical an

chemical characteristics of the runoff, and the ab

the system and the individual organism to assim

given constituent or a given mixture of constitu

Highway-runoff contaminants of particular inte

throughout the United States include deicers, nu

metals, industrial/urban-organic chemicals, sed

and agricultural chemicals from industrial, com

cial, residential, agricultural, and highway sour

Deicers are chemicals used to remove ice 

snow from roadways in the winter. Typically, sa

as sodium chloride and(or) calcium chloride are 

However, other compounds such as calcium ma

sium acetate (CMA) are sometimes used in area

the inorganic salts are a perceived problem. Loa

deicing chemicals per area of the road surface c

substantial on an annual basis, and they can cha

the geochemical conditions normally found in l

receiving waters (Granato, 1996; Bricker, 1999
4 Assessing Biological Effects from Highway-Runoff Constituents

Nutrients are constituents that are assimilated by 

organisms and that promote growth. In water-quality 

studies, the term generally applies to a number of measured constituents that contain nitrogen or phosphorus. 

Depending on the geochemistry of the runoff and the 

receiving water, nitrogen may occur as a nutrient in 

water as nitrite or nitrate anions, the ammonium cation, 

and(or) a number of natural organic compounds (Hem, 

1992; Bricker, 1999). Phosphorus may occur as phosphoric acid, anionic forms including orthophosphate, 

or as a component of organic compounds. Phosphorus 

is generally believed to be the limiting nutrient in many 

aquatic systems because natural geochemical processes 

(Hem, 1992; Bricker, 1999) more readily control phosphorus compounds. Loads of these nutrients and resultant effects on aquatic biota from stormwater can be 

substantial (Grizzard and others, 1980).

Metals are considered ubiquitous contaminants 

today, but were much less abundant on the surface 

before man began to disturb the Earth’s crust. There are 

a variety of biological mechanisms for sequestering 

and managing metals in the body because many metals 

are essential nutrients. The blood and tissues have a 

variety of carrier proteins, such as metallothionein 

(Suzuki, 1982), that regulate required inorganic nutrients, and metals generally show less tendency to accumulate in the body than many organic compounds. 

Metals, however, can pose important environmental 

concerns. One mechanism by which inorganic forms of 

metals tend to cause toxicological problems is known 

as isomorphic substitution—a process through which 

one element substitutes for another but may not be able 

to serve the same function. Organometallics, such as 

methylmercury, may interact with organisms in a 

manner that is similar to organic contaminants. 

Highway-related sources of metals include mobilization by excavation; vehicle wear; combustion of petroleum products; historic fuel additives, such as lead 

(Pb); maintenance materials, such as salts and deicers; 

abrasives; and catalytic-converter emissions.

Organic compounds in highway and road runoff 

that are derived from exhaust, fuel, lubricants, asphalt, 

and other synthetic chemicals are recognized as an 

environmental concern in many highway- and 

urban-runoff studies conducted over the last 20 years 

(Lopes and Dionne, 1998). These compounds are generally classified as semivolatile organic compounds 

(SVOCs) and volatile organic compounds (VOCs). 

Petroleum hydrocarbons, oil and grease, and polycyclic 

aromatic hydrocarbons (PAHs) are generally classified 

as SVOCs. Mono-aromatic petroleum compoun

the fuel additive Methyl tert-butyl ether are gen

classified as VOCs.

Polycyclic aromatic hydrocarbons (PAHs

group of organic compounds that can be formed 

incomplete combustion of a variety of organic s

stances, including petroleum. Although some PA

manufactured for specific uses, such as in pharm

cals, the source of most environmental PAHs is 

natural or anthropogenic combustion. This grou

compounds includes more than 100 individual c

cals that are usually found as complex mixtures 

environment (Agency for Toxic Substances and 

ease Registry, 1995). As their name implies, che

in this group are composed of more than one ar

ring. PAHs are of relatively low molecular weig

generally are volatile and relatively hydrophobi

a tendency to accumulate on airborne particulate 

or to associate with aquatic sediments. Commo

ples of individual compounds in this group incl

naphthalene, anthracene, phenanthrene, chrysen

benzo(a)pyrene.

The mono-aromatic compounds benzene

ene, ethylbenzene, and xylene (BTEX) are com

constituents of crude oil and petroleum product

also are produced as industrial solvents and as p

sors for pesticides, plastics, and synthetic fibers 

(Harwood and Gibson, 1997). The primary sour

BTEX compounds relative to highway pollution 

from spills and leakage of gasoline and other pet

products. Similar to the PAHs, the BTEX comp

generally are volatile and relatively hydrophobi

tend to be associated with storm-runoff sedimen

ground-water contamination.

Methyl tert-butyl ether (MTBE) is an oxy

compound that is added to gasoline to improve 

bustion and to reduce the levels of carbon mono

emissions from automobiles. The total emission 

MTBE from all sources into the environment in 

U.S. in 1992 was estimated to be 3 million poun

(U.S. Environmental Protection Agency, 1994). 

can be expected to increase as a result of the 19

Amendments to the Clean Air Act, which requi

addition of oxygenates to gasoline sold in urban 

that are not meeting target levels for carbon mo

emissions. In a summary of urban stormwater i

cities and metropolitan areas, MTBE was the 7t

frequently detected VOC. MTBE was detected 

about 7 percent of the 592 stormwater samples 

lected from the 16 cities and was detected at a r
Factors for Assessing Biological E

from 22 to 66 percent of samples taken in places where 

MTBE was in current use as a gasoline additive (Delzer 

and others, 1996). MTBE is quite volatile and would be 

expected to dissipate rapidly from soil or water surfaces (U.S. Environmental Protection Agency, 1993a). 

However, MTBE is about 40 times more soluble than 

the BTEX compounds and is less biodegradable than 

many common gasoline hydrocarbons. As a result, it 

is expected to be comparatively more persistent in 

ground water and in the shallow, fast-moving streams 

that are typical of urban and highway-runoff conveyances (Delzer and others, 1996). MTBE has been found 

in ground-water supplies at levels in excess of 

200 milligrams per liter (mg/L) in some locations (U.S. 

Environmental Protection Agency, 1993b), posing a 

potential exposure risk to humans and aquatic life.

Sediment comprises inorganic and organic 

material and can be transported by, suspended in, or 

deposited by stormwater. Suspended sediment is generally considered to be one of the most substantial 

nonpoint-source contaminants (Waters, 1995; 

Crawford and Mansue, 1996). Studies across the 

Nation have documented that sediment can have large 

effects on the biology of receiving waters, ranging 

from the burial of fish eggs to the destruction of the 

entire aquatic food chain (Waters, 1995; Simmons, 

1993). Many contaminants, including some metal ions, 

organic chemicals, and nutrients, are transported by 

sediment (Crawford and Mansue, 1996). Sediments 

have been associated with the destruction of aquatic 

habitat and a decrease in aquatic populations. Even relatively moderate sediment loading to an otherwise 

healthy stream can reduce the variety and abundance 

of aquatic life (Waters, 1995; Crawford and Mansue, 

1996; Simmons, 1993). Sediment loads can also cause 

engineering problems by decreasing the capacity of 

channels and impoundments.

Although they receive only limited use in vegetation control for highway maintenance, herbicides 

and other agricultural chemicals are frequent components of nonpoint-source pollution. When a specific 

product is used intentionally for highway-maintenance 

activities, it is a relatively straightforward process 

to estimate the risk associated with the use of that 

product. Regardless of the actual sources, herbicides, 

pesticides, and other agricultural products frequently 

occur in surface-water runoff and should be given 

consideration in evaluations of nonpoint-source 

pollution.

Other Factors

Although outside the scope of this report

factors may influence the health and abundance 

vidual organisms and biotic communities at a st

site, such as spills of hazardous substances, phy

habitat disturbance, and thermal pollution. Othe

factors of particular interest to the study and int

tion of apparent biological effects of highway-r

quality are contamination and habitat disturban

caused by periodic highway construction and m

nance, hazardous substance spills, and other 

construction/development in the study area. Kn

edge of the potential biological effects caused b

factors is important to assess results of a study t

included in a national or regional characterizati

highway runoff. The effects caused by a spill or 

disturbance from upstream development could 

tially overshadow effects caused by highway-ru

discharges into receiving waters.

The high concentrations of chemicals cau

episodic spills of fuel, lubricants, coolant, and o

chemicals are not normally considered to be ch

istic of highway-runoff constituent concentratio

Spills, however, should be documented because 

can affect measured water quality, and can affec

in receiving waters. Unlike vehicle emissions or 

cals that are intentionally applied during highw

struction and maintenance, the entry of contami

into the environment from spills of hazardous s

stances is much less predictable. Large amount

wide array of hazardous materials are routinely 

ported on the Nation’s highways. Examples inc

fuels, agricultural chemicals, industrial compou

and hazardous-waste products. About 2,400 che

spills on the Nation's highways are reported to F

authorities each year (National Response Cente

1999), and about 7,000,000 traffic accidents are 

reported by police to the National Highway Saf

Administration each year (Cerrelli, 1998, 1997)

McNeill and Olley (1998) noted the effects of s

"routine spills"caused by traffic accidents on 

highway-runoff water quality, and upon stream 

sites in their study area, and concluded that runo

management practice (BMP) structures should 

designed to retain these small spills for cleanup

Because both minor and major spills can bias in

tations about the quality of highway runoff and 

effects of highway runoff on biota, such events 

be tracked and noted as explanatory variables fo
6 Assessing Biological Effects from Highway-Runoff Constituents

data set collected at a given site. Once an accident 

occurs, the volume and types of contaminants that were 

released can usually be readily defined and appropriate 

measures for evaluating the nature and extent of the 

problem can be identified.

Although it is not specifically an environmentalcontaminant concern, the potential for physical habitat 

disturbance in a highway-runoff study area is of great 

interest for site selection. Sedimentation and soil erosion, loss and changes in vegetation, physical habitat 

alteration, and disturbance of wildlife transport corridors are all potential concerns. Some of the most substantial biological changes caused by development are 

directly or indirectly related to altered hydrology. 

Despite efforts to use BMPs to attenuate the hydrologic 

effects of development, increased peak flows and more 

flashy runoff will cause physical modifications to the 

channel shape, bed substrate, and banks of receiving 

waters, with corresponding effects on aquatic habitat 

and biota. Loss of forest canopy, increases in paved 

area, and shallow and(or) muddy detention areas also 

may cause thermal pollution problems, which can 

exacerbate chemical stressors on aquatic organisms in 

receiving waters. All these factors will vary from site to 

site, and will affect interpretation of cause-and-effect 

relations between highway-runoff quality and the 

health and abundance of aquatic organisms in receiving 

waters.

BIOLOGICAL ASSESSMENT 

TECHNIQUES

The presence of measurable quantities of contaminants in the environment is an indicator of potential exposure. Contaminant presence alone, however, 

does not necessarily indicate the occurrence of deleterious biological effects. Measures of exposure must be 

linked to measures of effect in order to establish a 

causal relationship. Biological responses to contaminant exposure are the result of a progression of events 

that can be described as follows: The contaminant must 

first be released into the environment. This may be the 

result of intentional discharge, emission, or application. 

Contaminant input may also be due to unintentional 

releases such as spills, leakages, or other accidents. A 

variety of physical, chemical, and biological processes 

then come into play to determine the ultimate distribution, longevity, availability, and chemical form of the 

contaminant in the environment. The first interaction of 

the contaminant with an organism occurs at the 

lecular level. If the degree of exposure is suffici

elicit a biochemical response, then there is the p

for effects at the tissue, organ, whole-organism, 

lation, and community levels of biological 

organization. 

Each level of biological organization has 

iency, or assimilative capacity, that allows it to m

injury. In order for a contaminant to elicit an eff

the higher levels of organization (e.g., populatio

community levels), it must first exceed the abili

the lower levels to attenuate the response. For ex

a 10 percent inhibition of an enzyme may or ma

cause an observable response at the tissue, orga

whole-organism level. The loss of a finite numb

individuals may or may not be observable as an 

effect on population status for some species. An

finally, due to redundancy of function, the decli

loss of some populations may, or may not, obse

affect the community if other species are able to 

those functional voids. 

These relationships and interdependencie

vide both promise and challenge to the use of b

cal responses as indicators of environmental he

Biochemical changes are the first biological res

that occur, therefore, they may be useful as earl

ing signals of contaminant problems. Effects at 

chemical level, however, may or may not result 

measurable responses at the higher levels of bio

organization. At the other end of the spectrum, 

different physical, chemical, or ecological stress

elicit apparently similar changes at the commun

level. As a result, it is often difficult to establish 

and effect at the higher levels of organization. T

challenge facing the environmental scientist is t

the comparative specificity of responses at the l

levels of biological organization to help establis

causal relationships with the more ecologically 

ingful community levels. Measurement of effec

several levels of biological organization can pro

better understanding of the significance of a con

nant's presence, and can help determine whethe

causal relationship exists. 

In the remainder of this section, a variety 

logical assessment techniques are discussed. No 

attempt has been made to provide an exhaustive 

Literally hundreds, if not thousands, of biologic

endpoints may be either directly, or associativel

affected by contaminant exposure. The techniqu

discussed below should be viewed simply as ex
Biological Assessment Techn

of biological tools that have been shown to be useful in 

evaluating environmental contaminants. Each technique has advantages and drawbacks that must be carefully considered in terms of its suitability to meet 

information needs for local, regional, and national 

studies. A number of the techniques are applicable to 

on-site use in receiving waters, whereas others are 

applied in the laboratory to samples collected in the 

field. In most situations, habitat assessments and analytical chemistry determinations should be made and 

documented in concert with biological measures to 

maximize opportunities for establishing cause and 

effect.

Biochemical, Physiological, and 

Histological Techniques

The principle underlying the use of subcellular 

biological indicators is that in order to elicit a toxic 

response, a chemical must first reach and interact with 

its biomolecular site of action. Whether that receptor 

site is a pore in a membrane, a transport protein on a 

fish gill, an enzyme in the liver of a bird, or any of a 

host of other possibilities, toxicity is always initiated 

on a molecular basis. Toxicants can interact with 

organisms on the molecular level in a wide variety of 

ways that challenge the maintenance of homeostasis 

(physiological stability). Metals can bind to various 

proteins disrupting membrane integrity, ion transport, 

and cellular metabolism. For example, mercury can 

bind to sulfhydryl groups on structural proteins 

(Donaldson, 1980), thereby disrupting membrane 

integrity. Copper (Cu) can interfere with ion regulation 

in fishes by affecting gill ATPase activity (Lorz and 

McPherson, 1976). Lead can deactivate δ-aminolevulinic acid dehydratase (ALAD), disrupting red blood 

cell metabolism (Goering, 1993). Organic contaminants also interact with organisms on the molecular 

level by disrupting membrane integrity and function, 

by displacing endogenous substrates of enzymes, or 

by diminishing energy reserves through increased 

demands on detoxification mechanisms. For example, 

pentachlorophenol can uncouple oxidative phosphorylation by interacting with the mitochondrial membrane 

(Moreland, 1980). Organophosphates and carbamates 

can displace acetylcholine and inhibit its hydrolytic 

cleavage by acetylcholinesterase (ACHe), thereby 

affecting nerve transmission (O’Brien, 1976). 

Widdows and Donkin (1991) report a variety of 

by which toxicants can diminish the energy rese

an organism to the detriment of survival. 

A wide range of biochemical and physiol

parameters are known to be responsive to chem

exposure. However, relatively few of the measu

biochemical and physiological responses are spe

certain chemical classes (for example, ACHe inh

by organophosphates and carbamates; ALAD in

tion by lead). The majority of responses are sens

a wide variety of chemical compounds and othe

ronmental stressors. Thus, with a few exception

biochemical and physiological measures are mo

useful as general indicators of organism health 

than specific indicators of individual chemical e

sure. In order to establish cause and effect, resp

that are general indicators of organism health m

used with other measures of chemical exposure

as chemical residue analysis. The following des

tions address examples of biochemical, physiol

and histological techniques that have been show

useful in evaluating environmental contaminant

Metal Sequestration and Regulation

Metallothioneins are a class of metal-bin

proteins that are found in a wide variety of orga

(Roesijadi, 1992) that are inducible after exposu

Cadmium (Cd), Copper (Cu), Mercury (Hg), an

(Zn) (Noel-Lambot and others, 1978). Metallot

neins are thought to reduce the toxicity of heavy

exposure by sequestration (Hamilton and Mehr

1986), with toxicity occurring only after its bin

capacity is exceeded (Brown and Parsons, 1978

Hepatic metallothionein levels in feral fishes fro

lakes contaminated with Cu, Zn, and Cd have b

closely correlated to metals exposure (Roch and 

1982). The inducible nature of metallothioneins 

gests that they represent a regulatory mechanism 

as such, provide a useful indicator of exposure 

tain metals. Other metal-binding proteins, such 

ferritin (Fe) and copper-chelatin (Cu), have bee

thoroughly studied in aquatic organisms.

Oxidative Metabolism

In organisms composed of more than one 

oxidative metabolism is important for catabolic

production, reduction of oxygen radicals and m

nance of oxidative homeostasis within cells, an
8 Assessing Biological Effects from Highway-Runoff Constituents

foreign compound (xenobiotic) metabolism. The cytochrome P450 monooxygenase (MO) system is one of 

the most extensively studied biochemical indicators of 

contaminant exposure and effects in fish and wildlife 

species (Stegeman and others, 1992). This inducible 

enzyme system has been shown to be responsive to a 

variety of environmental contaminants, including 

PAHs. The MO system plays a central role in detoxification of xenobiotic compounds by modifying their 

structure to a more readily eliminated form. The use of 

markers of oxidative metabolism in biological monitoring studies has been reviewed for aquatic organisms 

(Payne and others, 1987; Goksøyr and Förlin, 1992) 

and wildlife (Rattner and others, 1989). In feral organisms, a number of factors, such as age, sex, reproductive status, diet, disease, and general health conditions, 

can be confounding variables (Neal, 1980). However, 

successful quantitative use of MO activity in the field 

has been demonstrated by using caged organisms 

(Lindström-Seppä and Oikari, 1990) where factors 

affecting MO activity can be controlled or monitored 

during the period of exposure. An alternative means of 

controlling confounding factors is the use of in vitro 

systems. In these assays, extracts from environmental 

samples or SPMDs are tested in bioassays by using cell 

cultures. An example of such an in vitro model system 

for MO activity is the H4IIE hepatoma cell bioassay 

(Tillitt and others, 1991). In this bioassay, environmental contaminant extracts are used to dose the cells. After 

a period of incubation, ethoxyresorufin-o-deethylase 

(EROD) activity in the cells is measured. The MO 

response of the H4IIE cells is highly correlated to 

effects observed in the whole organism (Safe, 1987). 

Studies have demonstrated the ability of the H4IIE bioassay to be predictive of contaminant responses at 

higher levels of biological organization, such as egg 

mortality rates in PCB-exposed populations of fisheating birds (Tillitt and others, 1992).

Reproductive Parameters

Reproduction is another physiological function 

that has been shown to be sensitive to contaminant 

exposure (Thomas, 1990). The complexity of the reproductive process and the confounding influence of 

behavior, nutritional status, seasonality, and other variables make it difficult to ascertain the effects of contaminants on the reproductive success in feral 

populations. However, a number of biochemical 

parameters associated with reproduction have b

shown to be useful as indicators of contaminant 

sure, including levels of vitellogenin and sex-st

hormones (Thomas, 1990). Vitellogenin is the m

yolk protein precursor in non-mammalian verte

Under the influence of estrogen (van Bohemen 

ers, 1982), vitellogenin is synthesized in the liv

transported to the ovaries in the blood. Elevated 

of vitellogenin in male organisms have been lin

the presence of environmental contaminants (Fo

and others, 1996). Similarly, levels of sex-stero

mones (for example, estrogen, testosterone, and 

sors) have been shown to be related to reproduc

status in fish (Mylonas and others, 1997) and ha

shown to be responsive to a variety of environm

contaminants (Thomas, 1990; Folmar and other

1996).

Histopathology

A wide variety of histopathologic change

organisms can be used as indicators of contami

exposure and effects. As with many other biolo

endpoints, confounding variables at the tissue a

lular levels include other environmental stressor

as disease, parasitism, and normal seasonal vari

(Hinton and others, 1992). However, certain tis

have received considerable attention as indicato

contaminant effects. Liver tissue is a primary ta

interest as a result of its role in xenobiotic meta

Fatty infiltration, hepatocyte hypertrophy, vacuo

and other hepatic effects have been related to co

nant exposure (Couch, 1975). Similarly, the kid

has received considerable attention (Meyers and 

Hendricks, 1985) because of its role as a site of 

nation for many environmental contaminants. T

of aquatic organisms are particularly sensitive t

taminant exposure because they serve as an init

substantial site of uptake. Responses include ep

hyperplasia, lamellar fusion, and general necros

(Meyers and Hendricks, 1985; Jagoe and others

Finally, gonads (Hinton and others, 1992) and t

immune system (Anderson and Zeeman, 1995) 

been shown to be sensitive indicators of contam

effects.
Biological Assessment Techn

Tissue Analysis

Chemical analysis of contaminant concentrations 

in biological tissues is widely used as a measure of biological exposure to constituents of concern. Analysis of 

aquatic organisms’ tissues, when used in concert with 

chemical analysis of the water column and bed sediments, provide a direct measure of the bioavailability 

of the constituents of concern. When these chemical 

data are used in conjunction with other biological indicators, effects on individuals and populations of 

aquatic biota can also be examined. Although measured 

constituent concentrations in runoff, sediment, and 

biota would appear to provide a quantitative measure of 

a cause-and-effect relation for biota, the geochemistry 

of the receiving waters will affect bioavailability from 

season to season and site to site (Bricker, 1999), and 

biological characteristics of the different taxa studied 

will determine accumulation rates (Crawford and 

Luoma, 1993). The complexity of the Nation's freshwater environments, as well as differences in surrounding land use, physical and hydrologic features, and 

variations in flow temperature and water quality from 

season to season, complicate the use of tissue analysis 

in a consistent manner on a national or regional scale 

(U.S. Environmental Protection Agency, 1986). Contaminant concentrations are comparable only within 

the same species, and for organisms at the same life 

stage, reproductive condition, size, weight, and sex 

(Crawford and Luoma, 1993). Also, different constituents are accumulated selectively by different tissues 

within each organism, so the type of tissue used for 

analysis is another factor for consideration. Therefore, 

the consistency of results depends upon a program 

design that accounts for these chemical, physical, and 

biological factors.

Many factors must be considered when evaluating use of tissue analysis in studies that are to produce 

data for potential application in local, regional, or 

national synthesis studies (Crawford and Luoma, 

1993). These factors include: (1) measurable tissue 

concentrations that vary with environmental concentrations and exposure therein; (2) uptake of constituents 

that is relatively rapid in comparison to release; 

(3) tissue concentrations with low variability among 

individuals collected, to be representative of site conditions at a given time period; (4) availability of organisms that are hardy enough that can withstand 

contaminant concentrations of interest; (5) organisms 

that do not commonly travel beyond the immed

travel area; (6) organisms that are abundant and 

spread in the study area to withstand repeatable 

for analysis; (7) organisms that can be collected

that are large enough to meet sample size require

(8) organisms that are easy to find and hardy en

withstand captivity during controlled laboratory 

ies; and, (9) a species for which biological and 

logical information is available for comparison

Mollusks, fish, various aquatic invertebrates, an

cular plants have been successfully used in diff

applications for studies that include tissue analy

standard methods for the collection, processing

analysis of organisms within these groups have 

examined in terms of the suitability for a nation

thesis of biological, sediment, and water-quality 

studies (Crawford and Luoma, 1993).

Semipermeable Membrane Devices

The semipermeable membrane device (S

is an in-situ water-sampling device that mimics 

concentration of organic contaminants in lipids 

aquatic organisms (Huckins and others, 1993). 

Although not a biological assessment technique 

it is a powerful sampling technique for sequeste

environmental contaminants for biological asse

ments. The sampler operates passively and cons

thin film of neutral lipid enclosed in a flat, semi

able-membrane tube. Unlike living organisms t

metabolize and excrete many toxic organic con

nants, often eliminating the causal link between 

concentration and adverse effects, these time-w

sampling devices continue to concentrate organ

taminants from water to their maximum partitio

limits (exceeding 1 million fold for certain cont

nants). Also, only biologically available contam

concentrate in the sampler lipid because of the 

size of the pores in the polymeric membrane. T

selectivity contrasts with nearly all currently us

water-monitoring techniques. The compounds t

by the device can be analyzed by routine chemi

analysis or be used as a source of exposure in b

cal assessment procedures. Data from SPMDs, 

ever, must be calibrated in terms of the differen

equilibrium concentrations and the different rat

reaching equilibrium concentration for each con

of interest. 
10 Assessing Biological Effects from Highway-Runoff Constituents

Whole-Organism and

Single-Species Techniques

Whole-organism, or single-species, toxicity tests 

have been widely used to evaluate the effects of environmental contaminants. Mortality, growth, and reproduction are the typical responses measured by toxicity 

tests. Additionally, whole-organism exposures are often 

used to obtain the biological samples required for the 

biochemical, physiological, and histological assessments described above. A summary of historical application of toxicity testing to highway runoff is provided 

by Smith and Lord (1990). Toxicity-testing procedures 

have been developed for a wide range of species, 

including microbes, algae, vascular plants, and aquatic 

and terrestrial invertebrates and vertebrates. Additionally, tests have been designed that can be conducted onsite or in the laboratory with field-collected samples. 

The choice of species and life stage to be tested and 

appropriate routes of exposure are important considerations in whole-organism testing. Species considerations include sensitivity to the contaminants being 

evaluated, availability of healthy specimens for testing, 

ease of culture and maintenance, and local importance 

and relevance to the geographic area being evaluated. 

Generally, early life stages of organisms are more sensitive to contaminant exposure. The routes of exposure 

evaluated in toxicity studies may include water, food, 

air, physical contact, and maternal transfer. The choice 

of exposure route depends upon the properties and 

environmental distribution profiles of the contaminants 

being evaluated. Appropriate environmental samples 

for assessment may include field-collected water and 

sediments or contaminant fractions sequestered 

through sampling procedures, such as the previously 

described SPMDs. Toxicity testing has many benefits, 

but it also has some limitations that should be considered as a component of a national or regional waterquality assessment (Elder, 1990). The following 

descriptions address examples of whole-organism 

toxicity-testing techniques that have been shown to be 

useful in evaluating environmental contaminants. Tests 

of toxicity that are associated with field-collected 

sediments are most effective when used in concert 

with other contaminant-evaluation procedures. The 

Sediment Quality Triad is one such method that combines the power of laboratory sediment-toxicity 

assessment, field measures of benthic invertebrate 

community structure, and analytical chemistry 

uate potentially contaminated sites (Canfield, K

and others, 1994).

Microbial Assays

Microtox® and Mutatox® are microbial a

that use bioluminescent bacteria (Photobacteriu

phosphoreum) to detect the presence of cytotox

genotoxic environmental contaminants. The ass

be used with unprocessed water samples or with 

extracts of various environmental media, includ

water, sediment, and tissue. They can also be us

conjunction with SPMD extracts. Microtox® is 

evaluate cytotoxicity by quantifying reduction i

output as a result of death of the bacteria. The as

been shown to be sensitive to a wide range of to

(Kaiser and Palabrica, 1991; Jacobs and others, 

Mutatox® is similar in concept and procedure, b

a dark mutant strain of Photobacterium phosph

to detect the presence of DNA-damaging chemi

Genotoxicity of a sample is quantified by restor

of light production of bacterial cells upon rever

back to wild-type bacteria. It can be used with a 

suite of environmental media or SPMD extracts

assay has been shown to detect genotoxicity wi

100 chemicals (Johnson, 1992a,b; Ho and 

Quinn, 1993). The relative ease and efficiency o

the Microtox® and Mutatox® procedures make 

them ideally suited for screening large numbers 

environmental samples.

Algal Assays

Algal growth studies have been used to e

the effects of environmental contaminants and n

enrichment on aquatic algal communities. The m

commonly used species is the green alga Selena

capricornutum. A variety of responses can be m

sured, including optical density of exposed cult

oxygen production and(or) carbon dioxide upta

counts, gravimetric cell mass determinations, an

surement of chlorophyll (American Society for 

and Materials, 1997e). Increases or decreases in 

parameters, compared to control responses, are 

determine effects on algal growth.
Biological Assessment Techni

Aquatic Invertebrate Assays

Aquatic invertebrates have been widely used to 

evaluate environmental contaminants that are in receiving waters and aquatic sediments. For evaluation of 

waterborne contaminants, tests with Daphnia magna 

and Ceriodaphnia dubia are most commonly used 

(American Society for Testing and Materials, 1997b,d). 

With both species of aquatic invertebrates, assessments 

of contaminant effects on survival, growth, and reproductive success can be made. Aquatic sediments are 

often chosen as a medium for toxicity testing because 

they are recognized as contaminant sinks. Methods for 

evaluating contaminants that are in sediments have 

been developed for use with several aquatic invertebrate species, including the amphipod Hyalella azteca 

and the midge Chironomus tentans (American Society 

for Testing and Materials, 1997f). As with the waterborne testing procedures, effects on survival, growth, 

and reproduction can be measured. 

Early Life-Stage Toxicity

Studies with Fish

Early developmental stages are often the most 

sensitive to chemical stressors (McKim, 1977). Toxicity studies can be conducted in which fish are exposed 

to environmental contaminants in water and(or) food, 

or by injection of environmental-contaminant extracts. 

Toxicity studies can be conducted with a wide range of 

fish species and can provide information on the shortterm (acute) and long-term (chronic) toxicity of contaminants (American Society for Testing and Materials, 

1997a,c). Egg injection studies are particularly useful 

for evaluating the effects of contaminants on early 

developmental stages. The procedure involves injection 

of contaminants into freshly fertilized eggs and subsequent evaluation of mortality, hatchability, and developmental effects (Walker and others, 1994).

In-Situ Toxicity Assessment

In-situ exposures provide a method for assessing 

the survival and health of organisms in waters from the 

geographic area under evaluation. Duration of these 

tests can extend from 96 hours to 30 days. Early life 

stages of fish and(or) other organisms are exposed 

directly to water from the site of interest, using environmental chambers at the site (Finger and Bulak, 

1988; Hall and others, 1993). Measurements are made 

to evaluate effects on survival, growth, and behavior. 

This type of exposure study is particularly usefu

evaluating pollution effects on species that are i

nous to the site of interest. Results from in-situ 

sures provide information on the actual respons

organisms to mixtures of contaminants as they o

natural systems. When compared with laborator

ity studies with single chemicals, they can prov

insight into the additive or synergistic propertie

contaminant mixtures. Documentation of effect

survival and development of early life stages of 

also allows extrapolation to population-level eff

providing valuable information for assessing the 

contaminants on declining populations.

Population and Community

Techniques

Measures of contaminant effects at the po

tion and community levels focus on the structur

functional properties of the biotic components o

systems. Population and community surveys pr

direct measures of aquatic community structure 

function. Biological surveys can range from eva

tions of single indicator species to evaluations o

proportions of organisms found in an aquatic re

system. Obviously, both the level of resolution 

associated cost of the evaluation increase with th

of complexity of the survey. Application of biod

assessment techniques to transportation project

fully developed but may yield valuable informa

highway planners, scientists, and engineers invo

addressing environmental concerns (Bardman, 

Regional differences in species composition, ha

characteristics, thermal and hydrological regim

water-quality problems must be taken into cons

ation in the design of a nationally consistent eco

assessment program. Fortunately, however, a wi

ety of sources of biological population and com

information for many areas of the country is rea

available from the USGS, the USEPA, the U.S. 

and Wildlife Service, and many State wildlife a

ronmental protection agencies (Gurtz, 1994). Str

analysis techniques seek to determine the abund

and distribution of various taxa as indicators of 

tion and community stress. Presence or absence

numerical abundance or biomass, reproductive 

recruitment success, and spatial distribution of 

tor species are used to determine biotic status 

(Petersen, 1986; Sheehan and others, 1986). 
12 Assessing Biological Effects from Highway-Runoff Constituents

Functional analysis techniques focus more on the ecological processes of ecosystems, such as community 

respiration, nutrient cycling, decomposition rates, colonization rates, and trophic guild analysis (Cairns and 

others, 1972; Rapport and others, 1985; Crossey and 

LaPoint, 1988). 

Well-designed studies, using population and 

community techniques, are a valuable component of 

water-quality studies (Cuffney and others, 1993a,b). 

Population and community techniques relate directly to 

ambient conditions in receiving waters, incorporate a 

large variety of exposure pathways, eliminate the need 

to culture and maintain laboratory test organisms, and 

incorporate secondary effects that arise from unnatural 

changes in predator/prey relations. These techniques, 

however, present a challenge in interpretation of the 

magnitude of effects caused by changes in water quality with respect to effects caused by different natural 

and anthropogenic factors that will also change the 

structure and abundance of different biota in an aquatic 

system.

Measurements of effects at the higher levels of 

biological organization are important in terms of 

clearly demonstrating the ecological relevance of environmental impairment. However, current techniques 

are not specific to contaminant stress. Other forms of 

habitat degradation, interspecific competition, and 

nutrient alteration can cause similar effects at the population and community levels. Therefore, detailed documentation of the characteristics of the local stream 

habitat is necessary (Meador, Hupp, Cuffney, and 

Gurtz, 1993). Physical characteristics of the receiving 

waters, such as the geomorphic channel unit (pool, 

riffle, or run), riparian vegetation, bank stability, water 

temperature, stream depth, current velocity, and bedsediment particle-size distribution, will determine the 

natural ability of a stream reach to support a given population. Therefore, these factors should be assessed 

and documented upon site selection, should be measured concurrently with each assessment, and should 

be similar for control and study sites (Gurtz, 1994). 

Other natural factors such as extremes of flow, life 

cycles of aquatic taxa, and the accessibility of 

in-stream habitats, are important. The USGS National 

Water-Quality Assessment (NAWQA) Program 

annual community assessment work in the summ

low-flow period to avoid sampling soon after flo

events, to obtain samples of algae, aquatic inver

brates, and fish in a mature development stage, 

access stream sites when smaller streams and riv

be waded safely (Cuffney and others, 1993a,b; M

Cuffney, and Gurtz, 1993; Porter and others, 19

Gurtz, 1994). 

In order to establish cause and effect, it is 

ularly important to apply population and comm

techniques in concert with analytical chemistry 

minations and other biological measures of con

nant stress that may be more contaminant speci

Water temperature influences the metabolic and 

ductive rates of algae, benthic invertebrates, and 

Aquatic organisms are also sensitive to changes 

dissolved oxygen, pH and alkalinity, and other 

water-quality properties and constituents. There

an understanding of watershed geochemistry is 

tant to interpretation of population and commun

data.

Different elements of biomonitoring serv

rate but complimentary goals when population 

community techniques are applied to different k

assemblages, including algae, benthic invertebr

and fish. These different taxonomic groups resp

ferently to natural or anthropogenic disturbance

because of differences in habitat, food, mobility

ology, and life history; thus, an approach that u

more than one assemblage adds information tha

used to develop cause-and-effect relations (Gur

1994). Examples of population and community 

assessment techniques are described below.

Algal Population and Community 

Assessments

Algae are an important component of pop

and community assessments because they have 

life span (days to weeks), they readily exchange 

stituents with the water column and sediments, 

reflect local water quality because they are sede

(Porter and others, 1993; Gilliom and others, 19

Algae are operationally defined by physical 
Biological Assessment Techni

characteristics of appearance and size, and by the substrate upon which they thrive. Algal populations are 

typically characterized by the list of taxa present, by 

interpretation of the community structure, and by the 

algal biomass per unit area. The taxon-specific physiological requirements or tolerance for defined ranges of 

water-quality conditions are known for over 3,000 algal 

species; therefore, population and community data can 

be a good measure of water quality (Porter and others, 

1993). Algal populations can be sampled from natural 

surfaces, or—for more consistency between sites—

from artificial substrates emplaced in a receiving water. 

Even when the substrates are designed to closely 

mimic natural surfaces, the population structure may 

not represent the natural population in the surrounding 

habitat. This bias, however, may not be a liability 

when the study objective is to measure the effect of 

highway-runoff quality on algal populations at a 

number of sites. An example of protocols for design, 

sample collection, documentation, and QA/QC for 

population and community assessments of algae on a 

regional and national scale is provided by Porter and 

others (1993).

Aquatic Invertebrate Population and 

Community Assessments

Aquatic invertebrates are an important component of population and community assessments 

because these organisms have longer life spans 

(months to years) than algae, live in close association 

with streambed sediments, and are relatively sedentary 

and good indicators of local water quality (Cuffney and 

others, 1993a,b; Gilliom and others, 1995). Aquatic 

invertebrates are operationally defined as a diverse 

group of taxa that live in, on, or near streambed sediments, including aquatic insects, mollusks, crustaceans, and worms. Aquatic invertebrate populations are 

typically characterized by the list of taxa present, by 

interpretation of the community structure, by the biomass collected by standard methods within a predefined 

study area, and by visible deformities in the specimens 

collected. Aquatic invertebrates exhibit different tolerances to contaminants that are found in sediments and 

the water column (Hamilton and Saether, 1971; Hare 

and Carter, 1976; Wiederholm, 1984; Warwick, 

Some genera are relatively intolerant, and lowcontaminant levels eliminate them from the ben

community, while other genera are more toleran

would only be affected or completely disappear 

higher contaminant levels. Measures of the pres

absence, and abundance of various taxa can be 

an indicator of aquatic community status (Plafk

others, 1989). As with fish population assessme

indices have been developed for assessing aqua

habitat quality by the use of aquatic invertebrat

munity metrics (Hilsenhoff, 1987). Additionally

mities in chironomid larvae have been shown to 

related to the presence of contaminants in 

aquatic sediments (Hamilton and Saether, 1971

Cushman, 1984; Wiederholm, 1984; Warwick, 

1989). Reported deformities include thickening 

exoskeleton, enlargement and darkening of the 

capsule, asymmetric mouth-parts, missing or fu

eral teeth, and deformed antennae. Canfield, Sw

LaPoint (1994) provide a description of conside

regarding the use of benthic invertebrate assess

for evaluation of contaminated sediments. An e

of protocols for design, sample collection, docu

tion, and QA/QC for population and communit

assessments of aquatic invertebrates on a region

national scale is provided by Cuffney and other

(1993a,b).

Fish Population and Community 

Assessments

Fish are an important component of popu

and community assessments because these orga

have long life spans (years to decades), are of in

to the public, are potentially economically valua

highly mobile, and so are indicative of long-term 

watershed health (Meador, Cuffney, and Gurtz, 

Gilliom and others, 1995). Fish exist as a divers

group of species with different preferences for in

habitat. Fish populations are typically character

the list of species present, by physical measurem

of, and by visible anomalies on, the specimens 

lected. One of the more widely used methods fo

evaluating fish community assemblages is the I
14 Assessing Biological Effects from Highway-Runoff Constituents

Biotic Integrity (Karr, 1981; Karr and others, 1986). 

The scientist using the procedure utilizes a number of 

metrics, including such factors as species composition, 

trophic composition, and fish abundance and condition, 

to determine the structural and functional status of the 

fish community. Although the original index was developed for use in midwestern streams, alternative metrics 

have been developed for other regions of the country 

(Miller and others, 1988). An example of protocols for 

design, sample collection, documentation, and QA/QC 

for population and community assessments of fish on a 

regional and national scale is provided by Meador, 

Cuffney, and Gurtz (1993).

DATA-QUALITY CONSIDERATIONS

Data quality, compatibility, and utility are important considerations in biological assessments of environmental contaminants. Findings of an environmental 

assessment must also be readily transferable and useful 

to resource managers and regulators. To meet these 

objectives, supporting ancillary information must be 

available that (1) documents the methods and procedures that are used, (2) describes quality-assurance and 

quality-control procedures that are employed, and 

(3) adequately describes the environmental setting that 

is being evaluated in terms of geographic location, condition, and other confounding variables. Brief discussions of these topics are provided below; more 

thorough discussions are provided by Meador, Hupp, 

Cuffney, and Gurtz (1993); Meador, Cuffney, and 

Gurtz (1993); Gurtz and Muir (1994); and the 

Intergovernmental Task Force on Monitoring Water 

Quality (1995).

Documentation of Methods

Biological assessment techniques are continually 

being developed and refined. New techniques and 

improvements of existing techniques serve to enhance 

our understanding of the implications of environmental 

contaminants. However, this evolution of the science 

makes it increasingly difficult to compare data over 

time. This is of particular concern to long-term and 

broad-scale monitoring and assessment programs that 

draw upon the expertise of a wide range of scientists. 

The exclusive use of published and proven procedures 

would help alleviate this concern, but would im

scientific advancement. Environmental scientist

help resolve this dilemma by thoroughly docum

and describing any new techniques employed a

when practical, by conducting studies designed 

pare the results of new and existing methods (IT

1995). This will help to ensure temporal- and sp

data compatibility and comparability while con

to allow advancement of the science.

Quality Assurance and 

Quality Control

In terms of quality assurance and quality 

biological assessments provide a few unique cha

compared to chemical measurement of contami

residues (USEPA, 1995). The goals are the sam

provide sensitive, reproducible, and transferable 

There are, however, differences in the ways in w

certain performance criteria are addressed. The 

eters that are typically evaluated as indicators of 

include accuracy, precision, bias, detection limi

performance range, interference, and matrix app

ity. In analytical chemistry, these performance p

ters can be addressed by the use of internal stan

and spiked, blind, and blank samples (Jones, 19

Directly comparable procedures do not exist, ho

for many biological endpoints. Precision and bi

be addressed in the usual manner by subsampli

repeated measures of an endpoint, and compari

the results of different techniques. Accuracy, ho

is more difficult to determine given the frequent 

"true" values or applicable standards for biolog

endpoints. To indirectly address accuracy, envir

tal scientists draw upon comparisons of results 

obtained at reference sites, the use of reference 

cants, and an understanding of "normal" ranges 

values for biological endpoints. Detection limit

performance ranges are commonly a function o

range of possible values for a biological parame

rather than a measure of the ability of an instrum

procedure to detect a response. Finally, interfer

and matrix applicability considerations include 

ables, such as disease or other factors that can e

similar biological responses, factors affecting c

nant availability, or other confounding environm

variables. 
Data-Quality Considera

Description of Environmental 

Setting

A final consideration for assuring comparability 

and utility of the results of a biological assessment is an 

adequate description of the environmental setting for 

the site that is being evaluated. It is important to provide descriptive (or explanatory) information that precisely defines the geographic location of the sampling 

effort and that describes pertinent environmental variables that may affect the outcome of the assessment. 

An important factor to be considered in the design of 

biological assessments is the size of receiving waters. 

Important physical and biological characteristics 

change as small streams develop into large rivers. 

Therefore, it is important to compare biological characteristics among streams of similar size. Also, such factors as land-use practices in the watershed, the presence 

or absence of riparian vegetation, streambed substrate, 

geomorphological features, nutrient levels, and the natural geochemistry of the receiving water may influence 

the behavior of environmental contaminants and their 

availability to biota. Additionally, physical and hydrologic habitat characteristics can have a profound influence on the types and quantities of biota that are 

present. This is particularly important because an 

acceptable biological assessment technique must be 

used to discern between natural variability and the 

effects of anthropogenic influences in order to establish 

a cause-and-effect relationship. While these variables 

may seem obvious to the scientist performing the 

assessment, their documentation is essential to the ultimate users of the information (for example, resource 

managers, regulators, and the public).

Comparability Issues for a 

National Synthesis

Contaminants are only one of many poss

environmental stressors that can affect the statu

biota. The confounding variables identified abo

important considerations in any attempt to perfo

national synthesis or to build a national data ba

contaminant effects. To be useful, the results ob

from a biological assessment must be scientific

comparable (among investigators and laboratori

geographically transportable (across States or re

As such, the availability of supporting informat

discussed above, is essential to the process. The 

lated results of a toxicity test or a population as

ment are of little value without this supporting 

information. 

It would be difficult to rank each of the e

techniques discussed in this report in terms of it

to a national assessment. In fact, each has its in

strengths and weaknesses, and it is through the 

combinations of these techniques to provide mu

lines of evidence that they have their greatest ut

However, some general observations on the inh

strengths of the major categories can be made (t

Generally, as one moves from techniques applie

lower levels of biological organization (biochem

level), to techniques applied at the higher levels 

species and community levels), such factors as 

cal specificity and sensitivity tend to decrease. I

trast, responses observed at the higher levels of 

organization tend to have greater ecological rele

and regulatory utility. This relationship, once ag

indicates the importance of using a suite of tech

applied at a variety of levels of biological comp

Table 1. Comparative strengths of categories of biological assessment techniques

Category Relative

cost

Relative

sensitivity

Chemical

sensitivity

Ecological

relevance

Reg

u

Biochemical, physiological, and histological techniques................... Low High High Low Low

Whole-organism and single-species assay techniques ....................... Medium Medium Medium Medium Hig

Population and community techniques............................................... High Low Low High Me
16 Assessing Biological Effects from Highway-Runoff Constituents

BIOLOGICAL EFFECTS OF

HIGHWAY-RUNOFF QUALITY: 

A LITERATURE REVIEW

Relevant journal articles and reports were 

reviewed as part of this study on potential biological 

effects from highway runoff. These publications were 

identified in the National Highway-Runoff Water-Quality Data and Methodology Synthesis (NDAMS) data 

base as having an emphasis on the biological effects of 

highways and highway runoff on local ecosystems 

(Granato, 1997). This review is intended to examine 

existing information in terms of the factors necessary 

for assessing biological effects, available biological 

assessment techniques, and data-quality considerations that may be useful to highway practitioners. This 

is not an exhaustive review of the available highwayrunoff literature, nor is it a survey of the larger body of 

potentially relevant literature on the biological effects 

of urban runoff. This review, however, does represent a 

summary of reports that have a primary emphasis on 

biological effects of highway pollution that were 

readily obtainable in a 2-year literature search of much 

broader scope. Hopefully, the review will also provide 

information to assess current and future needs of highway practitioners in terms of the effects of highway 

runoff on local ecosystems. 

Of the 44 articles and reports reviewed, 32 were 

data/interpretive reports, which documented original 

research, 8 were summary or literature review reports, 

2 were abstracts from conference proceeding poster 

sessions, and 2 were general policy papers (table 2, at 

back of report). Of the 32 data/interpretive reports, 18 

were from studies done within the United States, and 

the remaining 14 were from Canada and Europe. 

Within the United States, the potential effects of highway-runoff quality were examined on different types of 

ecosystems in California, Colorado, Florida, Louisiana, 

Maryland, North Carolina, Ohio, Vermont, Virginia, 

Washington State, and Wisconsin. Fieldwork, reported 

by all of these interpretive studies, spanned a 26-year 

period from 1970 through 1996. About 80 percent of 

the fieldwork was done before the end of 1985. 

Biological studies commonly emphasize different species or taxonomic groups because of the different ways they respond to various environmental 

stresses. For example, differences in life span, mobility, 

and ecological niche among algae, aquatic invertebrates, and fish are useful for examining effects of contaminants at different temporal and spatial scales 

(Gilliom and others, 1995). Among the 32 data/

pretive research reports reviewed, algae were ex

in 11; plants, 6; aquatic invertebrates, 16; fish, 9

amphibians, 1; earthworms, 1; and other organi

Within each group, different species were exam

used as test specimens in the various interpretiv

reports.

Typically, ecological effects of highway-r

quality on receiving waters have been predicted 

statistical models of runoff-contaminant concen

and loadings based upon the event mean concen

of monitored storms (Horner and Mar, 1985; Sm

Lord, 1990; Driscoll and others, 1990; Young a

ers, 1996). These predictive approaches, which 

pare modelled loads and concentrations to publ

regulatory limits, indicate that there should be n

surable effects at sites with annual average daily 

(ADT) volumes below 30,000 vehicles per day 

(Smith and Lord, 1990; Driscoll and others, 199

Young and others, 1996). Biological assessmen

ever, have shown changes in individual organism

community structures, even at sites with relativ

traffic volumes (table 2). 

Although many studies have been done, t

significance of intermittent discharge of highwa

runoff on the ecology and quality of receiving w

not well documented (Dupuis, Kaster, and othe

1985; Maltby, Forrow, and others, 1995; Dupui

others, 1999). It is difficult to assess the effects 

way runoff in a natural setting because increase

ADT are associated with higher levels of backg

contaminant sources from surrounding land use

(Dupuis, Kobriger, and others, 1985).

Factors for Assessing 

Biological Effects 

Biological effects associated with highwa

runoff quality are dependent on the concentratio

availability of the constituents of interest in hig

runoff, on the concentrations and availability in 

ing waters, and on the long-term storage and av

ity in soils and sediments in the vicinity of the h

and in receiving waters. Concentrations and avai

of constituents in highway runoff depend on the 

cal and chemical characteristics of the roadway

ular sources, precipitation, and deposition from 

background sources in the study area (Young an

ers, 1996; Irish and others, 1996). Concentratio
Biological Effects of Highway-Runoff Quality: A Literature Re

availability of constituents in receiving waters depend 

on dilution by receiving waters (Dupuis, Kaster, and 

others, 1985; Horner and Mar, 1985; Driscoll and others, 1990; Cooper and others, 1996), the physical and 

chemical characteristics of local receiving waters 

(Driscoll and others, 1990; Bricker, 1999), the magnitude of background sources (Dupuis, Kaster, and others, 1985; Shively, and others 1986; Davis and George, 

1987), and biological uptake and processing in the 

local ecosystem (Birdsall and others, 1986; Baekken, 

1994; Maltby, Boxall, and others, 1995; Cooper and 

others, 1996; Schafer and others, 1998). Field research 

and resultant highway-runoff quality models indicate 

that, in general, event mean concentrations of pollutants in runoff and receiving waters are not acutely toxic 

(Dupuis, Kaster, and others, 1985; Driscoll and others, 

1990; Maltby, Forrow, and others, 1995; Dutka and 

others, 1998). However, studies also indicate that soils 

and sediments from highway runoff are a reservoir of 

contaminants that can affect the ecosystems near runoff 

discharge points (Portele and others, 1982; Gjessing 

and others, 1984; Mudre, 1985; Maltby, Forrow, and 

others, 1995; Boxall and Maltby, 1997).

Highway runoff contains a complex mixture of 

potentially adverse constituents. These constituents 

include deicers, nutrients, metals, organic chemicals, 

sediment, and potentially, herbicides and pesticides. 

Effects of these different contaminants depend upon 

study location, environmental setting, and the characteristics of the receiving waters (table 2). Cumulative 

effects on biological systems from highway-runoff 

quality include effects of all bioavailable contaminants 

and any interactions among them. 

Deicers were noted as a contaminant of concern 

in 15 of the studies reviewed (table 2). Several studies 

specifically mentioned deicers as a potential problem 

for aquatic ecosystems (Corbett and Manner, 1975; 

Crowther and Hynes, 1977; Dickman and Gochnauer, 

1978; Dupuis, Kaster, and others, 1985). Crowther and 

Hynes (1977), in a controlled experiment, noted a 

chronic effect defined as increased drift in streambed 

organisms in a freshwater trout stream when chloride 

concentrations exceeded 1,000 milligrams per liter 

(mg/L). Dickman and Gochnauer (1978) also did a 

controlled experiment by introducing 1,000 mg/L pure 

sodium chloride into an unpolluted stream during a 4-

week experiment to detect changes in stream microbiota. This study indicated that, when compared to an 

upstream control station, the abundance and diversity 

of algae was suppressed by the salt, but bacterial 

density increased because of suppression of pre

organisms. Adams-Kszos and others (1990) not

toxic effects in toxicity tests using bridge runof

salt concentrations exceeded about 11,000 mg/L

also noted possible synergistic effects between 

and metals that caused toxicity in bridge-runoff 

dilutions. Deicers contain many major and trace 

ments, and can contribute a large percentage of 

solute loads in highway runoff (Harned, 1988; G

1996). 

Metals, as environmental contaminants, w

mentioned in 32 of the studies reviewed (table 2

many studies, increased metal concentrations w

detected in the tissues of animals and plants exp

soils and sediments in highway environments (G

and Christensen, 1973; Corbett and Manner, 19

Dupuis, Kaster, and others, 1985; Birdsall and o

1986; Davis and George, 1987; Baekken, 1994; 

and others, 1996; Dupuis and others, 1999). Alt

these studies did not define the toxicity of the m

these organisms, several studies noted the poten

bioaccumulation of metals higher in the food ch

(Gish and Christensen, 1973; Birdsall and other

Cooper and others, 1996). 

The studies reviewed indicated biologica

mulation of highway-related metals not typicall

ied in the United States, including antimony fro

automobile brakes (Reifenhauser and others, 19

the platinum group elements (PGEs) platinum, 

dium, and rhodium that are associated with cata

converters in exhaust systems (Helmers, 1996; 

and others, 1998). In addition to these German 

PGEs have been measured in elevated concentr

in England (Pearce and others, 1997) and in San 

California (Hodge and Stallard, 1986). PGEs m

concern for future highway research because of 

widespread use of catalytic converters in autom

because PGEs have similar toxicities as copper, 

mium, cadmium, nickel, and zinc (Kaiser, 1980

because PGEs may be available as hydroxide or 

ride complexes (Wood, 1991). PGEs, however, 

normally prevalent in aquatic ecosystems, and i

tion about the chemistry and toxicity of these m

relatively sparse.

Volatile and semivolatile organic compou

(VOCs and SVOCs, respectively) in urban and 

way runoff are a major concern because measur

concentrations can exceed national drinking wa

and aquatic life standards and guidelines (Lope

and Dionne, 1998). One or more of these organ
18 Assessing Biological Effects from Highway-Runoff Constituents

compounds normally associated with vehicle exhaust, 

fuel, lubricants, and(or) asphalt were studied in 19 of 

the reports that were reviewed (table 2). The majority 

of highway-runoff investigations, done by the FHWA 

and the State-highway agencies, analyzed samples for 

volatile suspended solids (VSS), total organic carbon 

(TOC), and oil and grease. Studies that were conducted 

in the 1970’s and early 1980’s, however, did not define 

individual organic chemicals or the major classes of 

organics—VOCs, and SVOCs (including polycyclic 

aromatic hydrocarbons, PAHs)—in highway runoff, 

receiving waters, or biota (Dupuis, Kaster, and others, 

1985; Smith and Lord, 1990; Driscoll and others, 

1990). Many later studies of urban and highway runoff 

from the United States did detect PAHs at concentrations that exceed aquatic health standards (Lopes and 

Dionne, 1998). Several studies by a British research 

group found that PAHs were the most toxic of all the 

highway runoff constituents measured in highwayrunoff sediments collected from a British stream 

(Boxall and others, 1993; Maltby, Boxall, and others, 

1995; Maltby, Forrow, and others, 1995, Boxall and 

Maltby, 1997). A different European study found tissue 

enrichment of organic compounds in benthic invertebrates and fish that was up to five times higher than 

tissue concentrations at background sites (Baekken, 

1994). A recent study in California (Cooper and others, 

1996), however, did not detect PAHs in road runoff, 

receiving waters, and the tissue of aquatic biota at the 

detection limits of 5 parts per billion for the water samples and 2 parts per million for the tissue samples. 

Sediment (solid material that has been transported from its place of origin by erosion) is viewed as 

a major nonpoint-source contaminant throughout the 

United States (Davenport and others, 1991). Sediment 

is considered a problematic contaminant because it 

physically disrupts aquatic habitats and acts as a transport mechanism for other pollutants in aquatic ecosystems (Waters, 1995). Suspended solids and sediment 

were examined as a potential contaminant in 5 of the 

studies reviewed, and sediment was examined as a 

matrix for transport, storage, and release of other 

potential contaminants in 17 of the studies reviewed 

(table 2). In one study, effects of sediment from highway construction along a high mountain stream 

reduced the abundance and diversity of aquatic biota at 

four of seven sites (Cline and others, 1982). The effects 

varied from site to site depending on the local depositional environment and hydrologic effects, such as the 

periodic scouring action of high stream flows at some 

sites in this high-gradient stream. In many studi

iments were mentioned as a potential reservoir 

metals (van Hassel and others, 1980; Wanielista 

others, 1982; Shively and others, 1986) and for 

contaminants (Gjessing and others, 1984; Boxa

others, 1993; Lopes and Dionne, 1998). Ney an

Hassel (1983), concluded that highway-runoff s

ments were a source of pollutants to biota in Vi

streams because fish species associated with sed

had higher metal body burdens than species ass

with the water column. 

Other constituents in highway runoff (tab

that may adversely affect aquatic life include m

ions (in six of the studies reviewed), oxygen de

and oxygen deficit (five studies), nutrients (seve

studies), and herbicides/pesticides (four studies

Although major ions (and many trace elements 

metals) are natural components of natural water

necessary nutrients for aquatic organisms (Hem

a change in the chemical signature of the receiv

waters can affect the local aquatic community (B

1999). For example, Corbett and Manner (1975

a change from calcium-carbonate type to sodium 

chloride-type waters in some receiving streams 

Ohio. In another study, the release of sulphate i

way runoff changed nutrient dynamics and caus

eutrophication in a small Vermont lake by scave

the iron that had removed phosphate from the a

system (Morgan and others, 1984). Oxygen dem

from point sources and urban areas dominated t

logical changes in a study that included highway 

as one source (Davis and George, 1987). The inh

effects of highway-runoff constituents seemed t

uate biological oxygen demand in some laborat

experiments (Portele and others, 1982; Dupuis, 

and others, 1985). However, both stimulatory a

inhibitory effects of highway runoff have been 

recorded in the literature (Winters and Gidley, 1

Dupuis, Kaster, and others, 1985; Dutka and oth

1998). Highway runoff supplies nutrients to rec

waters, but the effect of these nutrients may be 

ated by metals and other contaminants in storm

(Grizzard and others, 1980). Potential problems 

herbicides and pesticides were discussed in sev

studies, but these studies concluded that proper 

agement and timing in the application of chemi

the right-of-way should reduce or eliminate adv

effects (Kobriger and others, 1984; Kramme an

Brosnan, 1985; Shively and others, 1986).
Biological Effects of Highway-Runoff Quality: A Literature Re

Biological Assessment 

Techniques

The biological assessment techniques recorded 

in the highway-runoff research included histopathological techniques in the form of tissue analysis of biological samples collected, bioassays in the form of field 

and laboratory toxicity tests, population and community assessments, and behavioral studies. Of the 44 

reports reviewed, 19 reported results of tissue analysis 

(table 2). The toxicity of soil sediment or runoff was 

assessed in 25 different reports. Plants, algae, aquatic 

invertebrates, and fish of different species were used as 

test subjects in the different toxicity tests. Community 

assessments were an integral part of about 16 of the 

studies reviewed. The drift of benthic species in the 

stream environment was assessed in two separate studies. Biological studies were supported by chemical 

analysis of soil water or sediment in about 35 of the 

studies reviewed. 

Use of tissue analysis is prevalent in the literature relating to highway-runoff quality. In many studies, tissue analysis indicates that contaminants in 

sediments and soils near the highway environment can 

be mobilized within the food chain (table 2). Several 

studies have detected uptake of contaminants into plant 

tissues, and in some cases, changes in the physical 

form and apparent health of plants at sites where sediment and soil had elevated levels of contaminants in 

the highway environment (Wanielista and others, 1982; 

Dupuis, Kaster, and others, 1985; McFarland and 

O'Reilly, 1992; Reifenhauser and others, 1995; Schafer 

and others, 1998). Gish and Christensen (1973) demonstrated potentially toxic concentrations of metals in 

earthworms collected from soils near highways. This 

may indicate the availability of these metals in soils or 

sediments that could be eroded by runoff during large 

storms. Ney and van Hassel (1983) noted that the 

amount of metal uptake depended upon fish species, 

the specific affinities of the different tissues within a 

fish, and site characteristics controlling the availability 

of metals in the local environment. Birdsall and others 

(1986) found that tissue concentrations of lead in tadpoles varied with differences in ADT among 20 sites in 

Maryland and Virginia. Generally, concentrations of 

different highway-runoff constituents in tissue samples 

collected from aquatic communities were elevated near 

highway outfalls when compared to background concentrations in the same species (van Hassel and others, 

1980; Dupuis, Kaster, and others, 1985; Baekke

1994; Maltby, Forrow, and others, 1995; Coope

others, 1996). 

Generally, bioassays of highway runoff d

indicate acute toxicity for aquatic life. In a num

algal assays, Winters and Gidley (1980) found b

stimulatory and inhibitory effects. They attribut

the inhibition to higher metal concentrations in 

samples. Wanielista and others (1982), however

detected only stimulatory effects in algal assays

Portele and others (1982) conducted assays on 

zooplankton, and rainbow-trout-fish fry, they fo

that soluble contaminants affected the algae and 

zooplankton and that suspended solids affected 

trout fry. In assays with heterotrophic microorg

algae, and salmon eggs, Gjessing and others (19

not detect acute toxicity in runoff from a two-la

Norwegian highway. Dupuis, Kaster, and others 

did field and(or) laboratory toxicity tests on a n

of species, including a number of micro- and m

invertebrates, algae, and fathead minnow. The fi

assays were done at three sites—two in rural Wi

and one in a rural watershed in North Carolina—

ADT counts below the predicted 30,000 VPD b

cal effect threshold. These sites were chosen to 

ine the effects of highway runoff without signifi

background contaminant loads (Dupuis, Kaster

others, 1985). Dupuis, Kaster, and others (1985

undiluted highway runoff from melting snow ne

rural highway with low traffic volume (7,400 V

and highway runoff from a busy urban freeway 

(120,000 VPD). Many of these laboratory bioas

however, were done with filtered runoff, or with 

that was slowly circulated from reservoir tanks 

which sediments had settled. These tests did pro

some sluggish behavior in the fish, but no meas

acute toxicity. High mortality in the invertebrat

accompanied by high mortality in control group

there was no clear indication of highway-runoff 

toxicity (Dupuis, Kaster, and others, 1985). The 

bioassays done by Dupuis, Kaster, and others (1

included a number of treatments with different 

ents and the metal chelating agent EDTA. Thes

did not indicate the toxicity of runoff but did in

an inhibitory effect caused by the metals in solu

series of reports from a study in England did no

onstrate acute toxicity of runoff or receiving wat

did determine that PAHs in highway sediments 

be toxic to benthic organisms exposed to these 

inants (Boxall and others, 1993; Maltby, Boxall
20 Assessing Biological Effects from Highway-Runoff Constituents

others, 1995; Boxall and Maltby, 1997). In a review of 

different studies on the toxicities of different deicers, 

including sodium chloride and calcium magnesium 

acetate (CMA), McFarland and O'Reilly (1992) noted 

some toxic effects on plants, invertebrates, and freshwater fish at very high deicer concentrations but lesser 

effects at concentrations expected in receiving waters. 

Adams-Kszos and others (1990) indicated that bridge 

runoff was toxic to sunfish when deicing chemical concentrations were high. Dutka and others (1998) used 

several different standardized bioassay methods on 

bridge runoff from several storms in Ontario, Canada. 

None of the tests produced acute toxicity, but different 

tests indicated genotoxicity for different storms. 

Because of the differing results from storm to storm 

and from test to test, Dutka and others (1998) concluded that a battery of tests was necessary to 

characterize the toxicity of runoff at highway sites. 

 Population and community techniques generally 

indicated local differences between control sites and 

study sites near highway-runoff discharge points. Corbett and Manner (1975) found that when compared to 

control sites, areas affected by highway runoff had 

fewer sensitive species of aquatic plants and animals. 

Dickman and Gochnauer (1978) documented changes 

in stream microbiota that were caused by sodium chloride in a controlled experiment in an otherwise unpolluted stream. Cline and others (1982) documented 

reductions in density, abundance, diversity, and taxonomic structure at four of seven sites that were caused 

by sediment from highway-construction activities. 

Wanielista and others (1982) demonstrated that areas 

affected by bridge drains had fewer species than control areas. Dussart (1984), however, found that areas 

affected by road runoff had higher productivity of algal 

and filamentous organisms. Dupuis, Kaster, and others 

(1985) reported ambiguous results at low traffic sites. 

In this study, some sites receiving highway runoff 

showed no changes in abundance or diversity, but other 

sites, sometimes along the same highway, did show 

effects from the highway runoff. Mudre (1985) also 

reported some effects, but had ambiguous results on a 

new highway with low traffic volumes (ADTs of about 

6,000 to 15,000 VPD). It was also demonstrated that 

the availability of pollutants at these sites was controlled by streamflow, organic carbon content of sediments, and the overland flow distance between the road 

and the stream (Mudre and Ney, 1986). Davis and 

George (1987) found that effects from urban runoff 

overshadowed the effects of highway runoff at several 

stations along a river with a rural-to-urban grad

land use. Baekken (1994) discovered that the di

and abundance of species were lower near a hig

outfall than at background sites, but these effec

localized around the individual discharge points

Maltby, Forrow, and others (1995) documented 

sensitive species and changes in the relative abu

of other species at four of seven sites downstrea

British highway. Cooper and others (1996) did 

detect changes in fish and most invertebrates, b

note reductions in stonefly populations in some 

downstream of the highway. In highway-runoff 

some of the more pronounced effects were appa

among benthic-invertebrate populations. This m

because benthic invertebrates have close associ

with streambed sediments (Cuffney and others, 

1993a,b), and different species have different an

acteristic sensitivities to minor changes in water 

(Kennen, 1998; Newton and others, 1998). In th

way studies, however, differences in population 

community structure may also have been partia

attributable to other explanatory variables that c

changes between control and study sites (Smith 

Kaster, 1983; Cooper and others, 1996). 

Data-Quality Considerations

Careful study design and thorough data-q

documentation are considered necessary to ove

the large variabilities in time and space, as well 

large uncertainties that are inherent in studies o

aquatic biology. For individual studies to be con

valid, current, and technically defensible (to me

information needs of the highway community), 

necessary to document (1) methods, (2) quality 

ance and quality control (QA/QC) information, 

thorough description of the environmental settin

(4) effects of seasonality (Granato and others, 1

is also necessary to standardize protocols for im

menting and documenting these four factors to 

bine existing data, or to design and implement a 

data-collection program for regional and(or) na

synthesis that will meet information needs and 

quality objectives (DQOs). For example, in a lit

review designed to assess potential effects of co

nants from bridges, Dupuis and others (1999) n

that several studies did not provide sufficient do

tation to support definitive conclusions for their 

National Cooperative Research Program study. 
Biological Effects of Highway-Runoff Quality: A Literature Re

implement a regional and(or) national synthesis, it is 

also important to study enough sites to characterize the 

various cause-and-effect relationships that may be 

prevalent in different environmental settings.

Detailed documentation of study methods is necessary to assess existing data sets to ensure that the data 

reported in different studies are comparable and meet 

DQOs for a regional or national synthesis (Granato and 

others, 1998). Methods of sample and data collection, 

analysis, and interpretation were at least partially 

documented in 34 of the 44 reports reviewed (table 2). 

It is difficult, however, to sufficiently document methods with the detail necessary for complete repeatability 

of experimental results; detailed documentation can 

be voluminous. For example, Dupuis, Kaster, and 

others (1985) used 406 pages of text to describe sites, 

methods, and results for studies at two streams and 

one lake. This report also was supplemented with a 

238-page report that described the methods used and 

the comparability among accepted methods (Dupuis, 

Kreutzberger, and others, 1985). Use of national protocols, therefore, may lead to consistent local, regional, 

and national data sets and will reduce the amount of 

documentation required in each report by establishing 

accepted methods at the national level.

 Published protocols are useful in that they establish standard methods, document these methods, and 

provide economy in the presentation of study results. 

For example, USGS's National Water-Quality Assessment (NAWQA) Program has established standard 

methods for virtually every aspect of its biological 

assessment components, including site characterization 

(Meador, Hupp, Cuffney, and Gurtz 1993; Fitzpatrick 

and others, 1998), tissue analysis (Crawford and 

Luoma, 1993; Nowell and Resek, 1994; Hoffman, 

1996), and community assessment techniques (Cuffney 

and others, 1993a,b; Meador, Cuffney, and Gurtz, 

1993; Porter and others, 1993). Required methods and 

resultant protocols should be driven by program objectives; however, method documentation, adequate methods comparison, systematic data-base structures, and 

collaborative efforts among agencies collecting environmental data will open up many opportunities for use 

of existing and intensive biological assessment data 

bases (Gurtz and Muir, 1994; ITFM, 1995). For example, the State of New Jersey has a network of more than 

700 stream sites used for macroinvertebrate community 

assessment that are distributed among different landuse categories in the six physiographic regions of the 

State (Kennen, 1998). This data set includes a number 

of minimally disturbed areas in each physiograp

region that can be used as regional reference/co

sites. 

Documented methods are an important p

necessary quality-assurance programs, and qua

control documentation ensures that methods me

quality objectives. Therefore, both quality assur

and quality control (QA/QC) are important com

nents of study design and documentation (Jones

Compared to performance standards for chemic

ratories, objective statements of method accurac

biological field methods are not quantitative (IT

1995). QA/QC factors typically reported for ch

studies such as precision, bias, performance ran

interferences, and method detection limits, how

can be defined in the context of biological asses

methods if methods and protocols are well estab

and method comparability studies are properly 

mented. For example, Lenz and Miller (1996) c

pared five different aquatic-invertebrate sample

collection methods at six sites in Wisconsin and 

that the equipment and methods used affect the 

of and the relative abundance of different specie

collected. 

Most of the highway studies with a biolo

component incorporated some form of QA/QC 

mentation because of the variability of biologic

tems; thus, the need for documented experimen

controls is recognized. There was some mention 

quality-assurance and quality-control measures 

but 12 of the 44 reports reviewed. Documentati

replicate samples was included in 21 of the repo

reviewed (table 2). Use of control sites, or exper

control samples was mentioned in 16 of the stu

reviewed. Use of experimental standards was do

mented in six studies. Other forms of QA/QC w

documented in 11 studies. For biological studie

ical component of the QA/QC and documentati

cess is proper selection of and complete 

characterization of the study sites. 

In studies of the biological effects of high

runoff quality, detailed descriptions of the envir

tal setting are necessary to interpret and standar

results. In most studies, the site characteristics w

described as confounding factors that influence

results. Cline and others (1982) identified site-h

lic factors as the controlling variables that deter

the effects of sediment on biota in a high-gradie

mountain stream. In a study of benthic inverteb

Smith and Kaster (1983) found that differences 
22 Assessing Biological Effects from Highway-Runoff Constituents

streamflow and the composition of stream substrate 

could overshadow effects of runoff on population characteristics at sites receiving runoff from highways with 

low traffic volume (ADTs less than 10,000 VPD). In 

another study, tissue lead content in tadpoles increased 

with increasing ADT on different highways among 20 

sites in Maryland and Virginia (Birdsall and others, 

1986). In this study, however, analysis of the differences between sites indicated that seasonality, microtopography (erosive or depositional environments), the 

residence time and episodic flushing of water and sediments, and differences in site sediment characteristics 

introduced variability in the relation between ADT and 

lead concentrations in tadpoles. The site characteristics 

of topography (stream gradient), flow velocity, sediment organic matter, and the overland flow distance 

from the pavement to the stream controlled the availability of highway-runoff related metals in sediment at 

six stream sites near a new highway in Virginia (Mudre 

and Ney, 1986). 

Descriptions of the study-site characteristics 

were inadequate for use in a quantitative national synthesis for most of the studies reviewed because this 

information must be assessed using constant methods 

and documented in consistent formats from site to site 

for regional or national interpretation. Dupuis, Kaster, 

and others (1985) fully documented the highway characteristics that they thought would be explanatory variables controlling the quality of storm-water runoff. 

These characteristics include traffic volume, highway 

type, number of lanes, existence of curbs, pavement 

material (concrete or asphalt), and the percent of paved 

and nonpaved area contributing to the highway-runoff 

drainage system. Dupuis, Kaster, and others (1985) 

also characterized many of the physical hydrologic features of the study sites, including historical average 

annual precipitation, precipitation and stream flow 

during the study period, watershed area, stream gradient, watershed land use, and other potential sources of 

point and nonpoint pollution that are commonly recognized as explanatory variables for biological studies. 

Many of the site characteristics necessary to compare 

results among sites, however, were not sufficiently documented in this or the other 44 highway-runoff reports 

reviewed. 

To examine the effects of highway-runoff waterquality runoff on biological systems at different sites, it 

is necessary to document many site characteristics that 

can be used to relate habitat to physical, chemical, and 

biological factors. Factors not characterized by most 

highway studies include elevation, sinuosity (to

graphic channel pattern), active channel width (

stream width at bank-full discharge), channel co

tions and features (such as runs, riffles, and poo

bank stability, water-management features, inst

fish cover, canopy cover, and detailed descriptio

the dominant substrate (boulders, cobbles, grave

silt, and(or) mud). Detailed diagrammatic mapp

stream features is also considered necessary for 

preting results of bioassessment studies (Meado

Hupp, Cuffney, and Gurtz, 1993; ITFM, 1995; 

Fitzpatrick and others, 1998; Newton and other

1998). To facilitate a national synthesis, other f

are also considered important, such as site ecor

location in latitude and longitude, classification 

nationally recognized system of stream-order n

watershed identification (such as the USGS hyd

unit code), watershed soils, surficial and bedroc

ogy, physiography, potential vegetation, and up

wetlands (Fitzpatrick and others, 1998). Fortun

methods for site assessment and characterizatio

well documented and much of the large-scale in

tion is available in existing geographic informat

tems if the latitude and longitude of a site is kno

(Meador, Hupp, Cuffney, and Gurtz, 1993; ITFM

1995; Fitzpatrick and others, 1998; Newton and 

1998). The USGS national bridge scour data ba

information about 27,000 bridge sites in 12 stat

also a good source of stream-site data, includin

land use, stream width, surface profile, vegetati

and bank material, channel profile, depth, erosi

environment, and location information (G.W. P

USGS, oral comun., 1999). 

It is necessary to have sufficient and cons

data from enough different sites to characterize 

atory variables to interpret biological and water-

data on a regional or national scale. Integration 

logical and water-quality data is often hindered 

of consistent data and the small number of sites 

which both types of data are available (Kennen

Driscoll and others (1990) developed a highway

quality model using precipitation, average annu

streamflow, and total hardness of surface waters 

predefined zones within the conterminous Unite

States to predict pollutant loadings and ecologic

effects from highway-runoff quality at any give

To determine the number of sites needed to esta

sufficient national data set, it is necessary to exa

the problem in terms of the number of regions n

to quantify each of these explanatory variables. 
Sum

define pollutant loadings, Driscoll and others (1990) 

used estimates of average concentrations from highway-runoff studies and estimated runoff volumes from 

9 rainfall zones defined by average annual storm characteristics, including duration, intensity, volume, and 

antecedent dry period (which have been revised to 15 

rainfall zones by the USEPA, 1992). Driscoll and 

others (1990) used 18 divisions of average annual 

streamflow to estimate dilution in receiving waters. 

These divisions were defined in a geometric progression from 0.05 to 5.00 cubic feet per second per square 

mile of drainage area, and when applied nationally, 

these divisions are used to define 30 zones in the conterminous United States. Driscoll and others (1990) 

estimate the geochemical availability and effect on 

aquatic organisms by pollutants in receiving waters 

using 6 divisions of hardness defined in 5 even increments from less than 60 to greater than 240 parts per 

million as calcium carbonate. This method of 

geochemical classification yields 24 different hardness 

zones within the conterminous United States. When 

these three variables are combined, they produce about 

140 distinct zones of precipitation, dilution, and 

geochemistry within the conterminous United States. 

To characterize effects on biota, it is also necessary to 

characterize habitat features. The ecoregion approach 

is often used for classification of similar habitat features on a national scale, and about 76 different ecoregions are defined as individual zones within the 

conterminous United States (Omernik, 1995; ITFM, 

1995). Superposition of these zones upon the zones of 

precipitation, dilution, and geochemistry defined, by 

Driscoll and others (1990), would greatly increase the 

number of distinct zones to be studied, and would still 

define site characteristics in a rather coarse manner.

Ecological systems are subject to variability in 

time, as well as to variability in space. Effects of seasonality were discussed in 16 of the 44 studies 

reviewed (table 2). There are two distinct approaches to 

the problem of seasonality. One approach is to control 

for seasonality by collecting all samples within prescribed seasonal conditions. This approach assumes 

that the biological community, if studied under similar 

conditions from year to year, will integrate adverse 

effects throughout the year. Investigations using this 

approach often involve sampling during summer lowflow conditions because aquatic organisms are at a 

good point in their life cycle for sampling and analysis, 

and the low flows facilitate sample collection in the 

streambed (M.R. Meador, USGS oral comun., 1999). 

Another approach is to attempt a detailed chara

tion of the effects of runoff during different sea

For example, examination of the effects of deic

chemicals on aquatic ecosystems would be the 

factor for sampling during the winter in highwa

runoff studies; in fact, 12 of the 16 studies that 

cussed seasonality also addressed deicers as a p

contaminant. 

There are also long-term temporal conside

when evaluating effects of highway runoff on b

Mudre and Ney (1986) demonstrated that the av

ity of metals (in highway-runoff sediment depo

was a function of total annual precipitation. Dur

study in Massachusetts, differences in temperat

precipitation caused loads of deicing chemicals 

from 50 percent to 200 percent of the median lo

a 5-year period (Granato, 1996). Studies should 

enough to document potential effects and(or) in

data within the context of long-term weather rec

the study area (Averett and Schroder, 1994; Gra

and others, 1998). Among the 32 data/interpreti

reports reviewed, the duration of fieldwork at ea

was greater than 1 year in 14 reports, was 1 year 

in 15 reports, and was not documented in 3 repo

(table 2). 

SUMMARY

Advancements in our understanding of th

mechanisms through which contaminants exert 

effects have stimulated interest in using biologi

techniques for environmental pollution assessm

monitoring. Although many biological endpoin

been shown to be responsive to contaminant ex

relatively few are specific to individual chemica

single biological endpoint can fully define the s

cance of environmental pollution at a given site

has its particular strength and weakness. Bioche

assays may be most useful for demonstrating di

effects from contaminants; studies with single s

may help minimize confounding environmental 

ables; and population and community assessmen

be the most appropriate techniques for estimatin

ecological significance of contaminant perturba

However, when used in conjunction with analyt

chemistry and habitat assessment as multiple lin

evidence, biological assessments can help provi

cause-and-effect linkages between environment

pollution and ecological impairment. 
24 Assessing Biological Effects from Highway-Runoff Constituents

A review of 44 reports on the biological effects 

of highway runoff on local ecosystems reveals several 

information gaps. The use of different methods from 

study to study and a general lack of sufficient documentation preclude making quantitative comparisons 

among different studies using the existing data. Qualitatively, the literature indicates that constituents from 

highway runoff and from highway-runoff sediments 

deposited in receiving waters near the highway are 

found in the tissues of aquatic biota, and that these 

sources may affect the diversity and productivity of 

biological communities, even though bioassays would 

suggest that highway runoff is not often toxic to 

aquatic biota. To provide the quantitative information 

needed, it is necessary to obtain information using 

standard methods, and to document study results in a 

manner that will be useful for a national or regional 

synthesis. 

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TABLE
34 Assessing Biological Effects from Highway-Runoff Constituents

Table 2. Documented metadata for reports examining the effects of highway runoff on biota in receiving water—Contin

Reference Report

type

Dates of data

collection Location/ environmental setting

Adams-Kszos and others, 1990............... D/I 09/1983–05/1985 New York/Lake Chautauqua, Bridge scupper s

Baekken,1994.......................................... D/I 1991 Oslo, Norway/2-lane highway, high hardness 

Norwegian lake

Bardman, 1997........................................ Policy NA Continental United States

Birdsall and others, 1986 ........................ D/I 07/1982–12/1982 Maryland and Virginia/highway, receiving wa

control sites

Boxall and others, 1993 .......................... P/A ND ND/streams affected by highways

Boxall and Maltby, 1997......................... D/I 10/1993, 01/1994–04/1994 England/streams crossed by M1 motorway

Cline and others, 1982 ............................ D/I 1975–77 Rt. 14 Colorado/high mountain stream

Cooper and others, 1996 ......................... D/I 03/1995–05/1996 California/streams adjacent to roadways

Corbett and Manner, 1975....................... R/S 1975 (approx.) Ohio/roadsides

Crowther and Hynes, 1977...................... D/I 12/1973–02/1975 Canada, Great Lakes region/rural “trout” strea

stream

Davis and George, 1987.......................... D/I 1978–80 England/river on rural-to-urban gradient with 

urban, and highway inputs

Dickman and Gochnauer, 1978............... D/I 07/1973–10/1973 Quebec, Canada/pristine stream draining hum

in a wilderness park

Dupuis, Kaster, and others, 1985 ............ D/I 07/1981–10/1983 (each 

study done in sequence 

about 1 year in duration)

Wisconsin, North Carolina/highways in rural 

lake and piedmont regions

Dupuis and others, 1999.......................... R/S ND worldwide studies

Dussart, 1984 .......................................... D/I 03/1976–04/1976 England/streams by a motorway

Dutka and others, 1998 ........................... D/I ND Burlington Ontario/highway-bridge runoff

Ellis and others, 1997.............................. R/S ND England/receiving waters

Gish and Christensen, 1973 .................... D/I 1970 Maryland and Virginia/highways, small stream

ponds (most near highways)

Gjessing and others, 1984 ....................... D/I 1981 Oslo, Norway/2-lane highway (laboratory ass

Helmers, 1996 ......................................... D/I 07/1992–08/1994 Germany/right-of-way of Autobahn A8

Table 2. Documented metadata for reports examining the effects of highway runoff on biota in receiving water

[ADT, average daily traffic; BMP, Best-management practice; D/I, data/interpretive; NA, not applicable; ND, not discussed; P/A, poster/abstract; 
Ta

Reference Chemical analyte(s) Chemical analysis 

matrix Seasonality

Adams-Kszos andothers, 1990............. deicers, properties, 

metals

water yes 

Baekken,1994....................................... whole-effluent toxicity biota, water ND

Bardman, 1997..................................... NA ND ND

Birdsall and others, 1986 ..................... metals biota, sediment ND

Boxall and others, 1993 ....................... metals, organics biota, sediment ND

Boxall and Maltby, 1997...................... organics sediment yes

Cline and others, 1982 ......................... total suspended solids water ND

Cooper and others, 1996 ...................... metals, organics biota, water ND

Corbett and Manner, 1975.................... deicers, majors, metals water yes (sodium chloride effects on wa

quality)

Crowther and Hynes, 1977................... deicers water yes

Davis and George, 1987....................... oxygen demand, metals biota, water ND

Dickman and Gochnauer, 1978............ deicers water ND

Dupuis, Kaster, and others, 1985 ......... deicers, metals, organic 

carbon

biota, sediment, water yes

Dupuis and others, 1999....................... deicers, metals, organics biota, sediment, water 

(dissolved and whole)

ND

Dussart, 1984 ....................................... NA (several mentioned 

but never tested)

biota yes (“study took place at the end o

winter, so the maximum possibl

impacts of events, such as road 

would be identifiable.” No comp

study was done in the summer.)

Dutka and others, 1998 ........................ whole-effluent toxicity water ND

Ellis and others, 1997........................... deicers, nutrients, metals, 

solids, organics, 

herbicides

biota, sediment, soil, water deicers

Gish and Christensen, 1973 ................. metals biota, soil ND

Gjessing and others, 1984 .................... organics, whole-effluent 

toxicity

suspended solids, water 

(filtered)

One November snowmelt event in

Helmers, 1996 ...................................... metals biota ND

PGE, platinum group element; R/S, review/summary; VPD, vehicles per day; ft, foot; mg/L, milligrams per liter; ppm, parts per million; %, percen
36 Assessing Biological Effects from Highway-Runoff Constituents

Horner and Mar, 1985 ............................. R/S 1977–82 Washington State/various highways

Karouna-Renier and Sparling, 1997........ D/I 06/1994 Maryland/retention ponds collecting stormwa

various land uses including 3 highway sites

Kobriger and others, 1984....................... R/S NA ND/wetlands

Kramme and Brosnan, 1985.................... D/I 07/1984–11/1984 Ohio/rural highway, grassy right-of way (I-71

semirural highway (I-90), rural, 2-lane road

Madigosky and others, 1991 ................... D/I 05/1989–06/1989 Louisiana/Roadside ditches in rural areas

Maltby, Boxall, and others, 1995 ............ D/I ND England/stream near M1 motorway

Maltby, Forrow, and others, 1995 ........... D/I 10/1990–07/1991, 04/1993 England/streams crossed by M1 motorway

McFarland and O’Reilly, 1992................ R/S ND ND/various field and laboratory studies

McHardy and George, 1985.................... D/I 01/1977–05/1979 England/river on rural-to- urban gradient with 

urban, and highway inputs

McNeill and Olley, 1998......................... D/I 03/1995–05/1996 Southwest Scotland, highway outfalls on sma

streams crossed by the A74 motorway 

Moore and Butler, 1994 .......................... R/S ND ND

Morgan and others, 1984 ........................ D/I 02/1981–12/1982 Vermont/lake surrounded by forest and limite

residential land use

Mudre, 1985............................................ D/I 1981–83 Virginia/6 streams with sites upstream, at the 

and downstream of a new highway (I-295)

Ney and van Hassel, 1983....................... D/I 04/1978–02/1979 Virginia/downstream of highway bridge

Portele and others, 1982.......................... D/I 1980–81 Seattle, Washington/highways

Reifenhauser and others, 1995................ P/A ND Germany/highways and roadways

Schafer and others, 1998......................... D/I ND Germany/plants grown in greenhouse in soils 

from roadside

Shively and others, 1986 ......................... D/I 01/1984–08/1985 Virginia/stream near highway, as well as urba

Smith and Kaster, 1983 ........................... D/I 06/1980–06/1981 South Wisconsin/State Route-15, rural stream

Smith, 1976 ............................................. R/S ND world-wide/roadside ecosystems (within 300 

roadway)

Thrasher, 1983......................................... Policy NA Continental United States/wetlands

van Hassel and others, 1980.................... D/I 04, 07, 11/1978 and 02/1979 Southwest Virginia/montain region

Table 2. Documented metadata for reports examining the effects of highway runoff on biota in receiving water—Contin

Reference Report

type

Dates of data

collection Location/ environmental setting
Ta

Horner and Mar, 1985 .......................... organics, oxygen 

demand, metals

water ND

Karouna-Renier and Sparling, 1997..... majors, metals, organics 

and properties 

sediment, pond water ND

Kobriger and others, 1984.................... deicers, metals, nutrients, 

organics, pesticides

biota, sediment, water deicers

Kramme and Brosnan, 1985................. herbicides, organics, 

properties

water (whole) ND

Madigosky and others, 1991 ................ metals tissue ND

Maltby, Boxall, and others, 1995 ......... metals, organics biota, sediment ND

Maltby, Forrow, and others, 1995 ........ metals, organics bed sediment, runoff Yes

McFarland and O’Reilly, 1992............. deicers, oxygen demand, 

metals

soil, water ND

McHardy and George, 1985................. metals tissue, water ND

McNeill and Olley, 1998...................... deicers, metals, organics, 

sediment

water deicers

Moore and Butler, 1994 ....................... metals, nutrients, 

organics, total 

suspended solids

water ND

Morgan and others, 1984 ..................... deicers, majors, metals, 

nutrients

atmospheric deposition, 

sediment cores, water

yes (limited)

Mudre, 1985......................................... metals biota, sediment ND

Ney and van Hassel, 1983.................... metals, properties biota ND

Portele and others, 1982....................... oxygen demand, metals, 

nutrients

biota, street sweepings, 

water

ND

Reifenhauser and others, 1995............. metals biota ND

Schafer and others, 1998...................... metals biota (plants), soil NA

Shively and others, 1986 ...................... deicers, metals, nutrients, 

organics, pesticides

sediment, water yes (discussed per parameter)

Smith and Kaster, 1983 ........................ deicers, metals, 

properties

biota, sediment, water yes

Smith, 1976 .......................................... metals atmosphere, biota, soil ND

Thrasher, 1983...................................... NA NA ND

van Hassel and others, 1980................. metals biota, sediment, water yes

Reference Chemical analyte(s) Chemical analysis 

matrix Seasonality
38 Assessing Biological Effects from Highway-Runoff Constituents

Wanielista and others, 1982 .................... D/I 05/1981–05/1982 Central Florida/bridges over streams

Winters and Gidley, 1980........................ D/I 1976–77 California/roadways over streams and cut-slop

Table 2. Documented metadata for reports examining the effects of highway runoff on biota in receiving water—Contin

Reference Biota studied Method Doc

me

Adams-Kszos and others, 1990............... fish toxicity testing yes

Baekken,1994.......................................... benthic invertebrates, bivalves, fish community assessment, tissue analysis yes

Bardman, 1997........................................ land (terrestrial) species community assessments ND

Birdsall and others, 1986 ........................ tadpoles tissue analysis yes

Boxall and others, 1993 .......................... benthic invertebrates toxicity testing (using sediment and 

sediment extracts)

ND

Boxall and Maltby, 1997......................... benthic invertebrates toxicity testing yes

Cline and others, 1982 ............................ benthic algae, lichen, benthic 

invertebrates, moss

community assessment yes

Cooper and others, 1996 ......................... benthic invertebrates, fish tissue analysis yes

Corbett and Manner, 1975....................... benthic fauna and vegetation community assessment ND

Crowther and Hynes, 1977...................... benthic invertebrates toxicity testing, field and lab 

assessment of drift

yes

Davis and George, 1987.......................... benthic invertebrates, plant tissue community assessment, tissue analysis yes

Table 2. Documented metadata for reports examining the effects of highway runoff on biota in receiving water—Contin

Reference Report

type

Dates of data

collection Location/ environmental setting
Ta

Wanielista and others, 1982 ................. deicers, majors, metals, 

nutrients, organics

biota, soil, water ND

Winters and Gidley, 1980..................... deicers, majors, metals, 

nutrients, organics

water study took place in the winters of 

1977

Reference Quality

assurance Comments

Adams-Kszos and others, 1990............... reagent control Found that a 50% dilution of bridge runoff was toxic to sunfi

especially for winter samples with salt concentrations gre

11,000 mg/L.

Baekken,1994.......................................... controls, replicates Tissue analysis and community assessment indicated chroni

adjacent to highway runoff outfall.

Bardman, 1997........................................ ND Described methods to assess potential effects of bisecting ha

transportation corridors.

Birdsall and others, 1986 ........................ replicates Lead concentration in frogs is correlated with traffic density 

sediment concentrations. Concentrations in frogs are high 

affect predators.

Boxall and others, 1993 .......................... controls Sediments from a site down gradient of a highway are toxic 

benthic invertebrate Gammarus pulex.

Boxall and Maltby, 1997......................... replicates PAHs are the major toxins in sediment extracts from sites af

highway runoff. Did spikes to determine that pyrene acco

approx. 45% of total toxicity (fluoranthene, 16%; phenant

3.5%)

Cline and others, 1982 ............................ reference sites, replicates Benthic communities and microinvertebrates were affected b

sediment from construction, reducing the density, abundan

diversity, but high flows in this high gradient stream remo

sediment deposits and therefore, limited damage spatially 

temporally.

Cooper and others, 1996 ......................... replicates, clean 

containers, container 

blanks, field and trip 

blanks, field reagent 

spike

The water quality of the stream, and analysis of tissues indic

the ecosystem was not affected by runoff from repaved 

asphalt/concrete pavement surfaces.

Corbett and Manner, 1975....................... control site Sensitive species of benthic fauna or vegetation were not fou

highway site.

Crowther and Hynes, 1977...................... controls, replicates Chloride pulses greater than or equal to 1,000 mg/L seemed 

threshold to cause increased drift of species in the field. S

concentrations of 2,500 mg/L produced about 20% mortal

laboratory.

Davis and George, 1987.......................... replicates Analysis of plant and animal tissue related to the chemistry 

water and subtle enough to relate to low-level secondary m

impacts.

Reference Chemical analyte(s) Chemical analysis 

matrix Seasonality
40 Assessing Biological Effects from Highway-Runoff Constituents

Dickman and Gochnauer, 1978............... algae, bacteria, other microbiota community assessment on artificial 

substrate

yes

Dupuis, Kaster, and others, 1985 ............ algae, fish, benthic invertebrates community assessment, field toxicity, 

lab toxicity, tissue analysis, drift

exten

Dupuis and others, 1999.......................... aquatic invertebrates, fish, plants community assessment, field toxicity, 

lab toxicity, tissue analysis

ND

Dussart, 1984 .......................................... algae community assessment yes

Dutka and others, 1998 ........................... assorted species a battery of toxicity tests, full strength 

and concentrated runoff

ND

Ellis and others, 1997.............................. aquatic invertebrates community assessment, tissue analysis ND

Gish and Christensen, 1973 .................... earthworms tissue analysis yes

Gjessing and others, 1984 ....................... algae, heterotrophic microorganisms 

(bacteria, fungi, protozoa), fish 

acute toxicity tests (full strength and 

several dilutions)

yes

Helmers, 1996 ......................................... plants tissue analysis yes

Horner and Mar, 1985 ............................. NA compare estimated concentrations to 

regulated standards

ND

Karouna-Renier and Sparling, 1997........ aquatic invertebrates toxicity test yes

Kobriger and others, 1984....................... numerous wetland species NA NA

Table 2. Documented metadata for reports examining the effects of highway runoff on biota in receiving water—Contin

Reference Biota studied Method Doc

me
Ta

Dickman and Gochnauer, 1978............... control sites, replicates, 

standard methods, 

intermethod 

comparison

Did controlled experiment in an unpolluted stream. Introduc

sodium chloride at 1,000 ppm for 4 weeks. Salt reduced a

and diversity of algae. Bacterial density was enhanced by 

suppression of predator organisms.

Dupuis, Kaster, and others, 1985 ............ experimental controls, 

dry and wet weather 

sampling, replicates, 

compared

Did not demonstrate acute toxicity of highway runoff. Did o

some effects but not conclusively. Observed effects limited 

locations.

Dupuis and others, 1999.......................... ND Questions traditional long-term continuous exposure method

toxicity tests. Notes that site specific criteria (based on loc

geochemistry) may improve evaluation of water-quality cr

Noted that several of the reports reviewed did not contain 

documentation to evaluate results.

Dussart, 1984 .......................................... replicates Motorway runoff can affect algal flora, but further analysis is 

identify the form of the effect.

Dutka and others, 1998 ........................... replicates, standard 

methods

Tested 9 bioassay methods using samples from the beginnin

of different storms. Found different results for different te

and between storms. Did not detect acute toxicity, but did 

chronic effects (genotoxicity).

Ellis and others, 1997.............................. ND Referenced several British studies where highway runoff fro

with an ADT greater than 25,000 VPD increased trace-me

organic chemical body burdens in aquatic invertebrates an

species diversity near highway outfalls. Reported immedi

affects for chemical spills, but delayed chronic effects from 

contaminated sediment.

Gish and Christensen, 1973 .................... controls, replicates Detected elevated concentrations of metals in worms and soi

highways. Concentrations decreased with distance from th

edge, and decreasing traffic volume. Determined that ther

bioconcentration from soil. Concentrations of lead in wor

toxic to predators in food chain.

Gjessing and others, 1984 ....................... controls, replicates Used runoff from a two-lane highway on several species. Tox

using fish eggs were done with highway sediments in a ta

slowly circulating tap water (may be questionable). None o

with runoff or sediments demonstrated acute toxicity.

Helmers, 1996 ......................................... calibration, standards, 

blanks, replicates

Found aluminum, cerium, lanthanum, neodymium, and zirco

grass tissues, as well as platinum that was linked to cataly

converter emissions of vehicles. Over a few years, concen

were increasing with increasing catalytic-converter use. A

and tin were also increasing, but attributable to asbestos-f

linings. 

Horner and Mar, 1985 ............................. ND Method for estimating effects based on regulatory limits and 

base of highway-runoff water-quality values.

Karouna-Renier and Sparling, 1997........ controls, replicates, 

blanks, spikes

Found that pond bottom sediments were not toxic, but test co

minimized formation of bioavailable forms of constituents 

chambers.

Kobriger and others, 1984....................... ND Feasibility to use wetlands to treat highway runoff.

Reference Quality

assurance Comments
42 Assessing Biological Effects from Highway-Runoff Constituents

Kramme and Brosnan, 1985.................... benthic invertebrates, plants bioassay/toxicity testing yes

Madigosky and others, 1991 ................... invertebrates tissue analysis yes

Maltby, Boxall, and others, 1995 ............ benthic invertebrates toxicity identification, evaluation, 

tissue analysis, lab toxicity tests

yes

Maltby, Forrow, and others, 1995 ........... 54 benthic invertebrate taxa, 29 species 

of aquatic fungi, 20 algal genera

community assessment yes

McFarland and O’Reilly, 1992................ algae, fish, plants toxicity testing ND

McHardy and George, 1985.................... algae tissue analysis yes

McNeill and Olley, 1998......................... benthic invertebrates community assessment yes

Moore and Butler, 1994 .......................... none NR ND

Morgan and others, 1984 ........................ algae, fish, phytoplankton, aquatic 

vegetation

community assessment yes, 

as

pr

lim

Mudre, 1985............................................ benthic invertebrates, fish community assessment, tissue analysis yes

Table 2. Documented metadata for reports examining the effects of highway runoff on biota in receiving water—Contin

Reference Biota studied Method Doc

me
Ta

Kramme and Brosnan, 1985.................... blanks, control treatment 

areas in close proximity 

experiment controls, 

standards, spikes 

Monitored water-quality impacts of the herbicides 2,4-D and 

and PAHs from road seal-coating material. Each chemical 

at one urban roadway site. Found that concentrations in ru

low or undetectable. Neither pesticide affected plant germ

rates but the 2,4-D in the runoff from the first postapplicat

did show some chronic effect in growth. Tests with the PA

however, did not indicate lethality for the microinvertebra

Daphnia magna.

Madigosky and others, 1991 ................... control site Noted significant increases in the concentrations of lead cad

aluminum in the tissues of crayfish collected from roadsid

in Louisiana when compared to a control site. The elevate

concentrations did not seem to affect the crayfish, but wer

enough to be a helth hazard to other organisms that use cr

food source.

Maltby, Boxall, and others, 1995 ............ replicates Stream water mixed with runoff was not toxic but experimen

that aromatic hydrocarbons are accumulated by biota in d

proportion to exposure concentrations. Identified PAHs as 

primary toxicant, therefore, population differences among 

the field studies could be caused by organism’s response to 

effects.

Maltby, Forrow, and others, 1995 ........... replicates, standards Demonstrated differences in sediment- and water-quality fro

upstream to downstream and resultant loss of pollution-se

taxa of macroinvertebrates and difference in relative abun

others. Hydraulics and sediment deposition caused by pot

highway runoff may affect communities.

McFarland and O’Reilly, 1992................ ND Did not find calcium magnesium acetate (CMA) to be partic

toxic.

McHardy and George, 1985.................... control site, replicates In comparison to stream water, algae accumulated metals at 

with significant positive correlations for cadmium and zin

concentrations generally increased along the rural to urba

with elevated metals near outfalls of sewage treatment plan

as highways, and other nonpoint sources.

McNeill and Olley, 1998......................... control sites At many sites, “normal” highway runoff did not have a subs

effect on downstream biota along this new highway with g

swale BMPs. However, problems with fine sediment and s

routine spills from auto accidents were noted as problems

Moore and Butler, 1994 .......................... ND There are limited data describing the effects of highway runo

receiving water bodies. Additionally, there is a lack of dat

describing water quality and the resultant aquatic impacts

Morgan and others, 1984 ........................ ND Phosphorus loading in a lake is controlled by sedimentation 

internal loading. The construction of I-91 in the watershed 

caused increased sulfate loading which has promoted inte

phosphorus recycling.

Mudre, 1985............................................ control sites, replicates, 

spikes

Detected elevated concentrations in tissues and some shifts i

community structures at sites along a relatively low-volum

(ADT: 6,000–15,000 VPD). Found substantial site-to-site 

that was caused by interrelated physical and chemical char

of these otherwise pristine stream sites.

Reference Quality

assurance Comments
44 Assessing Biological Effects from Highway-Runoff Constituents

Ney and van Hassel, 1983....................... fish (6 species) tissue analysis yes

Portele and others, 1982.......................... algae, fish, zooplankton bioassays/toxicity yes

Reifenhauser and others, 1995................ plants tissue analysis yes, 

de

Schafer and others, 1998......................... plants tissue analysis yes

Shively and others, 1986 ......................... benthics (many species and taxa) community assessment yes (

an

m

(b

as

m

Smith and Kaster, 1983 ........................... benthic invertebrates community assessment yes

Smith, 1976 ............................................. insects, plants, worms tissue analysis ND

Thrasher, 1983......................................... ND “ecological impact assessment” ND

van Hassel and others, 1980.................... benthic invertebrates, fish tissue analysis yes

Wanielista and others, 1982 .................... algae, plants plant tissue analysis, algal 

bioassay/toxicity

yes

Winters and Gidley, 1980........................ algae bioassay/toxicity testing (in lab) yes

Table 2. Documented metadata for reports examining the effects of highway runoff on biota in receiving water—Contin

Reference Biota studied Method Doc

me
Ta

Ney and van Hassel, 1983....................... replicates, spikes Found that fish species associated with sediments had higher 

contents than water-column species, and that different tiss

different affinities for different metals.

Portele and others, 1982.......................... clean containers, field and 

trip blanks

Soluble fraction of highway runoff adversely affected algae 

zooplankton. Suspended solids caused high mortality of r

trout.

Reifenhauser and others, 1995................ ND Examined use of grass cultures for localized heavy-metal de

rates.

Schafer and others, 1998......................... agricultural-soil 

controls

Plants in soil affected by highway runoff concentrate heavy m

PGE.

Shively and others, 1986 ......................... yes (no details) No significant effects on benthos were observed at either site

significant effects in water-quality parameters were invest

(except iron and lead).

Smith and Kaster, 1983 ........................... replicates, control site Monitored effects of a low-volume highway (ADT —7,000–

VPD). Effects of highway runoff were not distinguishable 

effects of habitat characteristics at each site. Need to mon

document physical stream characteristics, especially subst

stream current. Differences in measured biological param

trolled by site characteristics.

Smith, 1976 ............................................. ND Lead in particulate form. Found that concentrations decrease 

distance from the road. Lead concentrations increase with 

volume. Particulate load goes out exhaust into atmosphere 

dispersed into the roadside environment.

Thrasher, 1983......................................... NA Discusses alternatives to construction on wetlands, avoidanc

mitigation, and reconstruction.

van Hassel and others, 1980.................... replicates Tissue concentrations of source metals increased with increa

density. Stream water was not highly contaminated but se

were. (Sites ranged from an ADT of 50–15,000 VPD).

Wanielista and others, 1982 .................... replicates, standards, 

reagent controls

Lead levels were much higher in plants collected at bridge s

control sites. Bridge areas were dominated by fewer speci

controls. Stormwater runoff can become toxic to receiving 

bodies when mixed in high concentrations.

Winters and Gidley, 1980........................ replicates Heavy metals inhibited growth. Elevated levels of nutrients s

growth.

Reference Quality

assurance Comments
District ChiefMassachusetts—Rhode Island DistrictU.S. Geological SurveyWater Resources Division10 Bearfoot Rd.Northborough, MA 01532