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# Chapter 7 Contents

Chapter 7. Ecological Effects of Lead

________________________________ ________________________________ ___ 7

- 1

7.1. Introduction to Ecological Concepts

________________________________ ____________________________ 7

- 1

7.1.1. Ecosystem Scale, Function, and Structure

________________________________ ________________ 7

- 2

7.1. 2. Ecosystem Services

________________________________ ________________________________ _ 7

- 3

7.1.3. Critical Loads as an Organizing Principle for Ecological Effects of Atmospheric Deposition

________ 7

- 4

7.1.4. Ecosystem Exposure, Lag Time and Re

- entrainment of Historically Deposited Lead

_______________ 7

- 6

Table 7

- 1. Comparison among several metals: Time to achieve 95% of steady state metal concentration in soil; example in a temperate system

_______________________ 7

- 7
7.2. Terrestrial Ecosystem Effects

________________________________ ________________________________ __ 7

- 8

7.2.1. Introduction to Terrestrial Ecosystem Effects

________________________________ _____________ 7

- 8

7.2.2. Soil Biogeochemistry and Chemical Effects

________________________________ ______________ 7

- 9

7.2. 2.1. pH and CEC

________________________________ _______________________________ 7

- 9

7.2.2.2. Organic Matter

________________________________ ____________________________ 7

- 10

7.2.2.3. Aging 7

- 11

7.2.3. Bioavailability in Terrestrial Systems

________________________________ __________________ 7

- 12

7.2.3. 1. Plants 7

- 13

7.2.3.2. Invertebrates

________________________________ ______________________________ 7

- 19

7.2.3.3. Terrestrial Vertebrates

________________________________ ______________________ 7

- 22

7.2.3.4. Food Web

________________________________ ________________________________ 7

- 23

Table 7

- 2. Soil

- to

- tissue bioaccum ulation factors for various terrestrial plant, invertebrate, and vertebrate species

________________________________ ___ 7

- 25
7.2.4. Biological Effects

________________________________ ________________________________ __ 7

- 26

7.2.4.1. Plants and Lichen

________________________________ __________________________ 7

- 26

7.2.4.2. Invertebrates

________________________________ ______________________________ 7

- 30

7.2.4.3. Terrestrial Vertebrate s

________________________________ ______________________ 7

- 33

7.2.5. Exposure and Response of Terrestrial Species

________________________________ ____________ 7

- 35

7.2.6. Community and Ecosystem Effects

________________________________ ____________________ 7

- 37

7.2.7. Critical Loads in Terrestrial Systems

________________________________ ___________________ 7

- 41

7.2.8. So il Screening Levels

________________________________ _______________________________ 7

- 42

7.2.9. Characterization of Sensitivity and Vulnerability

________________________________ _________ 7

- 43

7.2.9.1. Species Sensitivity

________________________________ _________________________ 7

- 43

7.2.9.2. Nutritional Factors

________________________________ _________________________ 7

- 43
7.2.9.3. Soil Aging and S ite

- Specific Bioavailability

________________________________ _____ 7

- 44

7.2.9.4. Ecosystem Vulnerability

________________________________ ____________________ 7

- 44

7.2.10. Ecosystem Services

________________________________ ________________________________ 7

- 45

7.2.11. Summary of Effects in Terrestrial Systems

________________________________ ______________ 7

- 46

7.2.11.1. Biogeochemistry and Chemical Effects

________________________________ _________ 7

- 46

7.2.11 .2. Bioavailability and Uptake

________________________________ ___________________ 7

- 47

7.2.11.3. Biological Effects

________________________________ __________________________ 7

- 48

7.2.11.4. Exposure Response

________________________________ _________________________ 7

- 49

7.2.11.5. Community and Ecosystem Effects

________________________________ ____________ 7

- 49

7.2.11.6. C ritical Loads, Sensitivity and Vulnerability

________________________________ _____ 7

- 50

7.3. Aquatic Ecosystem Effects

________________________________ ________________________________ __ 7

- 50

7.3.1. Introduction to Aquatic Ecosystem Effects

________________________________ ______________ 7

- 50

7.3.2. Biogeochemistry and Chemical Effects

________________________________ _________________ 7

- 52

7.3.2.1. Other Metals

________________________________ ______________________________ 7

- 54

7.3.2.2. Biofilm

________________________________ ________________________________ __ 7

- 55

7.3.2.3. Carbonate

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- 55May 2011 ii DRAFT

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________________________________ _____________ 7

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7.3.3. Bioava ilability in Aquatic Systems

________________________________ ____________________ 7

- 57

7.3.3.1. Plants and Algae

________________________________ ___________________________ 7

- 61

Table 7

- 3. Bioconcentration factors for Pb in aquatic plants

___________________________ 7

- 65
7.3.3.2. Invertebrates

________________________________ ______________________________ 7

- 65

Table 7

- 4. Bioaccumulation fac tors for Pb in aquatic invertebrates

______________________ 7

- 69
7.3.3.3. Vertebrates

________________________________ _______________________________ 7

- 69

Table 7

- 5. Bioaccumulation factors for Pb in fish

________________________________ ____ 7

- 75
7.3.3.4. Food Web

________________________________ ________________________________ 7

- 76
7.3.4. Biological Effects

________________________________ ________________________________ __ 7

- 78

7.3.4.1. Plants and Algae

________________________________ ___________________________ 7

- 78

7.3.4.2. Invertebrates

________________________________ ______________________________ 7

- 81

7.3.4.3. Vertebrates

________________________________ _______________________________ 7

- 85

7.3.5. Exposure and Response of Aquatic Species

________________________________ ______________ 7

- 94

7.3.6. Community and Ecosystem Effects

________________________________ ____________________ 7

- 98

7.3.7. Critical Loads in Aquatic Systems

________________________________ ____________________ 7

- 101

7.3.8. Charac terization of Sensitivity and Vulnerability

________________________________ ________ 7

- 102

7.3.8.1. Seasonally

- Affected Physiological Changes

________________________________ ____ 7

- 102

7.3.8.2. Increased Nutrient Uptake

________________________________ __________________ 7

- 103

7.3.8.3. Temperature and pH

________________________________ _______________________ 7

- 103

7.3.8.4. Life Stage

________________________________ _______________________________ 7

- 104

7.3.8.5. Species Sensitivity

________________________________ ________________________ 7

- 104

7.3.8.6. Ecosystem Vulnerability

________________________________ ___________________ 7

- 106

7.3.9. Ecosystem Services

________________________________ _______________________________ 7

- 106

7.3.10. Summary of Aquatic Effects

________________________________ ________________________ 7

- 108

7.3.10.1. Biogeochemistry and Chemical Effects

________________________________ ________ 7

- 108

7.3.10.2. Bioavailability

________________________________ ___________________________ 7

- 108

7.3.10.3. Biological Effects

________________________________ _________________________ 7

- 110

7.3.10.4. Exposure and Response

________________________________ ____________________ 7

- 111

7.3.10.5. Community a nd Ecosystem Effects

________________________________ ___________ 7

- 111

7.3.10.6. Critical Loads, Sensitivity and Vulnerability

________________________________ ____ 7

- 112

7.4. Causality Determinations for Lead in Terrestrial and Aquatic Systems

________________________________ 7

- 113

Table 7

- 6. Summary of Pb causal determinations for plants, invertebrates and vertebrates

___ 7

- 113
7.4.1. Bioaccumulation of Lead in Terrestrial and Aquatic Biota as it Affects Ecosystem Services

_______ 7

- 113

7.4.2. Mortality

________________________________ ________________________________ ________ 7

- 115

7.4.3. Growth Effects

________________________________ ________________________________ ___ 7

- 116

7.4.4. Physiological Stress

________________________________ _______________________________ 7

- 117

7.4.5. Hematological Effects

________________________________ _____________________________ 7

- 118

7.4.6. Developmental and Reproductive Effects

________________________________ ______________ 7

- 118

7.4.7. Neurobehavioral Effects

________________________________ ____________________________ 7

- 120

7.4.8. Other Physiological Effects

________________________________ _________________________ 7

- 121

7.4.9. Community an d Ecosystem Level Effects

________________________________ ______________ 7

- 121 Chapter 7. References

________________________________ ________________________________ ___________ 7

- 124May 2011 7

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# Chapter 7. Ecological Effects of Lead

This chapter synthesizes and evaluates the most policy

- relevant science to help form the foundation 

### 1
for the review of the secondary (welfare

- based) NAAQS for Pb

. The Clean Air Act definition of welfare 

### 2
effects includes, but is not limited to, effects on soils, water, wildlife, vegetation, visibility, weather, and 

### 3
climate, as well as effects on materials, economic values, and personal comfort and well

- being. This 

### 4

ch apter discusses the effects of Pb on ecosystem components and processes and is organized into four 

### 5
sections. The introduction (Section 7.1) presents the organizing principles of this chapter and several 

### 6
basic concepts of metal ecotoxicology and ecosystem s ervices. Section 7.2 reviews the effects of Pb on 

### 7
terrestrial ecosystems ; how soil biogeochemistry affects Pb bioavailability, biological effects of Pb 

### 8
exposure and subsequent vulnerability of particular ecosystems, and critical loads for soils. A similar 

### 9
discussion of the effects of Pb on aquatic ecosystems is presented in Section 7.3, including water

- only 

### 10
exposures and sediment related effects. Both the terrestrial and aquatic system sections conclude with a 

### 11
discussion of alterations in ecosystem service functions as a consequence of Pb deposition. Finally, an 

### 12
integrative synthesis of effects of Pb across biota and causal determinations for Pb in both terrestrial and 

### 13
aquatic systems are presented in Section 7.4. Areas not addressed here include literature related to Pb shot 

### 14
or pellets and studies that examine human health

- related endpoints which are described in other chapters 

### 15

of this document. 

### 16



# 7.1. Introduction to Ecological Concepts

Metals, including Pb, occur naturally in the environment at measurable concent rations in soils, 

### 17
sediments, and water. Organisms have developed adaptive mechanisms for living with metals, some of 

### 18
which are required micronutrients (but not Pb). However, anthropogenic enrichment can result in 

### 19
concentrations that exceed the capacity of organisms to regulate internal concentrations, caus ing a toxic 

### 20
response and potentially death. Differences in environmental chemistry may enhance or inhibit uptake of 

### 21
metal from the environment, thus creating a spatial patchwork of environments that are at greater risk 

### 22

than other environment s. Similarly, organisms vary in their degree of adaptation to

, or tolerance of

, the 

### 23
presence of metals. These fundamental principles of how metals interact with organisms and ecosystems 

### 24

are described in detail in EPA ’ s F ramework for Metals Risk Assessment ( Fairbrother et al., 2007 )

. This 

### 25
section introduces critical concepts for understanding how Pb from atmospheric deposition may affect 

### 26

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and Environmental Research Online) at http://epa.gov/hero . HERO is a database of scientific literature used by U.S. EPA in the process of developing science assessments such as the Integrated Scie nce Assessments (ISA) and the Integrated Risk Information System (IRIS)

.May 2011 7

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### 1
and terrestrial ecosystems respond to Pb and how environmental chemistry interacts with organisms to 

### 2

affect e xposure and uptake

. 

### 3



## 7.1.1. Ecosystem Scale, Function, and Structure

An ecosystem is defined as the interactive system formed from all living organisms (biota) and 

### 4
their abiotic (chemical and physical) environment within a given area

. Ecosystems cover a hierarchy of 

### 5
spatial scales and can comprise the entire globe, biomes at the continental scale, or small, well



### 6
circumscribed systems such as a small pond ( U.S. EPA, 2008 ) . A pond may be a small but complex 

### 7
system with multiple trophic levels ranging from phytoplankton to several feeding guilds of fish plus fish



### 8
eating birds or mammals. A large lake, on the other hand, may be a very simple ecosystem, such as the 

### 9
Great Salt Lake in Utah that covers approximately 1,700 square mile s but contains only bacteria, algae, 

### 10
diatoms, and two invertebrate species. All ecosystems, regardless of size or complexity, share the 

### 11
commonality of multiple interactions between biota and abiotic factors, and a reduction in entropy 

### 12
through energy flow f rom photosynthetic organisms to top predators. This includes both structural (e.g., 

### 13
soil type and food web trophic levels) and functional (e.g., energy flow, decomposition, nitrification) 

### 14

attributes. 

### 15
Ecosystems are most often defined by their structure, a nd are based on the number and type of 

### 16
species present. Individual organisms of the same species are similar in appearance and genetics, and can 

### 17
interbreed and produce fertile offspring

. Interbreeding groups of individual organisms within the same 

### 18
species form populations, and populations of different species form communities. The community 

### 19
composition may also define an ecosystem type, such as a pine forest or a tall grass prairie. Pollutants can 

### 20
affect the ecosystem structure at any of these levels of bio logical organization ( Suter et al., 2005 )

. 

### 21

Individual plants or animals may exhibit changes in metabolism, enzyme activities, hormone function, or 

### 22
overall growth rates or may suffer gross lesions, tumors, deformities, or other pathologies. Effects on the 

### 23
nervous system of animals may cause behavioral changes that alter breeding behaviors or predator 

### 24
avoidance. However, effects on individuals must result in changes to their survival or reproductive output 

### 25
to have any effect on the population. Population level effects of pollutants include changes over time in 

### 26

a bundance or density (number of individuals in a defined area), age or sex structure, and production or 

### 27

sustainable rates of harvest ( Barnthouse, 2007 )

. Community level attributes affected by pollutants include 

### 28
species richness and abundance (also known as biodiversity), dominance of one species over another, or 

### 29
size (area) of the community. Pollutants may affect communities i n ways that are not observable in 

### 30
organisms or populations ( Bartell, 2007 ) , including: (1) effects resulting from interactions between 

### 31
species, such as altering predation rates or competitive advantage; (2) indirect effects, such as reducing or 

### 32May 2011 7

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– DO NOT C ITE OR QUOTE removing one species from the assemblage and allowing another to emerge ( Petraitis & Latham, 1999 ) ; 

### 1
and (3) alterations in trophic structure. 

### 2
Alternatively, ecosystems may be defined on a functional basis, such as rates of photosynthesis, 

### 3
decomposition, nitrification, or carbon cycling

. Pollutants may affect abiotic conditions (e.g., soil 

### 4
chemistry), which indirectly influences biotic structure and function ( Bartell, 2007 )

. Feedback loops or 

### 5
networks influence the stability of the system, and can be mathematically described through simplistic or 

### 6
complex process

, or energy flow

, models ( Bartell, 2007 ) . For example, the Comprehensive Aquatic 

### 7
Systems Model (CASM) is a bioenergetics

- based multi compartment model that describes the daily 

### 8
production of biomass (carbon) by populations of aquatic plants and animals over an a nnual cycle 

### 9

( DeAngelis et al., 1989 )

. CASM, originally designed to examine theoretical relationships between food 

### 10

web structure, nut rient cycling, and ecosystem stability, has since been adapted for risk assessments and 

### 11
has been applied to numerous lakes with a variety of pollutants ( Bartell, 2007 )

. Likewise, other theoretical 

### 12
ecosystem models are being modified for use in assessing ecological risks from pollutant exposures 

### 13

( Bartell, 2007 )

. 

### 14
Some ecosystems, and some aspects of particular ecosystems, are less vulnerable to long

- term 

### 15
consequences of pollutant exposure. Other ecosystems may be profoundly altered if a single attribute is 

### 16
affected. Thus, spatial and temporal definitions of ecosystem structure and function become an essential 

### 17
factor in defining impacted ecosystem services and critical loads of particular pollutants, either as single 

### 18
pollutants or in combination with other s tressors

. Both ecosystem services (Section 7.1.2) and critical 

### 19
loads (Section 7.1.3) serve as benchmarks or measures of the impacts of pollutants on ecosystems. 

### 20



## 7.1.2. Ecosystem Services

Ecosystem structure and function may be translated into ecosystem services ( Daily, 1997 )

. 

### 21
Ecosystem services are the benefits people obtain from ecosystems ( Millennium Ecosystem Assessment, 

### 22

2003 ) . Ecosystem services are defined as the varied and numerous ways that ecosystems are important to 

### 23
human welfare and how they provide m any goods and services that are of vital importance for the 

### 24
functioning of the biosphere

. This concept has gained recent interest and support because it recognizes 

### 25
that ecosystems are valuable to humans

, and are important in ways that are not generally app reciated 

### 26

( Daily, 1997 ) . Ecosystem services also provide a context for assessing the collective effects of human 

### 27
actions on a broad range of the goods and services upon which humans rely

. 

### 28
In general, both ecosystem structure and function play essential roles in providing goods and 

### 29
services. Ecosystem processes provide diverse benefits including absorption and breakdown of pollutants, 

### 30
cycling of nutrients, binding of soil, degradation of organic waste, maintenance of a balance of gases in 

### 31
the air, regulation of radiation balance and climate, and fixation of solar energy ( Daily, 1997 ; Westman, 

### 32

1977 ; WRI, 2000 ) . These ecological benefits, in turn, provide economic benefits and values to society 

### 33May 2011 7

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– DO NOT C ITE OR QUOTE ( Costanza et al., 1997 ; Pimentel et al., 1997 )

. Goods such as food crops, timber, livestock, fish and clean 

### 1
drinking water have market value

. The values of ecosystem services such as flood control, wildlife 

### 2
habitat, cycling of nutrients and removal of air pollutants are more difficult to measure ( Goulder & 

### 3
Kennedy, 1997 )

. 

### 4

Particular concern has develo ped within the past decade regarding the consequences of decreasing 

### 5
biological diversity ( Ayensu et al., 1999 ; Chapin et al., 1998 ; Hooper & Vitousek, 1997 ; Tilman, 2000 ; 

### 6

Wall, 1999 )

. Human activities that decrease biodiversity also alter the complexity and stability of 

### 7
ecosystems and change ecological processes

. In response, ecosystem structure, composition and function 

### 8

can be affected ( Chapin et al., 1998 ; Daily & Ehrlich, 1999 ; Levlin, 1998 ; Peterson et al., 1998 ; Pimm, 

### 9

1984 ; Tilman, 1996 ; Tilman & Downing, 1994 ; Wall, 1999 )

. Biodiversity is an important consideration at 

### 10
all levels of biological orga nization, including species, individuals, populations, and ecosystems

. Human



### 11
induced changes in biotic diversity and alterations in the structure and functioning of ecosystems are two 

### 12

of the most dramatic ecological trends of the past century ( U.S. EPA, 2004 ; Vitousek et al., 1997 )

. 

### 13

Hassan ( 2005 ) identified four broad categories of ecosystem services: 

### 14

 Supporting services are necessary for the pr oduction of all other ecosystem services

. Some 

### 15
examples include biomass production, production of atmospheric O 2 , soil formation and 

### 16
retention, nutrient cycling, water cycling and provisioning of habitat

. Biodiversity is a 

### 17
supporting service in that it is increasingly recognized to sustain many of the goods and 

### 18
services that humans enjoy from ecosystems. These supporting services provide a basis for an 

### 19

additional three higher

- level categories of services. 

### 20

 Provisioning services such as products ( Gitay et al., 2001 ) i.e., food (including game meat

, 

### 21

roots

, seeds, nuts

, and other fruit, spices

, fodder), fiber (including wood, textiles) and 

### 22
medicinal and cosmetic produ cts. 

### 23

 Regulating services that are of paramount importance for human society such as 

### 24

( 1 ) carbon sequestration, ( 2 ) climate and water regulation, ( 3 ) protection from natural hazards 

### 25

such as floods, avalanches, or rock

- fall ( 4 ) water and air purification, and ( 5 ) disease and pest 

### 26
regulation. 

### 27

 Cultural services that satisfy human spiritual and aesthetic appreciation of ecosystems and 

### 28
their components. 

### 29



## 7.1.3. Critical Loads as an Organizing Principle for Ecological Effects of Atmospheric Deposition

Critical loads were first defined for regulating emissions of sulfur and nitrogen oxides, but have 

### 30
since been applied to exposure of other pollutants, including metals ( Adams & Chapman, 2007 ) . A critical 

### 31May 2011 7

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– DO NOT C ITE OR QUOTE load is defi ned as

, “a quantitative estimate of an exposure to one or more pollutants below which 

### 1
significant harmful effects on specified sensitive elements of the environment do not occur according to 

### 2
present knowledge ” ( Nilsson & Grennfelt, 1988 ) . Because critical loads for Pb differ by ecosystem 

### 3
(aquatic

- water; aquatic

- sediment; terrestrial), dif fer by environmental chemistry properties that impact 

### 4
bioavailability, differ by species, and differ by endpoint of concern, they can be used as an organizing 

### 5
principle for linking atmospheric deposition with ecological impairment at multiple spatial scale s. There 

### 6
are two aspects to consider: ( 1) the critical load at a steady state in the environment (i.e., how much input 

### 7
is required to balance the rate of output), and ( 2) the time required to reach the critical load (i.e., the lag 

### 8
time between onset of exp osure and induction of measurable effects). This is particularly true for 

### 9
terrestrial ecosystems where changes in soil geochemistry

, as a result of either changing land use or 

### 10
ecological succession

, may significantly alter the amount of sequestration of Pb , thus changing its 

### 11
bioavailability and critical load if based on total metal. Ideally, therefore, critical loads for metals should 

### 12
be defined on the basis of bioavailable metal rather than total metal. This approach is being used in 

### 13
aquatic system s throug h the application of the biotic ligand model (BLM) ( Di Toro et al., 2001 ; Di Toro et 

### 14

al., 2005 ) , but is proving to be more difficult for modeling terrestrial s ystem s. 

### 15
For aquatic systems, a dynamic equilibrium exists between the surface water , the water column, 

### 16

and the sediment compartments (which must be defined when determining the critical load for each 

### 17

compartment ) . Although the sediment generally acts as a sink for pollutants in the w ater column, 

### 18
especially metals in parti cle form or as insoluble metal complexes, there may be some re

- entrainment 

### 19

from the sediments into the water column . This sediment

- water interface may change the solubility or 

### 20
bioavailability of the metal, thereby alt ering the critical load for the water column, particularly if 

### 21
expressed in the form of total metal. Note, however, that while sedimentation processes may change the 

### 22
time to steady state, they will not affect the ultimate critical load once steady state is achieved. 

### 23
The following pieces of information are required to calculate a critical load, each of which is 

### 24
discussed in more detail in the subsequent sections of this chapter: 

### 25

 Ecosystem at risk; 

### 26

 Receptors of concern (plants, animals, etc.); 

### 27

 Endpoints of concern (organism, population or community responses, changes in ecosystem 

### 28
services or functions); 

### 29

 Dose (concentration)

- response relationships and threshold levels of effects; 

### 30

 Bioavailability and bioaccumulation rates; 

### 31

 Naturally occurring (background) Pb (or other metal) concentrations; and 

### 32

 Biogeochemical modifiers of exposure. 

### 33May 2011 7

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– DO NOT C ITE OR QUOTE As stated in the 2008 ISA for Oxides of Nitrogen and Sulfur

- Ecological Criteria there is no single 

### 1

“ definitive ” critical load for a pollutant, partly because critica l load estimates reflect the current state

- of



### 2
knowledge and policy priorities, and also because of local or regional differences among ecosystems 

### 3

( U.S. EPA, 2008 )

. Changes in scientific understanding may include, for example, expanded information 

### 4

about dose

- response relationships, better understanding of bioavailability factors, and improved 

### 5
quantitative models for effects predictions. Changes in policy may include new mandates for resource 

### 6
protection, inclusion of per ceived new threats that may exacerbate the effects of the pollutant of concern 

### 7
(e.g., climate change), and a better understanding of the value of ecosystem services. 

### 8
In the short term, metal emissions generally have greater adverse effects on biota in aqua tic 

### 9
systems than in terrestrial systems because metals are more readily immobilized in soils than in sediment

. 

### 10
However, over the longer term, terrestrial systems may be more affected particularly by those metals with 

### 11
a long soil residence time, such as Pb. Thus, for a particular locale, either the terrestrial or the aquatic 

### 12

ecosystem at that site may have the lowe r critical load. Given the heterogeneity of ecosystems affected by 

### 13
Pb, and the differences in expectations for ecosystem services attached to diff erent land uses, it is 

### 14
expected that there will be a range of critical load values for Pb for soils and waters within the U.S. 

### 15



## 7.1.4. Ecosystem Exposure, Lag Time and Re

- entrainment of Historically Deposited Lead

Ecosystem exposure from atmospheric emissions of Pb depends upon the amount of Pb deposited 

### 16
per unit time. Ecosystem response will also depend upon the form in which the Pb is deposited, the areal 

### 17
extent of such deposition, and the modifying factors listed in the previous section. However, there is 

### 18
frequ ently a lag time between when metals are emitted and when an effect is seen, particularly in 

### 19
terrestrial ecosystems and, to a lesser extent, in aquatic sediments; water exposures result in more 

### 20
immediate system responses. This is because the buffering capa city of soils and sediments permits Pb to 

### 21

become sequester ed into organic matter, making it less available for uptake by organisms. The lag time 

### 22
from start of emissions to achieving a critical load can be calculated as the time to reach steady state from 

### 23

t he time when the Pb was initially added to the system

. Excluding erosion processes, the time required to 

### 24
achieve 95% of steady state is about 4 half

- lives (t 1/2 ) 1 ( Smolders et al., 2007 ) . Conversely, once 

### 25

emissions cease, the same am ount of time is required to reduce metal concentrations to background 

### 26

levels. 

### 27
Time to steady state for metals in soils is dependent upon rates of erosion, uptake by plants, and 

### 28
leaching or drainage from soils. Ignoring erosion, half

- life of metals can be p redicted ( Smolders et al., 

### 29

2007 ) for a soil as: 

### 30

1 Time required to reduce the initial concentration by 50% if metal input is zero.May 2011 7

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– DO NOT C ITE OR QUOTE 

### Kd R TF y d t

ρ + × ×

×

= 10000 69

. 0

2 / 1

Equation 7

- 1

where: 

### 1

d is the soil depth in m eters (m) 

### 2
y is the annual crop yield (t ons /ha

· y r ) 

### 3
TF is the ratio of the metal concentration in plant to that in soil 

### 4
R is the net drainage loss out of the soil depth of concern (m 3 /ha

· y r ) 

### 5 

## ρ is the bulk density of soil ( k g (dry weight) /L) 

### 6

Kd is the ratio of the metal concentration in soil to that in soil pore solution (L/kg) 

### 7 Metals removed by crops (or plants in general) comprise a very small fraction of the total soil metal 

### 8

and can be ignored for the purpose of estimating time to steady state. Thus, equation 7

- 1 is 

### 9
simplified to: 

### 10

Kd R d t

ρ 10000 69

. 0

2 / 1 

### × ×

=

Equation 7

- 2

and becomes a function of soil depth, the amount of rainfall, soil density, and soil properties that 

### 11

affect Kd . Pb has a relatively long time to steady state compared to other metals, as shown in 

### 12

Table 7

- 1

. 

### 13

Table 7

- 1. Comparison among several metals : T ime to achieve 95% of steady state metal concentration in soil; example in a temperate system

Metal Loading rate (g/Ha/Yr) Kd (L/Kg) Time (yr)

Se 100 0.3 1.3
Cu 100 480 a 1

, 860 a

Cd 100 690 a 2

, 670 a

Pb 100 19

, 000 a 73

, 300 a

Cr 100 16

, 700 a 64

, 400 a

a Mean Kd (ratio of total metal concentrations in soils to that in soil pore water ); and Time to achieve 95% of steady

- state concentration in soil. ( 49 Dutch soils ) ( de Groot et al., 1998 )

. Note: Based on a soil depth of 23 cm, a rain infiltration rate of 3

, 000 m 3 /ha

· yr, and the assumption that background was zero at the start of loading.

Source: Smolders, Fairborother et al. ( 2007 )

In aquatic systems, t 1/2 for Pb in the water column depends on the ratio of the magnitudes of the 

### 14

fluxes coming from and going in to the sediment, the ratio of the depths of the water column and sediment, 

### 15May 2011 7

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– DO NOT C ITE OR QUOTE and the sediment half

- life. Sediment t 1/2 is dependent upon the partic ulate and dissolved fractions and is 

### 1
calculated as for soils (Equation 7

- 2). 

### 2

Re

- entrainment of Pb particles via windblown dust from surface soils or dry sediments may occur. 

### 3

Amount and distance of re

- entrained particles and deposition rates are dependent upon w ind velocity and 

### 4
frequency; size, density, shape, and roughness of the particle; soil or sediment moisture; and terrain 

### 5
features including openness (including amount of vegetation), aspect relative to wind direction, and 

### 6
surface roughness. Resuspension is defined in terms of a resuspension factor, K, with units of m - 1 , or a 

### 7
resuspension rate (Λ), with units of sec

- 1 (Equation 7

- 3). The resuspension rate, Λ, is the fraction of a 

### 8
surface contaminant that is released per time and is defined by: 

### 9



### C R

= Λ

Equation 7

- 3

where: 

### 10

R is the upward resuspension flux ( μg/ m 2 / s ec ) 

### 11
C is the soil (or dry sediment) Pb concentration (μg/m 2 ) 

### 12 Such emissions may have local impacts, but are not likely to have long

- range effects, as particles 

### 13

generally remain low to the ground and are not lifted into the upper atmosphere. Although re



### 14

entrainment may alter the particle size distribution in a local area, it generally does not alter the 

### 15

bioavailable fraction, and deposited particles will be subject to the same biogeochem ical forces 

### 16

affecting bioavailability

. Therefore, exposure via re

- entrainment should be considered additive to 

### 17

exposure from atmospheric particulate deposition in terrestrial and aquatic ecosystems. 

### 18



# 7.2. Terrestrial Ecosystem Effects

7.2.1. Introduction to Terrestria l Ecosystem Effects

Numerous studies of the effects of Pb on components of terrestrial systems were reviewed in the 

### 19
2006 Pb AQCD. T he literature on terrestrial ecosystem effects of Pb , published since the 2006 Pb AQCD

, 

### 20

is considered with brief summaries fr om the AQCD where relevant. Section 7.2 is organized to consider 

### 21
uptake of Pb and effects at the species level, followed by community and ecosystem level effects. Soil 

### 22
biogeochemistry of Pb in terrestrial systems is reviewed in Section 7.2.2. Section 7.2.3 . considers the 

### 23
bioavailability and uptake of Pb by plants, invertebrates, and wildlife in terrestrial systems

. Biological 

### 24
effects of Pb on terrestrial ecosystem components including plants and lichen, invertebrates, and 

### 25
vertebrates (Section 7.2.4) are fol lowed by data on exposure and response of terrestrial species (Section 

### 26

7.2.5)

. Effects of Pb at the ecosystem scale are discussed in Section 7.2.6 . Section 7.2 concludes with a 

### 27May 2011 7

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– DO NOT C ITE OR QUOTE discussion of critical loads in terrestrial systems (Section 7.2.7), characteri zation of sensitivity and 

### 1
vulnerability of ecosystem components (Section 7.2.8), and effects on ecosystem services ( Section 7.2.9). 

### 2



## 7.2.2. Soil Biogeochemistry and Chemical Effects

According to data presented in the 2006 Pb AQCD, the fraction of soil metal that is directly 

### 3
available to plants is the fraction found in soil pore water, even though the concentration of metals in pore 

### 4

water is small relative to bulk soil concentration. The amount of Pb dissolved in soil solution is controlled 

### 5
by at least six variable s: ( 1) solubility equilibria ; ( 2) adsorption

- desorption relationship of total Pb with 

### 6
inorganic compounds (e.g., oxides of Al

, Fe

, Si

, Mn; clay minerals) ; ( 3) adsorption

- desorption reactions 

### 7
of dissolved Pb phases on soil organic matter ; ( 4) pH ; ( 5) cation exchange capacity (CEC) ; and ( 6) aging

. 

### 8
Adsorption

- desorption of Pb to soil solid phases is largely controlled by total metal loading. Therefore, 

### 9
areas with high Pb deposition will exhibit a lower fraction of total Pb partitioned to inorganic and organic 

### 10
matter. Decreasing soil pH, CEC, and organic matter have been strongly correlated to increases in the 

### 11
concentration of dissolved Pb species. Aging of metals in soils results in decreased amounts of labile 

### 12
metal as the Pb becomes incorporated into the soil solid phase ( McLaughlin et al., 2010 )

. Data from 

### 13
recent studies have further defined the impact of pH, CEC, organic matter (OM) , and aging on Pb 

### 14
mobilization and subsequent bioavailability in soils. 

### 15

7.2.2.1. pH and CEC

Models of metal bioavailability calibrated from 500+ soil toxicity tests on plants, invertebrates, and 

### 16
microbial communities indicated that soil pH and CEC are t he most important factors governing metal 

### 17
solubility and toxicity ( Smolders et al., 2009 ) . The variability of derived EC 50 values was most closely 

### 18

associated with CEC. Smolders et al. ( 2007 ) de termined that 12 to 18 months of artificial aging of soils 

### 19
amended with metal decreased the soluble metal fraction by about one order of magnitude. 

### 20
Miretzky et al. ( 2007 ) also showed that the conce ntration of mobile Pb was increased in acidic 

### 21
soils, and discovered that Pb adsorption to sandy loam clay was a function of both (1) Fe and Mn oxide 

### 22

interactions ; and (2) the formation of weak electrostatic bonds with charged soil surfaces. Similarly, the 

### 23
mobility of smelter

- produced metals in forest soils was found to be greater than in adjacent agricultural 

### 24

lands ( Douay et al., 2009 ) . The higher solubility was caused by the decreased soil pH o f the forest 

### 25
environments. Further, decreasing the soil pH via simulated acid rain events increased naturally occurring 

### 26
Pb bioavailability in field tests ( X. Hu et al., 2 009 )

. 

### 27
A sequential extraction procedure was employed by Ettler et al. ( 2005 ) to determine the relative 

### 28
bioavailability of different Pb fractions present in soils collected from a mining and smelt ing area in the 

### 29
Czech Republic. Five Pb fraction categories were identified: ( Fraction A ) exchangeable ; ( Fraction B ) acid 

### 30
extractable (bound to carbonates) ; ( Fraction C ) reducible (bound to Fe and Mn oxides) ; ( Fraction D ) 

### 31May 2011 7

- 10 DRAFT

– DO NOT C ITE OR QUOTE oxidizable (complexed with organic carbon) ; and ( Fraction E ) residual (silicates). Tilled agricultural soils 

### 1
were found to have decreased Pb, likely as a result of repeated cultivation, with the majority of Pb 

### 2
represented as the reducible Fraction C. Pb concentration in undisturbed forest soils, however, was largely 

### 3
present as the exchangeable fraction (A) , weakly bound to soil OM

. 

### 4

7.2.2.2. Organic Matter

Organic matter (OM) decreases bioavailability of Pb, but as it is turned over and broken down, 

### 5
pedogenic minerals become more important in Pb se questration ( Schroth et al., 2008 ) . Shaheen and 

### 6

Tsadilas ( 2009 ) noted that soils with higher clay content, OM , total calcium carbonate equivalent, and 

### 7
total free sesquioxides also exhi bited higher total Pb concentration, indicating that less Pb had been 

### 8
removed by resident plant species. Huang et al. ( 2008 ) examined the re

- mobilization potential of Pb in 

### 9

forest soils, and determined that mobilization of total Pb was strongly associated with dissolved organic 

### 10

matter (DOM) . Groenenberg et al. ( 2010 ) used a non

- ideal competitive adsorption Donnan model to 

### 11
explain the variability of OM binding affinity and uncertainties associated with metal speciation. They 

### 12
found that natural variations in fulvic acid binding properties were the most important variable in 

### 13
predicting Pb speciation. Guo et al. ( 2006 ) determined that the

- COOH and

- OH groups assoc iated with 

### 14

soil OM were important factors in Pb sequestration in soil, and Pb sorption was increased as pH was 

### 15
raised from 2 to 8. Because organic content increased the Pb sequestration efficiency of soils, OM content 

### 16

had a n inhibitory effect on Pb uptake by woodlouse species Oniscus asellus and Porcellio scaber ( Gál et 

### 17

al., 2008 ) . Vermeulen et al. ( 2009 ) demonstrated that invertebrate bioaccumulation of Pb from 

### 18

contami nated soils was dependent on pH and OM , but that other unidentified habitat

- specific differences 

### 19
also contributed. The relationship of bioaccumulation and soil concentration was modified by pH and 

### 20

OM , and also by habitat type. Kobler et al. ( 2010 ) showed that the migration of atmosphere

- deposited Pb 

### 21
in soil matrices was strongly influenced by soil type, indicating that cert ain soil types may retain Pb for 

### 22
longer periods of time. In humic forest soils, the highest Pb concentrations were measured in the humified 

### 23
bottom layer, whereas in soils characterized by well

- drained substrate and limestone bedrock, Pb 

### 24

concentration decre ased over time, likely as a result of water drainage and percolation. The authors 

### 25
theorized that the most significant Pb migration route was transportation of particulate

- bound Pb along 

### 26
with precipitation

- related flow through large soil pores. 

### 27

A number of recent laboratory studies have further defined the relationship of soil biogeochemical 

### 28
characteristics and Pb uptake by plants. Dayton et al. ( 2006 ) established significant negative correlations 

### 29
between log

- transformed Pb content of lettuce plants ( Lactuca sativa ), soil organic content, and CEC, and 

### 30

similar negative relationships were also confirmed for soil pH and amorphous Fe and Al oxide content. As 

### 31
part of a metal partitioning study, ( Kalis et al., 2007 ) determined that not only did metal concentration in 

### 32
the soil solution decrease as pH increased, but pH

- mediated metal adsorption at the root surface of Lolium 

### 33May 2011 7

- 11 DRAFT

– DO NOT C ITE OR QUOTE perenne determined root Pb concentration, with concentration in the shoot correlated with root 

### 1
concentration. Interestingly, Kalis et al. ( 2007 ) and Lock et al. ( 2006 ) also observed that the in flux of Pb in 

### 2

the water

- soluble fraction had an impact on soil pH. In addition, 1 μM humic acid decreased root Pb 

### 3

concentration in L. perenne plants grown in 0.1 and 1 μM Pb solution, likely as a result of Pb 

### 4
complexation and sequestration with the added O M ( Kalis et al., 2006 )

. 

### 5

7.2.2.3. Aging

Smolders et al. ( 2007 ) reviewed the effects of aging of Pb in soils on th e toxicity of Pb to plants 

### 6
and soil invertebrates, with aging defined primarily as leaching following initial influx, but also as 

### 7
binding and complexation. In nearly half of the Pb soil studies reviewed, observed dose

- response curves 

### 8

could not be establish ed following soil leaching, indicating that aged soils likely contain less bioavailable 

### 9
Pb. The authors concluded that competitive binding between soil ligands and biotic ligands on soil roots 

### 10
or invertebrate guts can be used to model the relationship of o bserved availability and toxicity of metals 

### 11
in soils. Because this concept is the basis of the Biotic Ligand Model (BLM) (Section 7.3.3), the authors 

### 12
proposed a terrestrial BLM approach to estimate the risk of metals to terrestrial organisms. However, 

### 13

Antu nes et al. ( 2006 ) noted that there were several key challenges involved in development of a terrestrial 

### 14
BLM applicable to plants, particularly the reliable measurement of free ion activities and ligand 

### 15
concentration in the rhizosphere, the identification of the organisms ’ ligands associated with toxicity, and 

### 16
the possible need to incorporate kinetic dissolution of metal

- ligand complexes as sources of free ion. 

### 17
Further, Pb in aged field soils has been observed to be less available for uptake into terrestrial organisms, 

### 18
likely as a result of increased sequestration within the soi l particles ( Antunes et al., 2006 )

. Magrisso et al. 

### 19

( 2009 ) used a bioluminescent strain of the bacterium Cupriavidus metallidurans to detect and quantify Pb 

### 20
bioavailability in soils collected adjacent to industrial and highway areas in Jerusalem, Israel, and in 

### 21
individual simulated soil components freshly spiked with Pb. The bacterium was genetically engineered 

### 22
to give off the bioluminescent reaction as a dose

- dependent response, and was inoculated in soil slurries 

### 23
for three hours prior to response evalua tion. Spiked soil components induced the bioluminescent 

### 24
response, and field

- collected components did not. However, the comparability of the simulated soils and 

### 25

their Pb concentration with the field

- collected samples was not entirely clear. Lock et al. ( 2006 ) compared 

### 26

the Pb toxicity to springtails ( Folsomia candida ) from both laboratory

- spiked soils and field

- collected Pb



### 27
contaminated soils of similar Pb concentrations. Total Pb concentrations of 3,877 mg Pb /kg dry weight 

### 28
and higher always caused significant effects on F. candida reproduction in the spiked soils. In f ield soils, 

### 29
only the soil with the highest Pb concentration of 14,436 mg Pb /kg dry weight significantly affected 

### 30
reproduction. When expressed as soil pore

- water concentrations, reproduction was never significantly 

### 31
affected at Pb concentrations of 0.5 mg/L, whereas reproduction was always significantly affected at Pb 

### 32
concentrations of 0.7 mg/L and higher, independent of the soil treatment. Leaching soils prior to use in 

### 33May 2011 7

- 12 DRAFT

– DO NOT C ITE OR QUOTE bioassays had only a slight effect on Pb toxicity to resident springtails, suggesting that among the 

### 1
processes that constitute aging of Pb in field soils, leaching is not particularly important with respect to 

### 2
bioavailability. 

### 3

Red

- backed salamanders ( Plethodon cinereus ) exposed to Pb

- amended soils (553, 1

, 700, 4

, 700, 

### 4

and 9

, 167 mg Pb /kg) exhib ited lowered appetite and decreased white blood cell counts at the two highest 

### 5
concentrations, as compared to controls {B, 2010, 379076} . However, salamanders tolerated field



### 6
collected, aged soils containing Pb concentration of up to 16,967 mg Pb /kg with n o significant deleterious 

### 7

effects. 

### 8
In summary, studies published during the past 5 years continue to substantiate the important role 

### 9
that soil geochemistry plays in sequestration or release of Pb

. Soil pH and CEC have long been known to 

### 10
be the primary cont rolling factors for amount of bioavailable Pb in soils, and a recent review of more than 

### 11
500 studies corroborates these findings ( Smolders et al., 2009 ) . Fe and Mn oxides are now known to al so 

### 12
play an important role in Pb sequestration in soils

. Pb binds to OM , although relatively weakly, and as the 

### 13

OM is broken down the Pb may be released into soil solution

. Leaching of metal through soil pores may 

### 14
be the primary route for loss of bioavailab le soil Pb; OM may reduce leaching and thus appear to be 

### 15
associated with Pb sequestration

. Aging of Pb in soils through incorporation of the metal into the 

### 16
particulate solid phase of the soil results in long term binding of the metal and reduced bioavailab ility of 

### 17
Pb to plants and soil organisms

. 

### 18



## 7.2.3. Bioavailability in Terrestrial Systems

Bioavailability was defined in the 2006 Pb AQCD as “ the proportion of a toxin that passes a 

### 19
physiological membrane (the plasma membrane in plants or the gut wall in animals) and reaches a target 

### 20
receptor (cytosol or blood). ” In 2007, EPA took cases of bioactive adsorption into consideration and 

### 21
revised the definition of bioavailability as “ the extent to which bioaccessible metals absorb onto, or into, 

### 22
and across biological mem branes of organisms, expressed as a fraction of the total amount of metal the 

### 23
organism is proximately exposed to (at the sorption surface) during a given time and under defined 

### 24

conditions ” ( Fairbrother et al., 2007 ) . Characteristics of the toxicant that affect bioavailability are: ( 1) 

### 25
chemical form or species ; ( 2) particle size ; ( 3) lability ; and ( 4) source. New information on sources of Pb 

### 26
in terrestrial ecosystems

, and their influence on subsequent bioavailability

, was reviewed in Chapter 3, 

### 27
while new information on the influence of soil biogeochemistry on speciation and chemical lability was 

### 28
presented in Section 7.2.2

. This s ection summarizes new literature on uptake and subsequent presence of 

### 29
Pb in tissues. Bioaccumulation factors (BAF ’ s) (i.e., the ratio of Pb concentrations in tissues of terrestrial 

### 30

biota to concentrations in soil or in food items) reported in this section are summarized in Tab le 7

- 2 . T he 

### 31
2006 Pb AQCD extensively reviewed the methods available for quantitative determination of the 

### 32May 2011 7

- 13 DRAFT

– DO NOT C ITE OR QUOTE mobility, distribution, uptake, and fluxes of atmospherically delivered Pb in ecosystems, and they are not 

### 1

reviewed in this secti on. 

### 2

7.2.3.1. Plants

The 2006 Pb AQCD noted that terrestrial plants accumulate atmospheric Pb primarily via two 

### 3
routes: direct stomatal uptake into foliage, and incorporation of atmospherically deposited Pb from soil 

### 4
into root tissue, followed by variable translocation to othe r tissues. Foliar Pb may include both 

### 5
incorporated Pb (i.e., from atmospheric gases or particles) and surficial particulate Pb deposition. 

### 6
Although the plant may eventually absorb the surficial component, its main importance is its likely 

### 7

contribution to t he exposure of plant consumers. This section will first review recent studies on uptake of 

### 8
Pb by plants through foliar and soil routes, and their relative contribution , followed by the consideration 

### 9

of translocation of Pb from roots to shoots, including a discussion of v ariability in translocation among 

### 10
species. 

### 11

Leaf and Root Uptake

Field studies carried out in the vicinity of Pb smelters have determined the relative importance of 

### 12
direct foliar uptake and root uptake of atmospheric Pb deposited in soils. Hu and Ding ( 2009 ) analyzed 

### 13
ratios of Pb isotopes in the shoot s of commonly grown vegetables and in soil at three distances from a 

### 14
point source (0.1, 0.2, 5 .0 km)

. Pb isotope ratios in plants and soil were different at two of those locations, 

### 15
leading the authors to the conclusion that airborne Pb was being assimilate d via direct leaf uptake. Soil Pb 

### 16
concentration in the rhizosphere at the three sites ranged between 287 and 379 mg Pb /kg ( S ite I), 155 and 

### 17
159 mg Pb /kg ( S ite II), and 58 and 79 mg Pb /kg ( S ite III, selected as the control site). The median shoot 

### 18

and root P b concentration s at each site were 36 and 47 mg Pb /kg, 176 and 97 mg Pb /kg, and 1.3 and 7 mg 

### 19

Pb /kg, respectively, resulting in shoot:root Pb ratios exceeding 1 .0 in S ite I ( for Malabar spinach [ Basella 

### 20

alba ]

, ratio = 1.6, and amaranth [ Amaranthus spinosus ]

, ratio = 1.1), and in S ite II ( for the weeds 

### 21
Taraxacum mongolicum

, ratio = 1.9, and Rostellaria procumbens

, ratio = 1.7). However, the two species 

### 22

studied at Site II were not studied at Site I or Site III. In the control site (Site III), no plant was foun d with 

### 23
a Pb shoot:root ratio greater than 1 .0 . Hu and Ding ( 200 9 ) concluded that metal accumulation was greater 

### 24
in shoot than in root tissue, which suggested both high atmospheric Pb concentration and direct stomatal 

### 25
uptake into the shoot tissue. 

### 26

Cui et al. ( 2 007 ) studied seven weed species growing in the vicinity of an old smelter (average soil 

### 27

Pb concentration of 4

, 020 mg Pb /kg) in Liaoning, China, to measure Pb accumulation rates in roots and 

### 28
shoots. Cutleaf groundcherry ( Physalis angulata ) accumulated the most Pb, with root and shoot 

### 29
concentration of 527 and 331 mg Pb /kg, respectively, and velvetleaf ( Abutilon theophrasti ) was the 

### 30
poorest absorber of Pb (root and shoot concentration of 39 and 61 mg Pb /kg, respectively). In all cases, 

### 31May 2011 7

- 14 DRAFT

– DO NOT C ITE OR QUOTE weed species near t he smelter accumulated more Pb than plants from non

- polluted environments (5 mg 

### 1

Pb /kg), indicating that aerially deposited Pb produced by smelting is bioavailable to plants. However, the 

### 2
ratio of root:shoot Pb concentration varied by species, and the autho rs presented no data to differentiate 

### 3
Pb taken up from soil from Pb incorporated via foliar uptake. 

### 4
Chrastny et al. ( 2010 ) characterized the Pb contaminat ion of an agricultural soil in the vicinity of a 

### 5
shooting range. Pb was predominantly in the form of PbO and PbCO 3 , and Pb was taken up by plants 

### 6
through both atmospheric deposition onto the plant and by root uptake. 

### 7
Because of their long life spans, trees can provide essential information regarding the sources of 

### 8
bioavailable Pb. A Scots pine forest in northern Sweden was found to incorporate atmospherically derived 

### 9
Pb pollution directly from ambient air, accumulating this Pb in bark, needles, and shoots ( Klaminder et 

### 10

al., 2005 ) . Nearly 50% of total tree uptake was determined to be from direct adsorption from the 

### 11
atmosphere. Further, Aznar et al. ( 2009 ) found that the Pb content of black spruce ( Picea mariana ) 

### 12
needles collected along a metal contamination gradient emanating from a Canadian sm elter in 

### 13
Murdochville, Quebec, showed a significant decrease in Pb concentration with increasing distance from 

### 14
the smelter. Interestingly, older needles were determined to accumulate larger quantities of Pb than 

### 15
younger ones, which the authors attributed t o “ incidental processing ” of atmospheric Pb. Foliar damage 

### 16
and growth reduction were also observed in the trees ( Aznar, Richer

- Lafleche, et al., 2009 ) . They were 

### 17
significantly correlated with Pb concentration in the litter layer, where Pb comes from atmospheric 

### 18
deposition and closely reflects it. In addition, there was no correlation between diminished tree growth 

### 19

and Pb concentration in the deeper mineral soil layers, strongly suggesting that only current atmospheric 

### 20
Pb was adversely affecting trees ( Aznar, Richer

- Laflèchea, et al., 2009 ) . Similarly, Kuang et al. ( 2007 ) 

### 21

noted that the Pb concentration in the inner bark of Pinus massoniana trees growing adjacent to a Pb

- Zn 

### 22
smelter in the Guangdong province of China was much higher (1.87 mg Pb /kg dry weight) than in 

### 23

reference

- area trees. Because concentration in the inner bark was strongly correlated with concentration in 

### 24
the outer bark, they concluded that the origin of the Pb was atmospheric. 

### 25
Dendrochronology (tree ring analysis) has become an increasingly important tool for measuring t he 

### 26
response of trees to Pb exposure ( Watmough, 1999 ) . The advent of laser ablation inductively coupled 

### 27
plasma mass spectrometry has made measurement of Pb concentration in individual tree rings possible 

### 28

( Watmough, 1999 ; Witte et al., 2004 ) . This allows for close analysis of the timing of Pb uptake relative to 

### 29
smelter activity and/or changes in soil chemistry. For example, Aznar et al. ( 2008 ) measured Pb 

### 30
concentration in black spruce tree rings to determine the extent and timing of atmospheric deposition near 

### 31
the Murdochville smelter. Variability in tree

- ring Pb content seemed to indi cate that trees accumulated 

### 32
and sequestered atmospheric Pb in close correlation with the rates of smelter emission, but that 

### 33
sequestration lagged about 15 years behind exposure. However, the ability to determine time of uptake 

### 34
from the location in growth r ings is weakened in species that transfer Pb readily from outer bark to inner 

### 35
bark. Cutter and Guyette ( 1993 ) identifi ed species with minimal radial translocation from among a large 

### 36May 2011 7

- 15 DRAFT

– DO NOT C ITE OR QUOTE number of tree species, and recommended the following temperate zone North American species as 

### 1

suitable for metal dendroch r onology studies: white oak ( Quercus alba ), post oak ( Q. stellata ), ea stern red 

### 2
cedar ( Juniperus virginiana ), old

- growth Douglas fir ( Pseudotsuga menziesii ), and big sagebrush 

### 3

( Artemisia tridentata ). In addition, species such as bristlecone pine ( Pinus aristata ), old

- growth 

### 4

redwood ( Sequoia sempervirens ), and giant sequoia ( S. gigantea ) were deemed suitable for local 

### 5
purposes. Patrick and Farmer ( 2006 ) determined that European sycamore ( Acer pseudoplat anus ) are not 

### 6
suitable for this type of dendrochronological analysis because of the formation of multiple annual rings. 

### 7
Pb in sapwood and heartwood is more likely a result of soil uptake than of direct atmospheric 

### 8

exposure ( Guyette et al., 1991 )

. D ifferentiati on of geogenic soil Pb in tree tissue from Pb that originated 

### 9
in the atmosphere requires measurem ent of stable Pb isotope ratios ( L. Patrick, 2006 ) . Tree bark samples 

### 10

collected fro m several areas of the Czech Republic were subjected to stable Pb isotope analysis to 

### 11
determine the source and uptake of atmospheric Pb ( Co nkova & Kubiznakova, 2008 ) . Results indicated 

### 12
that beech bark is a more efficient accumulator of atmospheric Pb than spruce bark. A decrease in the 

### 13

206 Pb/ 207 Pb ratio was measured in bark and attributed to increased usage of leaded gasoline between 1955 

### 14

and 1990; an increased 206 Pb/ 207 Pb ratio was ascribed to coal combustion ( Conkova & Kubiznakova, 

### 15

2008 ) . Similarly, Savard et al. ( 2006 ) compared isotope ratios of 206 Pb/ 207 Pb and 208 Pb/ 206 Pb in tree rings 

### 16
from spruce trees sampled at a control site near Hudson Bay, with those sampled near the Horne smelter 

### 17

active since 1928

, in Rouyn

- Noranda, Canada. The concentration of total Pb showed a major increase in 

### 18
1944 and a corresponding decrease of the 206 Pb/ 207 Pb ratios, suggesting that the smelter was responsible 

### 19
for the increased Pb uptake ( Savard et al., 2006 ) . The authors suggest ed that the apparent delay of 14 

### 20
years may have been attributable to the residence time of metals in airborne particles the buffering effect 

### 21
of the soils and, to a lesser extent, mobility of heavy meta ls in tree stems. Furthermore, through the use of 

### 22
the two different isotope ratios, Savard et al. ( 2006 ) were able to differentiate three types of Pb in tree 

### 23
rings: natural (derived from the mineral soil hori zons), industrial (from coal burning urban pollution), and 

### 24
mining (typical of the volcanogenic massive sul f ide ore deposits treated at the Horne smelter). 

### 25

Devall et al. ( 2006 ) measure d Pb uptake by bald

- cypress trees ( Taxodium distichum ) growing in a 

### 26
swamp near a petroleum refinery and along a bank containing Pb

- contaminated dredge spoils. They 

### 27
measured Pb in tree cores and showed greater uptake of Pb by trees in the swamp than by tree s growing 

### 28
on the dredge spoil bank, attributing the difference to exposure source (refinery versus dredge spoils) and 

### 29
differences in soil chemistry between the swamp and the dredge spoil bank ( Devall et al., 2006 )

. 

### 30
Similarly, Gebologlu et al. ( 2005 ) found no correlation between proximity to roadway and accumulated 

### 31
Pb in tomato and bean plants at sites adjacent to two state roads in Turkey (average Pb concentration 5.4 

### 32
and 6.0 mg Pb /kg), indicating that u ptake may be influenced by multiple factors, including wind 

### 33
direction, geography, and soil chemistry. Average Pb levels in leaves were 0.6 and 0.5 mg Pb /kg for 

### 34
tomato and bean plants, respectively, while fruit concentration averaged 0.4 mg Pb /kg for both s pecies. 

### 35
Conversely, if foliar contamination is due primarily to dust deposition, distance from a source such as a 

### 36May 2011 7

- 16 DRAFT

– DO NOT C ITE OR QUOTE road may be easily correlated with Pb concentration on the plants. For example, Ai

- Khlaifat and Al



### 1

Khashman ( 2007 ) collected unwashed date palm ( Phoenix dactylifera ) leaves at 3

- m trunk height from 

### 2
trees in Jordan to assess the extent of Pb contamination from the city of Aqaba. Whereas relatively low 

### 3

levels of Pb were detected in leaves collected a t background sites (41 mg Pb /kg), leaves collected 

### 4
adjacent to highway sites exhibited the highest levels of Pb (177 mg Pb /kg). The authors determined that 

### 5
Pb levels in date palm leaves correlated with industrial and human activities (e.g., traffic density ) ( Ai



### 6

Khlaifat & Al

- Khashman, 2007 ) . However, decreases in tissue Pb concentration with increasing distance 

### 7
from point sources can also follow from decreasing Pb in soil. Bindler et al

. ( 2008 ) used Pb isotopes to 

### 8
assess the relative importance of pollutant Pb versus natural Pb for plant uptake and cycling in Swedish 

### 9
forested soils. The Pb isotopic composition of needles/leave s and stemwood of different tree species and 

### 10
ground

- cover plants indicated that the majority of Pb present in these plant components was derived from 

### 11
the atmosphere, either through aerial interception or actual uptake through the roots. For the ground

- cove r 

### 12
plants and the needles/leaves, the 206 Pb/ 207 Pb isotopic ratios (1.12 to 1.20) showed that the majority of Pb 

### 13
was of anthropogenic origin. Stemwood and roots have higher 206 Pb/ 207 Pb ratio values (1.12 to 1.30) 

### 14
which showed the incorporation of some natura l Pb as well as anthropogenic Pb. For pine trees, the 

### 15
isotopic ratio decreased between the roots and the apical stemwood suggesting that much of the uptake of 

### 16
Pb by trees is via aerial exposure. Overall, it was estimated that 60

- 80% of the Pb in boreal for est 

### 17
vegetation originated from pollution; the Pb concentrations were, however, quite low

– not higher than 1 

### 18

mg Pb /kg plant material, and usually in the range of 0.01

- 0.1 mg Pb /kg plant material ( while soils had a 

### 19
range of 5 to 10 mg Pb /kg in the mineral horizons and 50 to 150 mg Pb /kg in the O horizons). Overall, the 

### 20
forest vegetation recycles very little of the Pb present in soils ( and thus does not play a direct role in the 

### 21
Pb biogeochemical cycle in boreal forest soils )

. 

### 22

Translocation and Sequestration of Lead in Plants

Although Pb is not an essential metal, it is taken up from soils through the symplastic route, the 

### 23
same active ion transport mechanism used by plants to take up water and nutrients and move them across 

### 24

root cell membranes ( U.S. EPA, 2006 ) . As with all nutrients, only the proportion of a metal present in soil 

### 25
pore water is directly available for uptake by plants. In addition, soil

- to

- plant transfer factors in soils 

### 26

enriched with Pb have been found to better correlate with bioavailable Pb so il concentration

, defined as 

### 27

DTPA

- extractable Pb, than with total Pb concentration ( U.S. EPA, 2006 )

. 

### 28
The 2006 Pb AQCD stated that most of the Pb absorbed from soil remains bound in plant root 

### 29

tissues either because ( 1) Pb may be deposited within root cell w all material, or ( 2) Pb may be 

### 30
sequestered within root cell organelles. Sequestration of Pb may be a protective mechanism for the plant. 

### 31
Recent findings have been consistent with this hypothesis: Han et al. ( 2008 ) observed Pb d eposits in the 

### 32
cell walls and cytoplasm of malformed cells of Iris lactea exposed to 0 to 10 mM Pb for 28 days. They 

### 33May 2011 7

- 17 DRAFT

– DO NOT C ITE OR QUOTE hypothesized that preferential sequestration of Pb in a few cells, which results in damage to those cells, 

### 1
helps in maintaining normal over all plant activities through the sacrifice of a small number of active cells. 

### 2
Similarly, macroscopic analysis of the roots of broad bean ( Vicia faba ) cultivated in mine tailings 

### 3
(average Pb concentration of 7,772 mg/kg) by Probst et al. ( 2009 ) revealed dark ultrastructural 

### 4

abnormalities that were demonstrated to be metal

- rich particles located in or on root cell walls. It is 

### 5
unclear whether the presence of these structures had any effect on overall plant health. 

### 6

Clark et al. ( 2006 ) investigated Pb bioavailability in garden soils in Roxbury and Dorchester, MA. 

### 7
The sources of Pb were considered to be Pb from paints and from leaded gasoline additives, with 40 to 

### 8
80% coming from paint. The average Pb concentration in foliar tissue of bean plants was 14 ± 5 mg Pb /kg 

### 9

while the con centration in the bean pod was only 20.6 mg Pb /kg. For mustard plants, there was a linear 

### 10
relationship ( R 2 =0.85) between Pb concentration in plant tissues and Pb concentration in the soil (both for 

### 11
plants grown in situ and those grown under greenhouse conditions). 

### 12
Murray et al. ( 2009 ) investigated the uptake and accumulation of Pb in several vegetable species 

### 13

(carrot [ Daucus carota ], radish [ Raphanus sativus ], lettuce [ L actuca sativa ], soybean [ Glycine max ], and 

### 14

wheat [ Triticum aestivu m ]) from metal

- contaminated soils, containing 10 to 40 mg Pb /kg and 

### 15

demonstrated that most Pb remained in the roots. No Pb was measured in the above

- ground edible 

### 16
soybean and wheat tissues, while carrots, the most efficient accumulator of Pb, contained a m aximum Pb 

### 17

tissue concentration of 12 mg/kg dry mass. Similarly, ( Cho et al., 2009 ) showed that green onion ( Allium 

### 18
fistulosum ) plants also take up little Pb when planted in soil spiked with Pb nitrate. No plant tissues 

### 19
contained a Pb concentration greater than 24 mg Pb /kg when grown for 14 weeks in soils of up to 3,560 

### 20

mg Pb /kg, and the majority of bioavailable Pb was determined to be contained within the roots. Chinese 

### 21
spinach ( Amaranthus dubius ) also translocates very little Pb to stem and leaf tissue, and uptake from Pb



### 22
containing soils (28 to 52 mg Pb /kg) is minimal ( Mellem et al., 2009 ) . Sonmez et al. ( 2008 ) reported that 

### 23
Pb accumulated by three weed species ( Avena sterilis

, Isatis tinctoria

, Xanthium strumarium ) grown in 

### 24

Pb

- spiked soils was largely conce ntrated in the root tissues, and little was translocated to the shoots 

### 25

( Sonmez et al., 2008 )

. 

### 26
Recent research has shown that Pb translocation to stem and leaf tissues does occur at significant 

### 27
rates in some species, including the legume Sesbania drummondii ( Pe ralta

- Videa et al., 2009 ) and 

### 28
buckwheat ( Fagopyrum esculentum ) ( Tamura et al., 2005 )

. Wang et al. ( 2006 ) noted that Pb soil

- to

- plant 

### 29
transfer factors were higher for leafy vegetables (Chinese cabbage, pak

- choi, and water spinach) than for 

### 30

the non

- leafy vegetable s tested (towel gourd, eggplant, and cowpea). Tamura et al. ( 2005 ) demonstrated 

### 31
that buckwheat is an efficient translocator of Pb. Buckwheat grown in Pb

- containing soils collected from 

### 32

a shoot ing range site (average 1M HCl extractable Pb= 6,643 mg Pb /kg) preferentially accumulated Pb in 

### 33
leaves (8,000 mg Pb /kg) and shoots (4,200 mg Pb /kg)

, over root tissues (3,300 mg Pb /kg). Although plant 

### 34
growth was unaffected, this level of leaf and shoot accu mulation is likely to have significant implications 

### 35
for exposure of herbivores. Similarly, Shaheen and Tsadilas ( 2009 ) reported that vegetables (pepper, okra, 

### 36May 2011 7

- 18 DRAFT

– DO NOT C ITE OR QUOTE and eggplant) grown in soils containing 24 to 30 mg Pb /kg total Pb were more likely to accumulate Pb in 

### 1
leaves ( range: undetected to 25 mg Pb /kg) rather than in fruits ( range: undetected to 19 mg Pb /kg); 

### 2
however, no significant correlation between soil Pb concentration and plant tissue Pb concentration could 

### 3

be established ( Shaheen & Tsadilas, 2009 )

. 

### 4
There is broad variability in uptake and translocation among plant species, and interspec ies 

### 5
variability has been shown to interact with other factors such as soil type. By studying multiple species in 

### 6

four Pb

- Zn mining sites in Yunnan, China, Li et al. ( 2009 ) demonstrated not only significant differences 

### 7
in uptake and translocat ion among the species studied, but also modification of the effect o n species by 

### 8
type of soil. Plants sampled represented nine species from four families

— Caryophyllaceae, Compositae, 

### 9
Cruciferae, and Pteridaceae. Overall, soil Pb concentration averaged 3,77 2 mg Pb /kg dry weight, with the 

### 10
highest site average measured at the Minbingying site (5,330 mg Pb /kg), followed by Paomaping (2,409 

### 11

mg Pb /kg), Jinding (1,786 mg Pb /kg), and Qilinkeng (978 mg Pb /kg). The highest average shoot Pb 

### 12
concentration (3,142 mg Pb / kg) was detected in Stellaria vestita (Caryophyllaceae) collected at 

### 13
Paomaping, while shoot concentration of Sinopteris grevilloides (Pteridaceae) collected from 

### 14
Minbingying exhibited the lowest shoot Pb concentration (69 mg Pb /kg). A similar trend was det ected in 

### 15

root tissues. S. vestita root collected from the Paomaping area contained the maximum Pb concentration 

### 16
measured (7,457 mg Pb /kg), while the minimum root Pb levels were measured in Picris hieraciodides 

### 17
(Pteridaceae) tissues collected from Jinping. These results indicate significant interspecies differences in 

### 18
Pb uptake, as well as potential soil

- specific differences in Pb bioavailability. S. vestita , in particular, was 

### 19

determined to be an efficient accumulator of Pb, with a maximum enrichment coeffi cient of 1.3. 

### 20
Significant correlations between soil Pb concentration and average shoot and root Pb levels were also 

### 21

established ( Y. Li et al., 2009 ) . Within plant species, the variability in uptake and translocation of Pb may 

### 22

extend to the varietal level. Antonious and Kochhar ( 2009 ) determined uptake of soil

- associated Pb for 23 

### 23
unique ge notypes from four species of pepper plants ( Capsicum chinense

, C. frutescens

, C. baccatum , and 

### 24

C. annum ). Soil Pb concentration averaged approximately 0.6 mg Pb /kg dry soil. No Pb was detected in 

### 25
the fruits of any of the 23 genotypes, except two out of sev en genotypes of C. baccatum , which had 0.9 

### 26
and 0.8 mg Pb /kg dry weight Pb in fruit. 

### 27
Fungal species, as represented by mushrooms, accumulate Pb from soils to varying degrees. Based 

### 28
on the uptake of naturally occurring 210 Pb, Guillen et al. ( 2009 ) established that soil

- associated Pb was 

### 29
bioavailable for uptake by mushrooms, and that the highest 210 Pb accumulation was observed in Fomes 

### 30
fomentarius mushrooms, followed by Lycoperdon perlatum

, Boletus aereus , and Macrolepiota procera

, 

### 31
indicating some spec ies differences. Benbrahim et al. ( 2006 ) also showed species differences in uptake of 

### 32
Pb by wild edible mushrooms, although they found no significant correlations between Pb content of 

### 33
mushrooms and soil Pb concentration. Pb concentrations in mushroom carpophores ranged from 0.4 to 

### 34
2.7 mg Pb /kg from sites with soil concentrations ranging from 3.6 and 7.6 mg Pb/kg dry soil

. 

### 35May 2011 7

- 19 DRAFT

– DO NOT C ITE OR QUOTE Recent studies substantiated findings from the 2006 Pb AQCD that plants store a large portion of 

### 1

Pb in root tissue. Pb soil

- to

- plant transfer factors are higher for leafy vege tables than for the non

- leafy 

### 2
vegetables ( G. Wang et al., 2006 ) and buckwh eat has recently been shown to be an efficient translocator 

### 3

of Pb from soil to above

- ground shoots ( Tamura et al., 2005 ) . However, there is broad variability in Pb 

### 4
uptake and translocation rates among plant species, and interspec ies variability has been shown to interact 

### 5
with other factors such as soil type. Field studies carried out in the vicinity of Pb smelters ( X. Hu et al., 

### 6

2009 ) show that Pb may accumulate in shoot tissue through direct stomatal uptake rather than by soil



### 7

root

- shoot translocation

. Dendrochronology has become more advanced in recent years and is a useful 

### 8

tool for m onitoring historical uptake of Pb into trees exposed to atmospheric or soil Pb

. Trees accumulate 

### 9
and sequester atmospheric Pb in close correlation with the rate of smelter emissions, although one study 

### 10

indicate d that sequestration can lag behind exposure from emissions by 15 years. Pb in the outer woody 

### 11
portion of the tree is more likely the result of direct atmospheric exposure, while Pb in sapwood is more 

### 12
likely a result of soil uptake

. This difference provides an important tool for analyzing source 

### 13

appo rtionment of Pb accumulation in plants ( Guyette et al., 1991 )

. 

### 14

7.2.3.2. Invertebrates

At the time of publicatio n of the 2006 Pb AQCD, little information was available regarding the 

### 15
uptake of atmospheric Pb pollution (direct or deposited) by terrestrial invertebrate species. Consequently, 

### 16
few conclusions could be drawn concerning the Pb uptake rate of particular spe cies although there was 

### 17
some evidence that dietary or habitat preferences may influence exposure and uptake. Recent literature 

### 18
indicates that invertebrates can accumulate Pb from consuming a Pb

- contaminated diet and from exposure 

### 19
via soil, and that uptake and bioaccumulation of Pb by invertebrates is lower than that observed for other 

### 20

metals. 

### 21

Snails

Cantareus asperses snails exposed to dietary Pb at 3.3, 86, and 154 mg/kg of diet ( spiked with Pb 

### 22

sulfate ) for up to 64 days were found to assimilate a signifi cant proportion of Pb, and feeding rates were 

### 23
unaffected by the presence of the metal ( Beeby & Richmond, 2010 ) . While bioconcentration factors 

### 24

(BCF ’ s) for Cd were observed to increase over the 6 4

- day study period, the rate of Pb assimilation 

### 25

remained consistent over time and n o evidence for a regulatory mec hanism for Pb was observed

. T he 

### 26
authors observed that, for additional Pb to be retained, snails would have to grow additional soft tissue. 

### 27

H elix aspersa snails rapidly accumulated Pb from contaminated soil (1,212 mg Pb /kg) and from eating 

### 28

contaminated lettuce (approximately 90 mg Pb /kg after 16 weeks ’ growth on Pb

- contaminated soil) 

### 29
during the first 2 weeks of exposure, at which point snail body burdens reached a plateau ( Scheifler, De 

### 30
Vaufleury, et al., 2006 ) . T here were no observed effects of Pb exposure or accumulation on survival or 

### 31May 2011 7

- 20 DRAFT

– DO NOT C ITE OR QUOTE growth in C. asperses or H. aspersa . In another study ( Ebenso & Ologhobo, 2009b ) , juvenile Achatina 

### 1

achatina snails con fined in cages on former Pb

- battery waste dump sites were found to accumulate Pb 

### 2
from both plant and soil sources. Soil Pb concentration averaged 20, 200, and 1,200 mg Pb /kg at the three 

### 3
main waste sites, while leaf tissues of radish ( R aphanus sativus ) gro wn at these sites averaged 7, 30, and 

### 4
68 mg Pb /kg dry weight, respectively. Although plant concentrations were low, they were correlated with 

### 5
elevated snail Pb tissue concentration. Pb concentration in snail tissues averaged 12, 91, and 468 

### 6

mg Pb /kg, respe ctively, at the three sites, which the authors stipulated were above the maximum 

### 7
permissible concentration of Pb for human consumption of mollusks, mussels, and clams (1.5 μg Pb/g 

### 8
tissue). Pb concentration in snail tissues generally is much lower than that of the soil substrates upon 

### 9
which they were reared, but higher than in other soil

- dwelling organisms. De Vaufleury et al. ( 2006 ) 

### 10
exposed H elix aspera snails to standardized (1999 European International Organization for 

### 11
Standardization methodology [ISO 11267:1999] ) artificial

- substrate soils containing 13, 26, 39, or 52 mg 

### 12

P b /kg for 28 days without supplemental food. After the exposure period, snail foot tissue contained 

### 13

increased levels of Pb

— 1.9, 1.7, and 1.5 μg Pb/g dry weight versus concentration averaging 0.4 mg/kg in 

### 14
control organisms. Viscera also exhibited increased Pb levels at the two highest exposures, with measured 

### 15
tissue concentration of 1.2 and 1.1 mg Pb /kg, respectively, as compared with control tissue Pb levels of 

### 16

0.4 mg Pb /kg. However, there was no significant increase in snail

- tissue Pb concentration when na tural 

### 17
soil was used in place of ISO medium, and there was no relationship between soil Pb concentration and 

### 18
snail tissue concentration, strongly suggesting the presence of soil variables that modify bioavailability. 

### 19

Notten et al. ( 2008 ) investigated the origin of Pb pollution in soil, plants, and snails by means of Pb 

### 20
isotope ratios. They found that a substantial proportion of Pb in both plants and snails was from current 

### 21
atmospheric exposure. 

### 22

Earthworms

Soil characteristics that interact with bioavailability of Pb may include biogeochemistry associated 

### 23

with different soil horizons, source of Pb, and proportion of soil:leaf litter. These have been studied 

### 24
principally with various species of earthworms. Bradham et al

. ( 2006 ) examined the effect of soil 

### 25
chemical and physical properties on Pb bioavailability. Eisenia andrei earthworms were exposed to 21 

### 26
soils with varying physical properties that were freshly spiked with Pb to give a s tandard concentration of 

### 27
2,000 mg/kg dry weight. Both internal earthworm Pb concentration and mortality rates increased with 

### 28
decreasing pH and CEC although the apparent role of CEC may only have been due to its correlation with 

### 29

other soil characteristics. These data corroborate that Pb bioavailability and toxicity are increased in 

### 30
acidic soils and in soils with a low CEC (Section 7.2.2). This finding was confirmed by Gandois et al 

### 31

( 2010 ) , who determined that the free

- metal

- ion fraction of total Pb concentration in field

- collected soils 

### 32
was largely predicted by pH and soil iron content. 

### 33May 2011 7

- 21 DRAFT

– DO NOT C ITE OR QUOTE The role of soil profile and preferred depth was studied using eight species of earthworms from 27 

### 1
locations in Switzerland, representing three ecophysiological groups ( Ernst et al., 2008 ) : epigeic (surface



### 2
dwelling worms), endogeic (laterally burrowing worms that inhabit the upper soil layers), and anecic 

### 3
(vertically burrowing worms that reach depths of 6 inches). For epigeic and aneci c earthworms, the total 

### 4
concentration of Pb in leaf litter and in soil, respectively, were the most important drivers of Pb body 

### 5
burdens. By contrast, the level of Pb in endogeic earthworms was largely determined by soil pH and CEC. 

### 6

As a result of these di fferences, the authors suggested that atmosphere

- sourced Pb may be more 

### 7
bioavailable to epigeic than endogeic species, because it is less dependent on modifying factors. Suthar et 

### 8

al. ( 2008 ) , on the other hand, found higher Pb bioaccumulation in the endogeic earthworm Metaphire 

### 9
posthuma than in the anecic earthworm species Lampito mauritii , and speculated that differences in Pb 

### 10
tissue level arose from differing life

- history stra tegies, such as feeding behaviors, niche preferences, and 

### 11
burrowing patterns, all of which exposed the endogeic species to greater Pb concentration. Accumulation 

### 12

studies conducted with Eisenia fetida earthworms documented the difficulty of extrapolating 

### 13

ac cumulation kinetic constants from one soil type to another, and showed that many soil physiochemical 

### 14
properties, including pH, OM , and CEC, among others, work in conjunction to affect metal bioavailability 

### 15

( Nahmani et al., 2009 ) . However, once taken up from the environment, more than half of the 

### 16
bioaccumulated Pb appears to be contained within earthworm tissue and cell membranes ( Li et al., 2008 )

. 

### 17
Despite significant Pb uptake by earthworms, Pb in earthworm tissue may not be bioavailable to 

### 18
predators. Pb in the earthworm Aporrectodea caliginosa was determined to be contained largely in the 

### 19
granular fraction (approximately 60% of total Pb), while the remaining Pb body burden was in the tissue, 

### 20

cell membrane, and intact cell fractions ( Vijver et al., 2006 ) . From this, the authors concluded that only a 

### 21
minority of earthworm

- absorbed Pb would be toxicologically available to cause adverse effects in the 

### 22
earthworms or in their predators. Ear thworm activity can alter Pb bioavailability and subsequent uptake 

### 23
by earthworms themselves and other organisms. The presence of earthworms may increase soil pH though 

### 24
the secretion of cutaneous mucus, and worm activity is generally associated with increas ed bioavailability 

### 25

of Pb. Sizmur and Hodson ( 2009 ) speculated that earthworms affect Pb mobility by modifying the 

### 26
availability of cations or anions. However, Coeurdassier et al. ( 2007 ) found that snails did not have a 

### 27
higher Pb content when earthworms were present, and that unexpectedly, Pb was higher in earthworm 

### 28
tissue when snails were present. 

### 29

Arthropods

Cicadas pupating in historically Pb

- arsenate

- treated soils accumulated Pb at concentrations similar 

### 30
to those reported previously for earthworms ( Robinson et al., 2007 ) . Likewise, tissue Pb levels measured 

### 31
in Coleoptera specimens collected from areas containing average soil concentration of 45 and 71 m g 

### 32

Pb /kg exhibited a positive relationship with soil Pb content, although abundance was unaffected ( Schipper 

### 33May 2011 7

- 22 DRAFT

– DO NOT C ITE OR QUOTE et al., 2008 ) . By contrast, the Pb sequestration rates that were observed in two woodlouse species, O. 

### 1

asellus and P. scaber , were species

- dependent ( Gál et al., 2008 ) . Both species were field collected at Pb



### 2
contaminated sites (average concentration, 245 mg Pb /kg dry weight; range, 21

- 638 mg Pb /kg dry 

### 3
weight), with O. asellus Pb levels averaging 43 mg Pb /kg over all sites, while P. scaber contained no 

### 4
detectable Pb residues. Pb concentration measured in granivorous rough harvester ants ( Pogonomyrmex 

### 5

rugosus ), in the seeds of some plant species they consume, and in surface soil, were all shown to decline 

### 6
with increasing distance from a former Pb smelter near El Paso, Texas, where soil leachable Pb at the 

### 7
three sites of ant collection ranged from 0.003 to 0.117 mg Pb /kg ( Del Toro et al., 2010 ) . Ants 

### 8
accumulated approximately twice as much Pb as was measured in seeds, but the study did not separate the 

### 9
effects of dietary e xposure from those of direct contact with soil or respiratory intake. 

### 10

7.2.3.3. Terrestrial Vertebrates

Tissue Pb residues in birds and mammals associated with adverse toxicological effects were 

### 11
presented in the 2006 Pb AQCD. In general, avian blood, liver, and kidn ey Pb concentrations of 0.2

- 3 μg 

### 12

Pb /dL, 2

- 6 mg Pb /kg wet weight, and 2

- 20 mg Pb /kg wet weight, respectively, were linked to adverse 

### 13
effects. A few additional studies of Pb uptake and tissue residues in birds and mammals conducted since 

### 14

2006 are reviewed he re. 

### 15
In a study of blood Pb levels in wild Steller ’ s eiders ( Polysticta stelleri ) and black scoters 

### 16

( Melanitta nigra ) in Alaska, the authors compiled avian blood Pb data from available literature to develop 

### 17

reference values for sea ducks ( Brown et al., 2006 )

. The background exposure reference value of blood 

### 18

Pb was <20 μg Pb /dl, with levels between 20 and 59 μg Pb /dl as indicative of Pb exposure. Clinical 

### 19
toxicity was in the range of 60

- 99 μg Pb /dl in birds while >100 μg Pb /dl results in acute, severe toxicity

. 

### 20
In measurement of blood Pb with a portable blood Pb ana lyzer, only 3% of birds had values indicating 

### 21
exposure and none of the birds had higher blood Pb levels or clinical signs of toxicity

. Tissue distribution 

### 22
of Pb in liver, kidney, ovary and testes of rain quail ( Coturnix coramandelicus ) following oral dosin g of 

### 23
0.5 mg/kg, 1.25 mg/kg or 2.5 mg/kg Pb acetate for 21 days indicated that Pb uptake was highest in liver 

### 24
and kidney and low in ovary and testes ( Mehrotra et al., 2008 ) . Resident feral pigeons ( Columba livia ) 

### 25
captured in the urban and industrial areas of Korea exhibited i ncreased lung Pb concentration, ranging 

### 26
from 1.6 to 1.9 mg Pb /kg wet weight ( Nam & Lee, 2006 ) . However, tissue concentration did not correlate 

### 27

with atmospheric Pb concentration, so the authors concluded that ingestion of particulate Pb (paint chips, 

### 28
cement, etc.) in the urban and industrial areas was responsible for the pigeons ’ body burden. Similarly, 

### 29
70% of American woodcock ( Scolopax minor ) chicks and 43% of American woodcock young

- of

- year 

### 30
collected in Wisconsin, U.S., exhibited high bone Pb levels of 9.6

- 93 mg Pb /kg dry weight and 1.5

- 220 

### 31

mg Pb /kg, respectively, even though radiographs of birds ’ gastrointestinal tracts revealed no evidence of 

### 32May 2011 7

- 23 DRAFT

– DO NOT C ITE OR QUOTE shot ingestion ( Strom et al., 2005 ) . Authors hypothesized that unidentified anthropogenic sources may 

### 1

have caused the observed elevated Pb levels. 

### 2

In addition to birds, soil

- dwelling mammals can also bioaccumulate atmospherically

- sourced Pb: 

### 3
Northern poc ket gophers ( Thomomys talpoides ) trapped within the Anaconda Smelter Superfund Site 

### 4
were shown to accumulate atmospherically deposited Pb. Gopher liver and carcass Pb concentration 

### 5
averaged 0.3 and 0.4 mg Pb /kg wet weight on low Pb soils (47 mg Pb /kg), 0.4 and 0.9 mg Pb /kg wet 

### 6
weight in medium Pb soils (95 mg Pb /kg) and 1.6 and 3.8 mg Pb /kg wet weight in high Pb soils (776.5 

### 7

mg Pb /kg) ( Reynolds et al., 2006 )

. 

### 8

Casteel et al. ( 2006 ) fou nd that bioavailability of Pb from environmental soil samples in swine ( Sus 

### 9

domestica ) depended on Pb form or type, with high absorption of cerussite and manganese

- Pb oxides and 

### 10
poor absorption of galena and anglesite. Juvenile swine (approximately 5

- 6 wee ks old and weighing 8

- 11 

### 11
kg) were fed Pb

- contaminated soils collected from multiple sources for 15 days (concentration range of 

### 12
1,270 to 14,200 mg Pb /kg) to determine the relative bioavailability. While Pb concentrations were 

### 13
roughly equivalent in blood, l iver, kidney, and bone tissues, individual swine exhibited different uptake 

### 14

abilities ( Casteel et al ., 2006 )

. 

### 15
Interestingly, dietary Ca deficiency (0.45 mg Ca daily versus 4 mg under normal conditions) was 

### 16
linked to increased accumulation of Pb in zebra finches ( Taeniopygia guttata ) that were provided with 

### 17
drinking water containing 20 mg/L Pb ( Dauwe et al., 2006 ) . Liver and bone Pb concentration were 

### 18
increased by an approximate factor of three, while Pb concentration in kidney, muscle, and brain tissues 

### 19
were roughly doubled by a Ca

- deficient diet. However, it is not known whether this level of dietary Ca 

### 20
deficiency is common in wild populations of birds. 

### 21

7.2.3.4. Food Web

In addition to the individual factors reviewed above, understanding the bioavailability of Pb along a 

### 22
simple food chain is essential for determining risk to terrestrial animals. While the bioavailability of 

### 23
ingested soil or particulates is relatively simple to measure and model, the bioavailability to secondary 

### 24
consumers of Pb ingested and sequestered by primary producers and primary consumers is more complex. 

### 25

Kaufman et al. ( 2007 ) caution that the use of total Pb concentration in risk asses sments can result in 

### 26
overestimation of risk to ecological receptors, and they suggest that the bioaccessible fraction may 

### 27
provide a more realistic approximation of receptor exposure and effects. This section reviews recent 

### 28

literature that estimates the bio accessible fraction of Pb in dietary items of higher order consumers, and 

### 29
various studies suggesting that Pb may be transferred through the food chain but that trophic transfer of 

### 30
Pb results in gradual attenuation, i.e., lower concentration at each success ive trophic level. 

### 31
Earthworm and plant vegetative tissue collected from a rifle and pistol range that contained average 

### 32
soil Pb concentration of 5,044 mg Pb /kg were analyzed for Pb content and used to model secondary 

### 33May 2011 7

- 24 DRAFT

– DO NOT C ITE OR QUOTE bioavailability to mammals ( Kaufman et al., 2007 ) . Earthworms were determined to contain an average of 

### 1

727 mg Pb /kg, and the Pb content of unwashed leaf tissues averaged 2,945 mg Pb /kg. Canonical 

### 2
correspondence analysis detected no relationship between earthworm and soil Pb concentration, but did 

### 3
show correlation between unwashed vegetation and soil concentration. The authors noted that th e 

### 4
relatively high Pb concentration of unwashed as opposed to washed vegetation indicated the potential 

### 5
importance of aerial deposition (or dust resuspension) in determining total vegetative Pb concentration. 

### 6
Based on the mammalian gastric model, they noted that 50% of vegetation tissue Pb and 77% of 

### 7
earthworm tissue Pb was expected to be bioavailable to consumers. The avian gizzard model indicated 

### 8

that 53% of soil Pb and 73% of earthworm Pb was bioaccessible to birds, and

, for both mammals and 

### 9

birds, the bi oaccessible fraction of Pb was a function of total Pb concentration. 

### 10
The transfer of Pb from soils contaminated by a Pb

- zinc mine was limited along a soil

- plant

- insect



### 11

chicken food chain ( Zhuang et al., 2009 ) . In soils averaging 991 mg Pb /kg, Rumex K

- 1 plants sequestered 

### 12
an average of 1.6 mg/kg wet weight Pb in the shoot tissue, while larvae of the leafworm Spodoptera litura 

### 13
accumulated an average Pb concentrati on of 3.3 mg Pb /kg wet weight S. litura

- fed chickens ( Gallus 

### 14
gallus domesticus ) accumulated 0.58 mg Pb /kg and 3.6 mg Pb /kg in muscle and liver tissue, respectively, 

### 15
but only liver Pb burden was increased significantly relative to controls. A large proporti on of ingested Pb 

### 16
was excreted with the feces. Likewise, an insectivorous bird species, the black

- tailed godwit ( Limosa 

### 17

limosa ) was shown to accumulate Pb from earthworms residing in Pb

- contaminated soils ( Roodbergen et 

### 18

al., 2008 ) . Pb concentration in eggs and feathers was increased in areas with high soil and earth worm Pb 

### 19
concentration (336 and 34 mg Pb /kg, respectively): egg Pb concentration averaged 0.17 mg Pb /kg and 

### 20
feather concentration averaged 2.8 mg Pb /kg. This suggests that despite a residence breeding time of only 

### 21
a few months, this bird species could accum ulate Pb when breeding areas are contaminated. 

### 22
Rogival et al. ( 2007 ) showed significant positive correlations between soil Pb concentration along a 

### 23
gradient (approximately 50 to 275 mg Pb /k g) at a metallurgical plant, and Pb concentration in both acorns 

### 24
(from Quercus robur ) and earthworms (primarily Dendrodrilus rubidus and Lumbricus rubellus ) collected 

### 25
on site. Acorn and earthworm Pb contents were, in turn, positively correlated with the Pb concentration in 

### 26
the liver, kidney, and bone tissues of locally trapped wood mice ( Apodemus sylvaticus ). 

### 27
The uptake and transfer of Pb from soil to native plants and to red deer ( Cervus elaphus ) was 

### 28
investigated in mining areas of the Sierra Madrona Moun tains in Spain ( Reglero et al., 2008 ) . The authors 

### 29
reported a clear pattern between plant Pb concentra tion and the Pb content of red deer tissues with 

### 30
attenuation (i.e., decreasing concentration) of Pb up the food chain. Interestingly, soil geochemistry likely 

### 31
was affected by mining activity as holm oak ( Quercus ilex ), gum rockrose ( Cistus ladanifer ), elml eaf 

### 32
blackberry ( Rubus ulmifolius ), and grass (Graminae) tissues collected from mining areas exhibited 

### 33
increased Pb levels (up to 98 mg Pb /kg in grasses and 21 mg Pb /kg in oak) despite the fact that total soil 

### 34
Pb concentration were not significantly greater than those of the non

- mining areas. 

### 35May 2011 7

- 25 DRAFT

– DO NOT C ITE OR QUOTE Positive relationships were observed between Cepaea nemoralis snail tissue Pb levels and Pb 

### 1

concentration measured in Urtica dioica leaves in field

- collected samples from areas characterized by 

### 2

metal soil contamination (approximately 200 to 400 mg Pb /kg) ( Notten et al., 2005 )

. Inouye et al ( 2007 ) 

### 3
found that several invertebrate prey of fence lizards, including Acheta domestica crickets, Tenebrio 

### 4

molitor beetles, and P. scaber isopods, accumulate Pb from dietary exposures (10, 50, 100, 250, 500 , 750, 

### 5

and 1

, 00 0 mg Pb /kg) lasting between 44 and 72 days. By day 44, Pb body burdens of crickets were 31, 50 

### 6

and 68 mg/kg (wet weight) at the three highest dietary exposures, respectively. Isopods and beetle larvae 

### 7
accumulated significantly less Pb, with average body burdens of 10, 15, and 14 mg Pb /kg following 56 

### 8
days of exposure, and 12, 14, and 31 mg Pb /kg following 77 days of exposure, respectively. For all 

### 9
invertebrates tested, Pb was sequestered partly in the exoskeleton, and partly in granules. Exos keleton Pb 

### 10
may be available to predators, but periodically returns to background level with each shedding. Granular 

### 11
Pb is unavailable. Trophic attenuation is thus likely. 

### 12
Overall, studies of Pb transfer in food webs have established the existence of pervas ive trophic 

### 13
transfer of the metal, but no consistent evidence of trophic magnification. It appears that on the contrary, 

### 14
attenuation is common as Pb is transferred to higher trophic levels. However, many individual transfer 

### 15
steps, as from particular plants to particular invertebrates, result in concentration, which may then be 

### 16
undone when stepping to the next trophic level. It is possible that whether trophic transfer is magnifying 

### 17
or attenuating depends on Pb concentration itself. Kaufman et al. ( 2007 ) determined that, at low 

### 18
concentrations of soil Pb, risk to secondary consumers (birds and mammals) was driven by the 

### 19

bioavai lability of Pb in worm tissues, while at high soil concentrations, bioavailability of soil

- associated 

### 20
Pb was more critical. The authors concluded that incorporation of bioavailability/bioaccessibility 

### 21

measurements in terrestrial risk assessments could lead to more accurate estimates of critical Pb levels in 

### 22
soil and biota. Finally, while trophic magnification does greatly increase exposure of organisms at the 

### 23
higher levels of the food web, these studies establish that atmospherically deposited Pb reaches sp ecies 

### 24
that have little direct exposure to it. For those species, detrimental effects are not a function of whether 

### 25
they accumulate more Pb than the species they consume, but of the absolute amount they are exposed to, 

### 26
and their sensitivity to it. 

### 27

Table 7

- 2

. Soil

- to

- tissue bioaccumulation factors for various terrestrial plant, invertebrate, and vertebrate species

Species Soil Concentration Tissue Type Tissue Concentration Bioaccumulation Factor Reference

Vegetable 379 mg/kg Root 47.3 mg/kg 0.12 X. Hu & Ding ( 2009 ) Vegetable 379 mg/kg Shoot 36.2 mg/kg 0.1 X. Hu & Ding ( 2009 ) P. angulata 4020 mg/kg Root 527.1 mg/kg 0.13 Cui et al. ( 2007 ) P. angulata 4020 mg/kg Shoot 331.1 mg/kg 0.08 Cui et al. ( 2007 ) A. theophrasti 4020 mg/kg Root 38.7 mg/kg 0.01 Cui et al. ( 2007 ) A. theophrasti 4020 mg/kg Shoot 61.4 mg/kg 0.02 Cui et al. ( 2007 )May 2011 7

- 2 6 DRAFT

– DO NOT C ITE OR QUOTE Species Soil Concentration Tissue Type Tissue Concentration Bioaccumulation Factor Reference

S. vestita 2409 mg/kg Shoot 3142 mg/kg 1.3 Y. Li et al. ( 2009 )
S. vestita 2409 mg/kg Root 7457 mg/kg 3.1 Y. Li et al. ( 2009 ) S. grevilloides 5330 mg/kg Shoot 69 mg/kg 0.01 Y. Li et al. ( 2009 )
D. carota 40.1 mg/kg Root 12.2 mg/kg 0.3 Murray et al. ( 2009 )
A. fistulosum 3560 mg/kg Whole plant 23.7 mg/kg 0.007 Cho et al. ( 2009 )
C. baccatum 0.6 mg/kg Fruit 0.86 mg/kg 1.4 Antonious & Kochhar ( 2009 )
A. achatina 1200 mg/kg Whole body 468.5 mg/kg 0.39 Ebenson & Ologhobo ( 2009b )
A. achatina 200 mg/kg Whole body 91.4 mg/kg 0.46 Ebenson & Ologhobo ( 2009b )
A. achatina 20 mg/kg Whole body 12.2 mg/kg 0.61 Ebenson & Ologhobo ( 2009b ) H. asper s a 39 mg/kg Viscera 1.1 mg/kg 0.03 De Vaufleury et al. ( 2006 ) H. asper s a 52 mg/kg Viscera 1.2 mg/kg 0.02 De Vaufleury et al. ( 2006 )
L. terrestris 710 mg/kg Whole body 20.6 mg/kg 0.03 Darling & Thomas ( 2005 )
O. asellus 245 mg/kg Whole body 43 mg/kg 0.18 G á l et al. ( 2008 ) T. talpoides 777 mg/kg Liver 1.63 mg/kg 0.002 Reynolds et al. ( 2006 ) T. talpoides 777 mg/kg Whole body 3.85 mg/kg 0.005 Reynolds et al. ( 2006 )
L. limosa 336 mg/kg Egg 0.17 mg/kg 0.0005 Roodbergen et al. ( 2008 )
L. limosa 336 mg/kg Feather 2.79 mg/kg 0.008 Roodbergen et al. ( 2008 )



## 7.2.4. Biological Effects

Various adverse effects can be observed in exposed terrestrial species following uptake and 

### 1

accumulation of Pb. While many of the responses are specific to organism type, induction of antioxidant 

### 2
activities in response to Pb exposure has been reported in plants, invertebrates, and vertebrates. In this 

### 3
section, the observed biological effects caused by exposure to atmosphe re

- derived Pb will be discussed, 

### 4

while the results of dose

- response experimentation will be addressed in Section 7.2.5

. Because 

### 5

environmental releases of Pb often include simultaneous release of other metals, it can be difficult to 

### 6
identify Pb

- specific eff ects in field studies, with the exception of effects from leaded gasoline and some 

### 7
Pb smelter deposition

. Many laboratory studies that expose organisms to natural soils (or to biosolids



### 8
amended soils) also include exposure to multiple metals

. There is some information about mechanisms of 

### 9
metal interactions, such as through competition for binding locations on specific enzymes or on cellular 

### 10
receptors, but generally such interactions (particularly of multiple metals) are not well understood 

### 11

( ATSDR, 2004 )

. Despite a few well

- known examples of metal antagonism (e.g., Cu and Mo or Cd and 

### 12
Zn), it is common practice to assume additivity of effects ( Fairbrother et al., 2007 )

. Because this review is 

### 13
focused on effects of Pb, studies reviewed for this section and the following include only those for which 

### 14
Pb was the only, or primary, metal to which the organism was exposed

. 

### 15

7.2.4.1. Plants and Lichen

Pb exposure has been linked to decreased photosynthesis in affected plants, significant induction of 

### 16
antioxidant activities, genetic abnormalities, and decreased growth. Induction of antioxidant responses in 

### 17
plants has been shown to increase tolerance to m etal soil contamination, but at sufficiently high levels, 

### 18May 2011 7

- 27 DRAFT

– DO NOT C ITE OR QUOTE antioxidant capacity is exceeded, and metal exposure causes peroxidation of lipids and DNA damage, 

### 1
eventually leading to accelerated senescence and potentially death ( Stobrawa & Lorenc

- Plucinska, 2008 )

. 

### 2

Effects on Photosystem and Chlorophyll

The effect of Pb exposure on the structure and function of plant photosystem II was studied in giant 

### 3

duckweed, Spirodela polyrrhiza ( Ling & Hong, 2009 ) . Although this is an aquatic plant, photosystem II is 

### 4
present in all plants, and effects on photosystem II observed in any plant species are likely to occur in all 

### 5
of them. The Pb concentration of extracted photosystem II particles was found to increase with increasing 

### 6

environ mental Pb concentration, and increased Pb concentration was shown to decrease emission peak 

### 7
intensity at 340 nm, amino acid excitation peaks at 230 nm, tyrosine residues, and absorption intensities. 

### 8
This results in decreased efficiency of visible light abs orption by affected plants. The authors theorized 

### 9

that Pb 2+ may replace either Mg 2+ or Ca 2+ in chlorophyll or the oxygen

- evolving center, inhibiting 

### 10
photosystem II function through an alteration of chlorophyll structure. Consistently with these results, Wu 

### 11

et al. ( 2008 ) demonstrated that Pb exposure interfered with and decreased light absorption by spinach 

### 12

( Spinacia oleracea ) plants. Spinach seeds were soaked in 5, 12, or 25 mM Pb Cl 2 for 48 hours prior to 

### 13
germination, and following 42 days of growth, plants were sprayed with Pb Cl 2 solutions. Chloroplast 

### 14
absorption peak intensity, fluorescence quantum yield at 680 nm, and whol e

- chain electron transport rate 

### 15
all decreased with Pb exposure, as did photosystem II photoreduction and oxygen evolution. Liu et al. 

### 16

( 2010 ) observed that chlorophyll a and b content in wheat grown in soils spiked with Pb nitrate rose with 

### 17
length of exp osure until 14 days, at which point chlorophyll decreased. At exposures of 0.1 and 0.5 mM 

### 18
Pb in hydroponic solution for 50 days, concentration of chlorophyll a and b was decreased in radish ( R. 

### 19

sativus ) ( Kumar & Tripathi, 2008 ) . Changes in chlorophyll content in response to Pb were also observed 

### 20
in lichen and moss species following exposures intended to simul ate atmospheric deposition ( Carreras & 

### 21
Pignata, 2007 )

. Usnae amblyoclada lichen was exposed to aqueous Pb solutions of 0.5, 1, 5, and 10 mM 

### 22
Pb nitrate; chlorophyll a concentration was shown to decrease with increasing Pb exposure. However, the 

### 23
ratio of lichen dry weight to fresh weight increased following Pb exposures. As compared to other metals, 

### 24

P b caused less physiological damage, which the authors attributed to the metal ’ s high affinity for binding 

### 25
to and sequestration within cell walls. 

### 26
The effect of Pb exposure on chlorophyll content of the moss and liverwort species Thuidium 

### 27

delicatulum

, T. sparsifolium , and Ptychanthus striatus was investigated following simulated atmospheric 

### 28
exposures of 10 - 10 to 10 - 2 M Pb ( Shakya et al., 2008 ) . Both chlorophyll a and total chlorophyll content of 

### 29
the mosses ( T. delicatulum and T. sparsifolium ) decreased with increasing Pb exposure, but the effect was 

### 30

not stati stically significant. For the liverwort, Pb exposure resulted in significant decreases in content of 

### 31
chlorophyll a , chlorophyll b , and total chlorophyll. Further, the total chlorophyll content of Hypnum 

### 32May 2011 7

- 28 DRAFT

– DO NOT C ITE OR QUOTE plumaeforme mosses was decreased by 5.8% following ex posure to 10 mM Pb, while lower exposures 

### 1
slightly elevated chlorophyll content. 

### 2

Response of Antioxidants

Increased antioxidant activity is a common response to Pb exposure, although this endpoint may 

### 3
not necessarily be an indication of deleterious effect s on plant vitality. Increases in reactive oxygen 

### 4
species with increasing exposure to Pb have been demonstrated in broad bean ( Vicia faba ) ( C

. - R. Wang et 

### 5

al., 2010 ; C.

- R. Wang, Wang, Tian, Yu, et al., 2008 ; C. Wang, Y. Tian, et a l., 2010 ) and tomato 

### 6
( Lycopersicon esculentum ) ( C.

- R. Wang, Wang, Tian, Xue, et al., 2008 ) , where they were accompanied by 

### 7
proportional increases in superoxide dismutase (SOD), glutathione, guaiacol peroxidase, as well as lipid 

### 8
peroxidat ion, and decreases in catalase. Reddy et al. ( 2005 ) found that horsegram ( Macrotyloma 

### 9
uniflorum ) and bengalgram ( Cicer arietinum ) plants exposed to Pb solutions of 200, 500, and 800 mg 

### 10

Pb /kg exhibited increased antioxidant activity: at exposures of 800 mg Pb /kg, root and shoot SOD activity 

### 11

increased to 2

– 3 times that of controls, and induction was slightly higher in M. uni florum . Similarly, 

### 12
catalase, peroxidase, and glutathione

- S

- transferase activities were elevated in Pb

- stressed plants, but were 

### 13
again higher for M. uniflorum . Antioxidant activities were also markedly greater in the root tissues than 

### 14

the shoot tissues of t he two plants, and were very likely related to the higher Pb accumulation rate of the 

### 15
roots. The effectiveness of the up

- regulation of antioxidant systems in preventing damage from Pb uptake 

### 16
was evidenced by lower malondialdehyde (MDA) (a chemical marker o f lipid peroxidation) concentration 

### 17

in M. uniflorum versus C. arietinum , indicating a lower rate of lipid peroxidation in response to M. 

### 18
uniflorum ’ s higher antioxidant activity. 

### 19
Gupta et al. ( 2010 ) contrasted responses of two ecotypes of Sedum alfredii (an Asian perennial 

### 20
herb), one an accumulator of Pb and the other not. Glutathione level was increa sed in both, and root and 

### 21
shoot lengths were decreased following long

- term exposures to Pb up to 200 μM. However, the 

### 22
accumulator plants exhibited greater SOD and ascorbate peroxidase activity, likely as a result of greater 

### 23
Pb uptake and a concurrent incre ased detoxification capacity. Similar results were reported by Islam et al. 

### 24

( 2008 ) : following Pb exposures of 200 μM, catalase, ascorbic acid, and glutathione levels of another 

### 25

Chinese herb, Elsholtzia argyi , were increased, while SOD and guaiacol peroxidase activities decreased. 

### 26
Microscopic analysis a lso showed that affected plants exhibited abnormal chloroplast structures. The 

### 27
response of glutathione was further confirmed in wheat ( Liu et al., 2010 ) grown in soils spiked with Pb 

### 28
nitrate. Evidence of increasing lipid peroxidation (MD A accumulation) with increasing Pb exposure was 

### 29

also found in mosses ( Sun et al., 2009 ) and lichens. Lichens field

- collected from the trunks of poplar 

### 30

( Populus tremula ) trees in eastern Slovakia were chemically analyzed for metal concentration arising 

### 31
from exposure to smelter pollution ( Dzubaj et al., 2008 ) . These concentrations (ranging from 13 to 1

, 523 

### 32

mg Pb /kg dry weight) were assessed in relation to physiological variables, including chlorophyll a and b

, 

### 33May 2011 7

- 29 DRAFT

– DO NOT C ITE OR QUOTE carotenoids, photosystem II activity, CO 2 gas exchange (respiration), and MDA content. Lichen Pb levels 

### 1
were significantly correlated only with MDA content. 

### 2

Gr owth

There is evidence of effects of Pb on higher growth processes as well. Both growth and carotenoid 

### 3
and chlorophyll content of Brassica juncea (mustard) plants were negatively affected by Pb exposure 

### 4

( John et al., 2009 ) . Pb treatments of 1,500 μM (as Pb acetate solution) decreased root lengths and stem 

### 5
heights by 50% after 60 days. Exposure to 600 μM Pb and greater decreased carotenoid content, while 

### 6
chlorophyll a was decrea sed at Pb exposures of 450 μM and higher. However, no effects were seen in 

### 7
growth or chlorophyll production of maize ( Zea mays ) following growth in smelter ash

– spiked soils 

### 8
containing 1,466 mg Pb /kg (and 18.6 mg Cd /kg) ( Komarek et al., 2009 ) . Pb concen trations of 7,331 

### 9
mg/kg (98.0 mg/kg Cd) were required to elicit chlorosis and the expected decreased in growth. 

### 10
Chinese cabbage ( Brassica pekinensis ) exposed to Pb

- containing soils (0, 4, and 8 mM/kg dry 

### 11
weight) exhibited depressed nitrogen assimilation as measured by shoot nitrite content, nitrate reductase 

### 12
activity, and free amino acid concentration ( Xiong et al., 2006 ) . The authors planted germinated cabbage 

### 13
seeds in soils spiked with Pb acetate to give fin al soil concentration of 0.2, 0.4, and 0.8 mg/kg dry weight 

### 14
total Pb and collected leaf samples for 11 days. At exposures of 0.4 and 0.8 mg/kg, leaf nitrite content was 

### 15
decreased by 29% and 20%, while nitrate content was affected only at the highest Pb exp osure (70% of 

### 16
control levels). Free amino acid content in exposed plants was 81% and 82% of control levels, 

### 17
respectively. B. pekinensis shoot biomass was observed to decrease with increasing Pb exposures, with 

### 18
biomass at the two highest Pb exposures repres enting 91% and 84% of control growth, respectively. 

### 19

Genetic and Reproductive Effects

Exposure to Pb also resulted in genetic abnormalities, including bridges, condensed bivalents, and 

### 20
laggards, in the meiotic cells of pea plants ( Lathyrus sativus ) ( Kumar & Tripathi, 2008 ) . Seeds were 

### 21
germinated in soils amended with Pb nitrate at concentrations of 25, 50, 100, 200, and 300 mg Pb /kg, and 

### 22
concentrations of 100 mg Pb /kg and greater were found to be genotoxic or detrimental to pea viability. 

### 23

Cenkci et al. ( 2010 ) exposed fodder turnip ( B. rapa ) to 0.5 to 5 nM of Pb nitrate for 6 days and showed 

### 24
decreased genetic template stability (as quantified by random amplified pol ymorphic DNA profiles) and 

### 25
decreased photosynthetic pigments. 

### 26
Pb exposure also decreased germination rate and growth, and increased pollen sterility in radish 

### 27
grown for 50 days in hydroponic solutions containing 0.5 mM Pb ( Kumar & Tripathi, 2008 ) Plants 

### 28
decreased growth, curling and chlorosis of young leaves, and decreased root growth. In addition, Gopal 

### 29

and Rizvi ( 200 8 ) showed that Pb exposure increased uptake of phosphorus and iron and decreased sulfur 

### 30
concentration in radish tops. 

### 31May 2011 7

- 30 DRAFT

– DO NOT C ITE OR QUOTE Interestingly, as in zebra finch (Section 7.2.3.3), Ca was found to counteract the toxic effects of Pb 

### 1
in both monocotyledon and dicoty ledon plant seedlings, with tomato ( Lycopersicon esculentum ), rye 

### 2
(Lolium sp.), mustard, and maize plants exhibiting increased tolerance to Pb exposures of 5, 10, and 20 

### 3
mg/L in the presence of Ca concentration of 1.2 mM and higher ( Antosiewicz, 2005 )

. 

### 4

7.2.4.2. Invertebrates

Exposure to Pb also causes antioxidant effects, reductions in survival and growth, as well as 

### 5
decreased fecundity in terrestrial invertebrates as summarized in the 2006 Pb AQCD. In addition to these 

### 6
endpo ints, recent literature also indicates that Pb exposure can cause significant neurobehavioral 

### 7

aberrations, and in some cases, endocrine

- level impacts. Second

- generation effects have been observed in 

### 8
some invertebrate species. 

### 9
The morphology of γ

- aminobuty ric acid (GABA) motor neurons in Caenorhabditis elegans 

### 10
nematodes was affected following exposure to Pb nitrate for 24 hours ( Du & Wang, 2009 ) . The authors 

### 11
determined that exposures as low as 2.5 μM Pb nitrate could cause moderate axonal discontinuities, and 

### 12
observed a significant increase in the number of formed gaps and ventral cord gaps at Pb nitrate 

### 13
exposures of 75 and 200 μM. Younger C. elegans larvae were more likely to exhibit neurobehavioral 

### 14
toxicity symptoms in response to Pb exposure (2.5 μM) ( Xing, Guo, et al., 2009 ) . Neural degeneration, as 

### 15
demonstrated by dorsal and ventral cord gaps and neuronal loss was also more pronounced in young 

### 16

larval C. elegans than in older larvae and adults ( Xing, Rui, et al., 2009 )

. C. elegans nematodes exposed 

### 17
to Pb concentration as low as 2.5 μM for 24 hours also exhibited significantly altered behavior 

### 18
characterized by decreased head thrashes and body bends. Exposures of 50 μM Pb and greater decreased 

### 19

the number of nematode forward turns ( D. Y. Wang & Xing, 2008 ) . Chemotaxis towards NaCl, cAMP, 

### 20

and biotin was also decreased in C. elegans nematodes exposed to Pb concen tration greater than 2.5 μM 

### 21

( Xing, Du, et al., 2009 ) . Th is evidence suggests that Pb may exert neurotoxic action in invertebrates as it 

### 22

does in vertebrates. However, it is unclear how these behavioral aberrations would affect fitness or 

### 23

survival ( D. Y. Wang & Xing, 2008 )

. 

### 24
Younger individuals also appear to be more sensitive to the reproductive effects of Pb exposure. 

### 25

Guo et al. ( 2009 ) showed that concentrations of 2.5, 50, and 100 μM Pb had greater significant adverse 

### 26
effects on reproductive output when early

- stage larval C. elegans were exposed. Adult C. elegans 

### 27
exhibited decreased brood size only when exposed to the highest Pb concentration. 

### 28
The progeny of C. elegans nematodes exposed to 2.5, 75, and 200 μM Pb nitrate exhibited 

### 29
significant indications of multi

- generational toxicity ( D. Y. Wang & Peng, 2007 ) . Life spans of offspring 

### 30
were decreased by increasing parental Pb exposure, and were comparable to the reductions in parental 

### 31

life

- spans. Similarly, diminished fec undity was observed in the progeny of C. elegans exposed to Pb (9%, 

### 32
19%, and 31% reductions of control fecundity, respectively), although the decrease was smaller than in 

### 33May 2011 7

- 31 DRAFT

– DO NOT C ITE OR QUOTE the exposed parental generation (reductions of 52%, 58%, and 65%, respectively). Sign ificant behavioral 

### 1
defects affecting locomotion were also observed in the offspring, but these impacts were not determined 

### 2

to be concentration

- dependent. 

### 3

E. andrei earthworms exposed to 21 different soils, each containing 2,000 mg/kg freshly added Pb, 

### 4

for 28 days exhibited highly variable mortality, ranging from 0% to 100%, ( Bradham et al., 2006 ) . Pb 

### 5
body burden of exposed worms ranged from 29 to 782 mg Pb /kg. Internal Pb concentration was also 

### 6
negatively correlated to reproductive output. C EC and pH were found to be the principal soil 

### 7

characteristic s determining the differences in those effects, although the apparent role of CEC may only 

### 8

have been due to its correlation with other soil characteristics

. Low soil Pb concentration (5 mg/kg) also 

### 9
decreased the protein content of E. fetida earthworms during a 7

- day exp osure ( M. Li et al., 2009 ) . Higher 

### 10
Pb concentration had no effect on protein production. However, cellulase activity was increased by the 7

- 

### 11
day exposures to Pb at all exposure concentrations (31%, 13%, and 23% of control activity at exposures 

### 12
of 5, 50, and 500 mg Pb /kg, respectively), indicating that Pb exerted a detrimental effect on worm 

### 13
metabolism. By contras t, Svendsen et al. ( 2007 ) found that L. rubell us earthworms exposed for 42 days to 

### 14

field

- collected smelter

- polluted soils containing average Pb concentration of 106, 309, and 514 mg Pb /kg 

### 15
dry weight exhibited normal survival and cocoon production rates, even though they accumulated more 

### 16

Pb with increa sed environmental concentration. The much smaller effect may be explained by the 

### 17
increased aging time undergone by field soil, causing a larger fraction of the total Pb to be complexed and 

### 18
sequestered by organic and inorganic compounds. 

### 19
As in plants, induc tion of antioxidant activity is affected by exposure to Pb in invertebrates. The 

### 20
induction of antioxidant activity was correlated to standard toxicity measurements in Theba pisana snails 

### 21

( Radwan et al., 2010 ) . Topical application of Pb solutions (500 to 2,000 μg Pb per animal) to snails 

### 22

resulted in dec reased survival, increased catalase and glutathione peroxidase activities, and decreased 

### 23
glutathione concentration. The 48

- h ou r LD 50 concentration was determined to be 653 μg per snail. Snail 

### 24
glutathione content was decreased at exposures of 72.2% of the 4 8

- h ou r LD 50 value, while Pb exposure at 

### 25

40% of the 48

- h ou r LD 50 value induced catalase and glutathione peroxidase activities. Further, decreased 

### 26
food consumption, growth, and shell thickness were observed in juvenile A. achatina snails exposed to 

### 27

Pb

- contam inated (concentration greater than 134 mg/kg) diet for 12 weeks ( Ebenso & Ologhobo, 2009a )

. 

### 28
A similar depression of growth was observed in sentinel juvenile A. achatina snails deployed at Pb



### 29
polluted sites in the Niger Delta region of Nigeria. Although snail mortality was not increased 

### 30
significantly by exposure to soil Pb up to 1,200 mg/kg, a concentration

- dependent relationship was 

### 31
established for growth, with significant reduction observed at 12

- week exposures to 20 mg Pb /kg ( Ebenso 

### 32
& Ologhobo, 2009b ) . However, consumption of field

- collected Pb

- polluted U. dioica leaves containing 3 

### 33
mg/kg sto pped all reproductive output in C. nemoralis . Snails also exhibited diminished food 

### 34
consumption rates when offered leaves with both low (1.5 mg Pb /kg) and high Pb content, but the 

### 35
mechanism of the dietary aversion was not defined ( Notten et al., 2006 )

. 

### 36May 2011 7

- 32 DRAFT

– DO NOT C ITE OR QUOTE Chronic dietary exposure to Pb was also examined in post

- embryonic oribatid mites ( Archegozetes 

### 1
longisetosus ) ( Kohler et al., 2005 ) . Both algae and bark samples were soaked in 100 mg/L Pb as Pb nitrate 

### 2
and provided as diet and substrate, respectively, to larval mites. In addition to elevated heat shock proteins 

### 3
(hsp70), 90.8% of the protonymphs exhibited significant leg deformities, including abnormal claws, 

### 4
shortened and thickened legs, and translocated setae. Although not specifically discussed, it is very likely 

### 5
that these deformities would decrease mite mobility, prey capture, and reproductive viability. 

### 6

Lock et al. ( 2006 ) compared the toxicity of both laboratory

- spiked soils and field

- collected Pb



### 7
contaminated soils to springtails ( F. candida ). The 28

- day EC 50 values derived for F. candida ranged from 

### 8

2,060 to 3, 210 mg Pb /kg in leached and unleached Pb

- spiked soils, respectively, whereas field

- collected 

### 9
soils had no significant effect on springtail reproduction up to (but not including) 14,436 mg Pb /kg ( Lock 

### 10

et al., 2006 ) . Consequently, leaching soils prior to use in bioassays had only a slight effect on Pb to xicity 

### 11
to resident springtails, and did not provide an appropriate model for field

- weathered, Pb

- contaminated 

### 12
soils. This indicates that physiochemical factors other than leaching may be more important determinants 

### 13
of Pb bioavailability

. A 4

- week exposure to Pb

- amended soils containing up to 3,200 mg Pb /kg had no 

### 14
significant adverse effect on Sinella curviseta springtail survival or reproduction ( Xu et al., 2009 )

. 

### 15
Carabid beetles ( Pterostichus oblongopunctatus ) inhabiting soils contaminated by pollution from a 

### 16

Pb

- Zn smelter (containing 136 to 2,635 mg Pb /kg) were field

- collected and then laboratory

- reared for two 

### 17
generations ( Lagisz & Laskowski, 2008 ) . While fecundity was positively correlated to soil metal 

### 18
concentration (e.g., more eggs were produced by females collected from contaminated areas), the 

### 19
hatching rate of eggs diminished with increasing soil metal contamination. For the F1 generat ion, females 

### 20
produced by parents inhabiting highly polluted areas exhibited decreased body mass. The authors stated 

### 21
that these results indicate that invertebrates inhabiting metal

- (or Pb

- ) contaminated soils could face 

### 22

“ significantly altered life

- history parameters. ” 

### 23
Several studies suggest that Pb may disrupt hormonal homeostasis in invertebrates. Shu et al. 

### 24

( 2009 ) reported that vitellogenin production in both male and female S. litura moths was disrupted 

### 25
following chronic dietary exposure to Pb. Adult females reared on diets containing 25, 50, 100, or 200 

### 26

mg Pb /kg exhibite d decreased vitellogenin mRNA induction, and vitellogenin levels were demonstrated 

### 27
to decrease with increasing Pb exposure. Conversely, in a study by Zheng and Li ( 2009 ) , vitellogenin 

### 28

mRNA wa s detected at higher levels in males exposed to 12 and 25 mg Pb /kg, although vitellogenin 

### 29
levels were not affected. Similarly, the sperm morphology of the Asian earthworm ( Pheretima guillelmi ) 

### 30
was found to be altered significantly following 2

- week exposure to soils containing 1,000, 1,400, 1,800, 

### 31
and 2,500 mg Pb /kg ( Zheng & Li, 2009 ) . Common deformities were swollen head and head helices, while 

### 32
head bending was also recor ded in some cases. These deformities were observed following exposures to 

### 33

concentration below the 14

- day LC 50 (3,207 mg Pb /kg) and below the concentration at which weight was 

### 34
diminished (2,800 mg Pb /kg). Experimentation with the model organism Drosophila i ndicates that Pb 

### 35May 2011 7

- 33 DRAFT

– DO NOT C ITE OR QUOTE exposure may increase time to pupation and decrease pre

- adult development, both of which are 

### 1

endocrine

- regulated ( Hirsch et al., 2010 )

. 

### 2

7.2.4.3. Terrestrial Vertebrates

According to the 2006 Pb AQCD, commonly o bserved effects of Pb on avian and mammalian 

### 3
wildlife include decreased survival, reproduction, and growth, as well as effects on development and 

### 4
behavior. More recent experimental data presented here expand and support these conclusions, and also 

### 5

indicate that Pb can exert other effects on exposed terrestrial vertebrates, including alteration of hormones 

### 6

and other biochemical variables. 

### 7

Red

- backed salamanders ( Plethodon cinereus ) exposed to Pb

- amended soils (553, 1,700, 4,700, 

### 8
and 9,167 mg Pb /kg) by Bazar et al. ( 2010 ) exhibited lowered appetite and decreased white blood cell 

### 9
counts at the two highest concentrations, but tolerated field

- collected, aged soils containing Pb 

### 10

co ncentrations of up to 16,967 mg Pb /kg with no significant deleterious effects. The white blood cell 

### 11

count of adult South American toads, Bufo arenarum was also decreased by weekly sublethal 

### 12
intraperitoneal injections of Pb acetate at 50 mg Pb/kg body weigh t ( Chiesa et al., 2006 ) . The toads also 

### 13
showed altered serum profiles and increased number of circulating blast cells. Final toad blood Pb levels 

### 14
were determined to be 8.6 mg Pb /dL , although it is unclear whether this is representative of Pb 

### 15

concentration observed in field B. arenarum populations exposed to Pb. The authors suggested that, based 

### 16
on these findings, long

- term environmental exposure to Pb could affect toad immune respon se. In western 

### 17
fence lizards ( S. occidentalis ), sub

- chronic (60

- day) dietary exposure to 10 to 20 mg Pb /kg per day 

### 18
resulted in significant sublethal effects, including decreased cricket consumption, decreased testis weight, 

### 19
decreased body fat, and abnormal posturing and coloration ( Salice et al., 2009 ) . Long

- term dietary Pb 

### 20
exposures are thus likely to decrease lizard fitness. 

### 21
Even in cases of high environmental Pb exposures, however, linking Pb body burdens to adverse 

### 22
biological effects can be difficult. Pb tissue concentration in field

- collected urban blackbirds ( Turdus 

### 23

merula ) were determined to be 3.2 mg Pb /kg, 4.9 mg Pb /kg, and 0.2 mg Pb /kg wet mass in breast 

### 24
feathers, washed tail feathers, and blood, respectively ( Scheifler, Coeurdassier, et al., 2006 ) . Although 

### 25
these levels were significantly higher than those measured in rural blackbirds, elevated Pb tissue 

### 26
concentration was not significantly correlated to any index of body condition . On the other hand, 

### 27
Hargreaves et al. ( 2010 ) showed that Pb tissue concentration of female arctic shorebi rds was negatively 

### 28
correlated with reproductive success. Maternal blood Pb levels were negatively associated with hatching 

### 29
success in black bellied plovers ( Pluvialis squatarola ) and ruddy turnstones ( Arenaria interpres ), and 

### 30
with nest duration in all spec ies tested. There was no significant correlation between adult whole

- blood or 

### 31
feather Pb concentration and Pb levels in produced eggs. 

### 32May 2011 7

- 34 DRAFT

– DO NOT C ITE OR QUOTE The long

- term effect of atmospheric Pb deposition on pied flycatcher ( Ficedula hypoleuca ) 

### 1
nestlings was determined in nat ive communities residing in the Laisvall mining region of Sweden 

### 2

( Berglund et al. , 2010 ) . Moss samples indicated that Pb deposition in study areas ranged between 100 and 

### 3

2,000 mg Pb /kg dry weight during operations and 200 and 750 mg Pb /kg when operations ceased. A 

### 4
simultaneous slight reduction was observed in pied flycatcher blood P b levels, from 0.4 to 0.3 mg Pb /kg). 

### 5
However, clutch size was decreased in pied flycatchers inhabiting the mining area both during and after 

### 6
mining operations, and mean nestling mortality was 2.5 and 1.7 higher after mining operations in the 

### 7
mining region than in reference areas, respectively. The authors noted that Pb deposition in the mining 

### 8
region remained elevated even after mining operations ceased, and that stable Pb isotope analysis 

### 9
suggested that smelter Pb remained available to pied flycatcher thro ugh the transfer of historically 

### 10
deposited Pb in soil to prey items. 

### 11
The level of corticosteroid hormones in field populations of white stork nestlings ( Ciconia ciconia ) 

### 12
in a mining area affected by Pb and other metals was positively correlated with blood Pb levels ( Baos e t 

### 13

al., 2006 ) . The effect was more pronounced for single nestlings than for multiple

- chick broods. 

### 14
Surprisingly, average blood Pb levels in chicks inhabiting reference areas was 91 μg/L ( ± 51), which was 

### 15
higher than blood Pb levels from the mining area (44 ± 34 μg/L). However, the correlation between blood 

### 16
Pb levels and the corticosteroid stress response in white stork nestlings was observed in both groups of 

### 17
birds. Burger and Gochfeld ( 2005 ) exposed herring gull ( Larus argentatus ) c hicks to Pb acetate via an 

### 18
intraperitoneal injection of 100 mg Pb/kg body weight, to produce feather Pb concentration approximately 

### 19
equivalent to those observed in wild gulls. Pb

- exposed gulls exhibited abnormal behaviors, including 

### 20
decreased walking and f ood begging, erratic behavioral thermoregulation, and diminished recognition of 

### 21

caretakers. 

### 22
Again, dietary or other health deficiencies unrelated to Pb exposure are likely to exacerbate the 

### 23

effects of Pb. Ca

- deficient female zebra finches ( T. guttata ) had a suppressed secondary humoral immune 

### 24
response following 28

- day exposures to 20 mg/L Pb in drinking water ( Snoeijs et al., 2005 ) . This 

### 25
response, however, was not observed in birds fed sufficient Ca. Although a significant finding, these data 

### 26
are difficult to interpret under field conditions where the o verall health of avian wildlife may not be easily 

### 27

determined. 

### 28
Chronic Pb exposures were also demonstrated to adversely affect several mammalian species. 

### 29
Young adult rats reared on a diet containing 1

, 500 mg/kg Pb acetate for 50 days demonstrated less 

### 30
plas ticity in learning than non

- exposed rats ( McGlothan et al., 2008 ) , indicating that Pb exposure caused 

### 31
significantly altered neurological function. Yu et al. ( 2005 ) showed that dietary Pb exposure affected both 

### 32
the growth and endocrine function of gilts ( S. domestica ). Consumption of 10 mg Pb/kg diet resulted in 

### 33
lower body weight and food intake after 120 days of dietary exposure; Pb exposure decreased final weight 

### 34
by 8.2%, and average daily food intake of Pb

- exposed pigs was decreased by 6.8% compared to control 

### 35
intake. Additionally, concentration of estradiol, lutenizing hormone, and pituitary growth hormone were 

### 36May 2011 7

- 35 DRAFT

– DO NOT C ITE OR QUOTE decreased (by 12%, 14%, and 27% versus controls, respectively), while blood Pb level was increased by 

### 1
44% to an average 2.1 μg/dL. In cattle grazing near Pb

- zinc smelters in India, blood Pb levels were 

### 2
positively correlated with plasma levels of the thyroid hormones thyroxin e (T4) and tri

- i o do thyronin e 

### 3

(T3) and the hepatic biomarkers alanine transaminase and aspartate transaminase ( Swarup et al., 2007 )

. 

### 4
Total lipids, total protein and albumin levels were decreased in the same animals. 

### 5

Pb

- treated oocytes of buffalo ( Bubalus bubalis ) assessed in vitro at concentrations ranging from 0.5 to 

### 6

1

, 000 μg/dL in one

- day cultures indicated a significant decline in viability of oocytes at 100 μg/dL ( Nandi 

### 7

et al., 2010 ) . Dose

- dependent effects on oocyte v iability, morphological abnormalities, cleavage, 

### 8

b l astocyst yield and blastocyst hatching were observed in Pb

- treated oocytes with maturation significantly 

### 9
reduced at 250 μg/dL and 100% oocyte death at 3

, 200 μg/dL. Similarly, the reproductive viability of red 

### 10
deer ( C. elaphus ) inhabiting a Pb

- contaminated mining area of Spain was shown to be altered, with 11% 

### 11
and 15% reductions in spermatozoa and acrosome integrity observed in male deer from the mining area 

### 12
compared with those residing in reference areas ( Reglero et al., 2009 )

. 

### 13



## 7.2.5. Exposure and Response of Terrestrial Species

Given that exposure to Pb may adversely affect organisms at the indiv idual, population, or 

### 14
community level, determining the rate and concentration at which these effects occur is essential in 

### 15
predicting the overall risk to terrestrial organisms. However, data from controlled studies using a single 

### 16
compound are scarce relati ve to field studies, which in turn often investigate effects of multiple metal 

### 17
contaminants and afford too little control on interacting variables to be of use in establishing a general 

### 18

dose

- response function

. This section updates available information der ived since the 2006 Pb AQCD, 

### 19
summarizing several dose

- response studies with soil invertebrates. No new exposure

- response 

### 20
information was available for plants, birds, or mammals. 

### 21

Dose

- dependent responses in antioxidant enzymes were observed in adult L. maur itii earthworms 

### 22
exposed to soil

- associated Pb contamination (75, 150, 300 mg Pb /kg) ( Maity et al., 2008 ) . By day seven 

### 23
of exposure, glutathione

- S

- tran sferase activity and glutathione disulfide concentration were positively 

### 24
correlated with increasing Pb exposures, while glutathione concentration exhibited a negative dose



### 25
response relationship with soil Pb concentration. However, these trends had become i nsignificant by the 

### 26
end of the total exposure period (28 days), as a result of normalization of antioxidant systems following 

### 27
chronic exposure. This strongly suggests that changes to earthworm antioxidant activity are an adaptive 

### 28
response to Pb exposures. 

### 29
Both survival and reproductive success of E. fetida earthworms showed concentration

- dependent 

### 30
relationships with soil Pb concentration during the course of standard 14

- and 56

- day toxicity tests ( Jones 

### 31

et al., 2009 ) . Five levels of Pb soil concentration were prepared for the acute 14

- day study via spiking 

### 32

with Pb nitrate

— 0, 300, 711, 1,687, and 2,249 mg Pb /kg, while soil concentration of 0, 355, 593, 989, and 

### 33May 2011 7

- 36 DRAFT

– DO NOT C ITE OR QUOTE 1,650 mg Pb /kg were used in chronic (56

- day) earthworm bioassays. A 14

- day acute LC 50 of 2,490 mg 

### 1

Pb /kg was determined from the dose

- response relationship, while the approximate 5 6

- day NOEC (no 

### 2
observed effect concentration) and EC 50 values were about 400 mg/kg and 1,000 mg/kg Pb, respectively. 

### 3

Currie et al. ( 2005 ) observed mortality of E. fetida after 7 and 14 days in spiked field soil at seven levels 

### 4
of Pb (0 to 10

, 000 mg Pb /kg). They reported LC 50 values of 2,662 mg Pb /kg at 7 days and 2,589 

### 5

mg Pb /kg at 14 days or 2,827 mg Pb /kg at both 7 and 14 days, depending on the number of worms in the 

### 6
experimental enclosure. 

### 7

Other studies have shown no correlation between Pb concentration in either earthworm tissue or 

### 8
soil, and earthworm survival rate. Although the Pb content of E. fe tida held in metal

- contaminated soils 

### 9
containing between 9.7 and 8,600 mg Pb /kg was positively correlated with Pb concentration of soil, there 

### 10
was no statistical relationship with earthworm survival during a 42

- day exposure period ( Nahmani et al., 

### 11

2007 ) . However, Pb concentration in soil leachate solution was significantly correlated with decreased 

### 12
earthworm survival and growth (linear regression: R 2 = 0.64, p< 0.0001). The 42

- day Pb EC 50 for E. fetida 

### 13
growth was 6,670 mg Pb /kg. 

### 14
Langdon et al. ( 2005 ) exposed three earthworm species ( E. andrei

, L. rubellus , and A. caliginosa ) 

### 15

to Pb nitrate

- amended soils at concentrations of 1,000 to 10,000 mg Pb /kg to determine species variability 

### 16
in uptake and sensitivity. Twenty

- eight

- day LC 50 values for the three species were 5,824 mg Pb /kg, 2,867 

### 17

mg Pb /kg, and 2,747 mg Pb /kg, respectively, indicating that L. rubellus and A. caliginosa are significantly 

### 18

more vulnerable to Pb contamination than E. andrei , a common laboratory species

. This is comparable to 

### 19
previous findings by Spurgeon et al. ( 1994 ) who reported 14

- day LC 50 of 4,480 mg Pb /kg and 50

- day 

### 20

LC 50 of 3,760 mg Pb /kg for E. fetida , another standard laboratory test species

. In the more recent study of 

### 21
E. fetida sensitivity summarized above, Jones ( 2009 ) reported LC 50 values for E. fetida that are similar to 

### 22

those for L. rubellus and A. caliginosa

. It is likely that these apparent species differences are a result of 

### 23

differential bioavailabi lity of the Pb in test soils. However, the Pb body burden of all three species in the 

### 24
study by Langdon et al. ( 2005 ) increased with increasing environmental concentration, and there were no 

### 25
species differences in Pb tissue content. When given a choice between treated and untreated soils, all 

### 26
worm species exhibited significant avoidance of Pb

- contaminated s oils, and altering pH (and, 

### 27
consequently, Pb bioavailability) had no impact on avoidance ( Langdon et al., 2005 ) . Field earthworms 

### 28
may thus be able to reduce their exposure to Pb through behavior. 

### 29
The individual and population

- level responses of the springtail Paronychiurus kimi to Pb were 

### 30
determined by Son et al. ( 2007 ) using artificial soils, following the 1999 European ISO methodology. The 

### 31

7

- day Pb LC 50 was determined to be 1,322 mg Pb /kg dry weight, while the 28

- day reproduction E C 50 was 

### 32
established as 428 mg Pb /kg. The intrinsic rate of population increase was lower at a Pb soil concentration 

### 33
of 1,312 mg/kg, and the authors estimated that, at this level, P. kimi populations would be extirpated. The 

### 34

authors noted that, in this case , the reproductive endpoint overestimated the population

- level risk for P. 

### 35

kimi springtails exposed to Pb, and proposed that more specific measures of population

- level endpoints 

### 36May 2011 7

- 37 DRAFT

– DO NOT C ITE OR QUOTE (such as the reduction in intrinsic rate of increase) be used to determine risk to populations. Menta et al. 

### 1

( 2006 ) showed that a nominal soil concentration of 1,000 mg Pb /kg decreased the reproductive output of 

### 2

two collembolans, Sinella coeca and F. candida . Pb concentration of 50, 100, and 500 mg Pb /kg slightly 

### 3
but significantly depressed S. coeca adult survival, while F. candida survival was statistically unaffected 

### 4
by Pb exposure. 

### 5
In addition to species variability, physical and chemical factors affecting Pb bioavailability were 

### 6

also demonstrated to significantly influence the toxicity of Pb to terrestrial species. As noted previously in 

### 7
Section 7.2.2, laboratory

- amended artificial soils provide a poor model for predicting the toxicity of Pb



### 8
contaminated field soils, because aging and leaching processes, along with variations in physiochemical 

### 9
properties (pH, CEC , OM ), influence metal bioavailability. Consequently, toxicity values derived from 

### 10

exposure

- response experimentation with laboratory

- spiked soils probably overestimate true 

### 11

envi ronmental risk, with the possible exception of highly acidic sandy soils. Because toxicity is 

### 12
influenced by bioavailability of soil biogeological and chemical characteristics, extrapolation of toxic 

### 13

concentrations between different field

- collected soils wi ll be difficult. Models that account for those 

### 14
modifiers of bioavailability, such as the terrestrial biotic ligand model proposed by Smolders et al. ( 2009 )

, 

### 15
have proven difficult to develop due to active physiologica l properties of soil organisms affecting either 

### 16
uptake (such as root phytochelatins) or sequestration of Pb (such as granule formation in root tissues and 

### 17
earthworms, or substitution of Pb for calcium in bones). 

### 18



## 7.2.6. Community and Ecosystem Effects

According to the 2006 Pb AQCD, natural terrestrial ecosystems near significant Pb point sources 

### 19
(such as smelters and mines) exhibited a number of ecosystem

- level effects, including decreased species 

### 20
diversity, changes in floral and faunal community composition, and d ecreasing vigor of terrestrial 

### 21
vegetation. These findings are summarized in Table AX7

- 2.5.2 of the Annex to the 2006 Pb AQCD ( U.S. 

### 22

EPA, 2006 ) . More recent literature explored the interconnected effects of Pb contamination on soil 

### 23
bacterial and fungal commun ity structure, earthworms, and plant growth, in addition to impacts on soil 

### 24
microbial community function. 

### 25
Inoculation of maize plants with Glomus intraradices arbuscular mycorrhizal fungi isolates 

### 26
decreased Pb uptake from soil, resulting in lower shoot Pb concentration and increased plant growth and 

### 27

biomass ( Sudova & Vosatka, 2007 ) . Similarly, Wong et al. ( 2007 ) showed t hat the presence of arbuscular 

### 28
mycorrhizal fungi improved vetiver grass ( Vetiveria zizanioides ) growth, and while Pb uptake was 

### 29
stimulated at low soil concentration (10 mg Pb /kg), it was depressed at higher concentration (100 and 

### 30
1,000 mg Pb /kg). Bojarczuk and Kieliszewska

- Rokicka ( 2010 ) found that the abundance of 

### 31
ectomycorrhizal fungi was negatively correlated wit h the concentration of metals, including Pb, in the 

### 32
leaves of silver birch seedlings. Arbuscular mycorrhizal fungi may thus protect plants growing in Pb

- 

### 33May 2011 7

- 38 DRAFT

– DO NOT C ITE OR QUOTE contaminated soils. Microbes too may dampen Pb uptake and ameliorate its deleterious effects: biomass 

### 1

o f plants grown in metal

- contaminated soils (average Pb concentration 24,175 mg Pb /kg dry weight) 

### 2
increased with increasing soil microbial biomass and enzymatic activity ( Epelde et al., 2010 ) . However, 

### 3
above certain Pb concentration, toxic effects on both plants and microbial communities may prevent these 

### 4
ameliorating effects. R.Y. Yang et al. ( 2008 ) found that both the mycorrhizal colonization and the growth 

### 5

of Solidago canadensis were negatively affected by soil Pb contami nation. They suggested that, more 

### 6
generally, Pb

- mediated alterations in plant

- fungal dynamics may be the cause of ecological instability in 

### 7
terrestrial vegetative communities exposed to metals. 

### 8
The presence of both earthworms and arbuscular mycorrhizal fu ngi decreased the mobility of Pb in 

### 9
mining soils undergoing phytoremediation ( Ma et al., 2006 ) . Inoculation with both ea rthworms and fungi 

### 10
increased plant growth at sites contaminated with mine tailings compared to that observed at sites with 

### 11
75% less Pb contamination. Most likely, this was a result of the decrease in bioavailable (DTPA



### 12

extractable and ammonium acetate

- extr actable) Pb to 17% to 25% of levels in areas without the 

### 13
earthworm and arbuscular mycorrhizal fungi amendments. The presence of earthworms in metal



### 14

contaminated soils decreased the amount of water

- soluble Pb ( Sizmu r & Hodson, 2008 ) , but despite this 

### 15
decrease, ryegrass accumulated more Pb from earthworm

- worked soils than soils without worms present. 

### 16
Sizmur and Hodson speculated that increased root dry biomass may explain the increased uptake of Pb in 

### 17
the presence of earthworms. By contrast, Coeurdassier et al. ( 2007 ) found that Pb was higher in 

### 18

earthworm tissue when snails were present, but that snails did not have a higher Pb content when 

### 19
earthworms were present. 

### 20
Microbial communities of industrial soils containing Pb concentrations of 61, 456, 849, 1,086, and 

### 21
1,267 mg Pb /kg dry weight were also improved via revegetation with n ative plants, as indicated by 

### 22
increased abundances of fungi, actinomycetes, gram

- negative bacteria, and protozoa, as well as by 

### 23
enhanced fatty acid concentration ( C. B. Zhang et al., 2006 ) . Increased plant diversity ameliorated the 

### 24
effects of soil Pb contamination (300 and 600 mg Pb /kg) on the soil microbial community ( R. Y. Yang et 

### 25

al., 2007 )

. 

### 26
The effect of Pb on microbial community function has been quantified previously using functional 

### 27
endpoints such as respiration rates, fatty acid production, and soil acid phosphatase and urease activities, 

### 28

whi ch may provide a better estimate of ecological impacts than microbial diversity or abundance 

### 29
measurements. When the microbial properties of metal

- contaminated urban soils were compared to those 

### 30
of rural soils, significant differences were detected in basal community respiration rates and microbial 

### 31

abundance ( Y. Yang et al., 2006 ) . The urban soils studied contained multiple metal contaminants, but 

### 32

microbial biomass was t he only measured endpoint to be significantly and negatively correlated to Pb 

### 33
concentration. This suggests that anthropogenic Pb contamination may adversely affect soil microbial 

### 34
communities, and alter their ecological function. Most studies of metal

- induc ed changes in microbial 

### 35
communities have been conducted using mixtures of metals. Akerblom et al. ( 2007 ) tested the effects of 

### 36May 2011 7

- 39 DRAFT

– DO NOT C ITE OR QUOTE six metals (Cr, Zn, Mo, Ni, Cd, and Pb) individually. All tested metals had a similar effect on the species 

### 1
composition of the microbial community. Exposure to a high Pb concentration (52 mg Pb /kg) negatively 

### 2
affected respiration rate s. Total phospholipid fatty acid content was determined to negatively correlate 

### 3
with increasing Pb exposure, indicating alteration of the microbial community. ( S. Khan et al., 2010 )

, 

### 4
found that following a 2

- week exposure to three levels of Pb (150, 300, and 500 mg Pb /kg), both acid 

### 5
phosphatase and urease levels (measures of soil microbial activity) decreased significantly, although they 

### 6
had recovered by the ninth week. In addition, the number of culturable bacteria was also decreased, but 

### 7
only at the highest exposure concentration tested. 

### 8

Soil bacteria co mmunity structure and basal respiration rates were examined in natural soils with 

### 9
pH values ranging from 3.7 to 6.8 ( Lazzaro et al., 2006 ) . Six soil types of differing pH were treated with 

### 10
Pb nitrate concentrations of 0.5, 2, 8, and 32 mM. Basal respiration was adversely affected in two soil 

### 11
types tested at the highest Pb tr eatment (32 mM), and in a third at the two highest Pb treatments (8 and 32 

### 12
mM). Terminal Restriction Fragment Length Polymorphism analysis indicated that bacterial community 

### 13
structure was only slightly altered by Pb treatments. While pH was correlated with the amount of water



### 14
soluble Pb, these increases were apparently not significant enough to affect bacterial communities, 

### 15
because there were no consistent relationships between soil pH and respiration rate or microbial 

### 16
community structure at equivalent soil Pb concentration. 

### 17
Pb exposure negatively affected the prey capture ability of certain fungal species. The densities of 

### 18
traps constructed by nematophagous fungi decreased in soils treated with 0.15 mM Pb chloride ( Mo et al., 

### 19

2008 ) . Nematophagous fungi are important predators of soil

- dwelling nematodes, collecting their prey 

### 20
with sticky nets, branches, and rings. This suppression caused a subsequent reduction in fungal nematode 

### 21
capturing capa city, and could result in increased nematode abundance. 

### 22
High concentration of soil metals were linked to a significant reduction in soil microorganism 

### 23
abundance and diversity. Soil columns spiked with Cu, Zn, and Pb acetate (total Pb concentration of 278 

### 24
to 838 mg Pb /kg, depending on depth) exhibited a 10

- to 100

- fold decrease in microbial abundance, with 

### 25
specific microbe classes (e.g., actinomycetes) seemingly more affected than others ( Vaisvalavicius et al., 

### 26

2006 ) . Concurrently, decreases in soil enzymatic activity were also observed, with saccharase activity 

### 27
decreased by 57

– 77%, dehydrogenase activity by 95

– 98%, and urease activity 65

– 97%. Although this 

### 28
suggests that Pb contamination may alter the nu trient cycling capacity of affected soil communities, it is 

### 29
difficult to separate the impact of Pb from the contributions of copper and zinc that were added with the 

### 30

Pb. 

### 31

The microbial communities of soils collected from a Pb

- Zn mine and a Pb

- Zn smelter wer e 

### 32
significantly affected by Pb and other metals (e.g., Cd) ( Q. Hu et al., 2007 ) . At a mine site, Pb 

### 33
concentration of 57 to 204 mg Pb /kg and Cd concentration of 2.4 to 227 mg Cd /kg decreased the number 

### 34

of bacteria

- forming colonies extracted from soils. Principal component analysis of microbial community 

### 35

structure demonstrated that different communities were associated with different metal soil concentration. 

### 36May 2011 7

- 40 DRAFT

– DO NOT C ITE OR QUOTE Similarly, soil microbial communities exposed to metal contamination from a smelter site (soil Pb 

### 1
concentration ranging from 30 to 25,583 mg Pb /kg dry weight) showed decreased bacterial functional 

### 2
diversity (although fungal functional diversity increased) and no effects o n soil respiration rates were 

### 3

observed ( Stefanowicz et al., 2008 ) . This led the authors to conclude that bacterial diversity is a more 

### 4
sensitive endpoint and a better indicator of metal exposure than fungal diversity or microorganism 

### 5
activity. In a similar study, Kools et al. ( 2009 ) showed that soil ecosystem variables measured after a 6

- 

### 6

month ex posure to metal

- contaminated soil indicated that Pb concentration (536 or 745 mg Pb /kg) was an 

### 7
important driver of soil microbial species biomass and diversity. 

### 8

Pb

- resistant bacterial and fungal communities were extracted regularly from soil samples at a 

### 9

s hooting range site in southern Finland ( Hui et al., 2009 ) . While bioavailable Pb concentration averaged 

### 10
100 to 200 mg Pb /kg as determined by water extraction, the total Pb concentrations measured on site were 

### 11

30,000 to 40,000 mg Pb /kg. To determine Pb tolerance, bacterial colonie s extracted and cultured from 

### 12
shooting range and control soils were grown on media containing either 0.4 or 1.8 mM Pb. While bacteria 

### 13
isolated from control soil did not proliferate on high

- Pb media, shooting

- range soil microbe isolates grew 

### 14
on high

- Pb medi a and were deemed Pb tolerant. The authors noted that bacterial species common in 

### 15
control samples were not detected among the Pb

- tolerant species isolated from shooting

- range soils. Thus, 

### 16
it was concluded that even long

- term exposure to minimally bioavaila ble Pb can alter the structure of soil 

### 17
decomposer communities, which could in turn decrease decomposition rates. 

### 18
Microbial communities associated with habitats other than soils are also affected by exposure to 

### 19
atmospherically deposited Pb. Alder ( Alnus nepalensis ) leaf microorganism populations were greater in 

### 20

number at non

- affected sites than at sites adjacent to a major Indian highway with increased Pb pollution 

### 21

( Joshi, 2008 ) . The density, species richness, and biomass of testate amoebae communities grown on 

### 22
Sphagnum fallax mosses were significantly decreased fo llowing moss incubation in Pb solutions of either 

### 23
625 or 2,500 μg Pb /L ( Nguyen

- Viet et al., 2008 ) . More importantly, species richness and density were 

### 24
negatively correlated with Pb concentration accumulated within the moss tissue. The structure of 

### 25

microbial communities associ ated with lichen surfaces was affected by lichen trace

- element 

### 26
accumulation, including Pb content. Lichens collected from industrial areas had elevated Pb concentration 

### 27
(10 to 20 mg Pb /kg versus 5 to 7 mg Pb /kg in urban and rural areas, respectively) and h oused bacterial 

### 28
communities characterized by increased cyanobacteria biomass ( Meyer et al., 2010 )

. 

### 29
Following a 28

- day exposure to field

- collected soils contaminated with metals (including Pb at 426 

### 30

mg Pb /kg), both population growth and individual g rowth of the earthworm L. rubellus were diminished 

### 31

( Klok et al., 2006 ) . The authors proposed that, although these reductions were unlikely to result in 

### 32
extirpation, avian predators such as the godwit ( Limosa limosa ) that feed heavily on earthworms may be 

### 33
affected adversely by a reduction of available earthworm biomass. 

### 34
During the past 5 years, there has been increasing interest in the effects of Pb and other metals on 

### 35
the functional aspects of soil microbial communities. Most studies show that Pb decreases diversity and 

### 36May 2011 7

- 41 DRAFT

– DO NOT C ITE OR QUOTE function of soil microorganisms

. However, in an example of ecological mutualism, plant

- associated 

### 1
arbuscular mycorrhizal fungi protect the host plant from Pb uptake and fungal viability seems to be 

### 2
protected by the plants. Similarly, soil microbial communities (bacterial species as well a s fungi) in Pb



### 3
contaminated soils are improved by revegetation

. A few studies have reported on effects of Pb to 

### 4
populations of soil invertebrates. They demonstrated that Pb can decrease earthworm population density, 

### 5
although not to levels that would result in local extinction

. There have been no recently reported studies 

### 6
on the potential effects of Pb on terrestrial vertebrate populations or communities, or possible indirect 

### 7
effects through reduction of prey items such as earthworms

. However, it is well kno wn from historical 

### 8
data that Pb can have a widespread and dramatic effect on populations of waterfowl exposed to spent shot 

### 9

( Beye r et al., 1996 ) and may be negatively affecting loons in the northeastern U. S. through ingestion of 

### 10
Pb fishing sinkers ( Scheuhammer & Norris, 1996 )

. Studies at shooting ranges and downwind of smelters 

### 11
previously reported in the 2006 Pb AQCD demonstrate effects of dispe rsed Pb on terrestrial soil and plant 

### 12
communities with resulting decreases in secondary consumer vertebrate species. 

### 13



## 7.2.7. Critical Loads in Terrestrial Systems

The critical load is the environmental concentration predicted or estimated from available literatur e 

### 14
data, above which adverse effects to organisms are likely to occur. It is based on the relative toxicity of 

### 15
the compound to species or ecosystem processes of concern, and an estimate of residence time in the soil 

### 16
environment. The concept of critical load is discussed in more detail in Section 7.1. 3 of this chapter and 

### 17

in Section 7.3 of the 2006 Pb AQCD ( U.S. EPA, 2006 )

. 

### 18

Hall et al. ( 2006 ) used the critical load approach to conduct a national risk assessment of 

### 19
atmospheric Pb deposition for the U. K. While specific regions were determined to have low critical load 

### 20
values for Pb (central England, the Pennines, and southern Wales), the authors noted that this approach 

### 21
can be significantly biased, as available ecotoxicological data used in the modeling were from studies that 

### 22
were not conducted in soils representative of all U.K. soils. De Vries et al. ( 2009 ) similarly observed that 

### 23
the uncertainty inherent in a critical load approach to Pb risk assessment is inf luenced by the critical 

### 24
concentration of dissolved metal and the absorption coefficients of exposed soils. However, this approach 

### 25
did indicate that for forest soils in the Netherlands, 29% of the areas would be expected to exceed the 

### 26

critical load, based o n currently available toxicity data and Pb pollution data ( de Vries & Groenenberg, 

### 27

2009 ) . Similarly, although Pb soil concentration in the Bologna Province of Italy were far below 

### 28

concentra tions harmful to soil organisms, current atmospheric Pb deposition rates suggest that critical 

### 29
load exceedances are likely in the future, unless annual Pb emissions are decreased ( Morselli et al., 2 006 )

. 

### 30
In some cases, risk assessment for Pb predicts risks to terrestrial animals at environmental 

### 31
concentrations that fall below natural background levels, and uncertainties associated with standard 

### 32
toxicity testing can become magnified through the ris k assessment process, degrading the reliability of 

### 33May 2011 7

- 42 DRAFT

– DO NOT C ITE OR QUOTE estimates of hazardous environmental concentrations. Smolders et al. ( 2009 ) concluded from their 

### 1
comparison of field soils, spiked soils, and artificially aged spiked soils that corrections for aging and 

### 2
interacting soil properties in spiked soils will make predicted

- no

- effect concentrations rise above 

### 3
background. Buekers et al. ( 2009 ) proposed the use of a Tissue Residue Approach as a risk estimation 

### 4

method for terrestrial vertebrates that does n ot predict risks at background levels, and has smaller 

### 5
uncertainty. This approach eliminates the need for quantitative estimation of food intake or Pb species 

### 6
bioavailability. Blood Pb no

- adverse

- effect concentration (NAEC) and lowest adverse effects 

### 7

conce ntration (LAEC) derived from 25 literature studies examining the effects of Pb exposure on growth, 

### 8
reproduction, and hematological endpoints were used to construct a series of species sensitivity 

### 9

distributions for mammals and birds. For mammals, the hazard ous concentration for 5% of species (HC5) 

### 10
values obtained from the species sensitivity distributions ranged from 11 to 18 μg Pb /dL blood ; HC5 

### 11
values for birds ranged from 65 to 71 μg/dL. The authors proposed the use of 18 and 71 μg/dL as critical 

### 12

threshold values for mammals and birds, respectively, which are below the lowest NAEC for both data 

### 13
sets used, and are above typical background Pb values. 

### 14
Short of conducting expensive in vivo toxicity studies, it is difficult to determine environmental Pb 

### 15

toxicit y given the variation of physiochemical and soil properties that alter bioavailability and toxicity. 

### 16

Furman et al. ( 2006 ) proposed the use of a physiologically based ext raction test to predict risks posed to 

### 17
waterfowl from environmental Pb contamination. The extraction process was modeled after gastric and 

### 18
intestinal conditions of waterfowl, and was used to gauge the bioavailability of Pb from freshly amended 

### 19
and aged con taminated soils. The concentration of Pb extracted through the use of the physiologically 

### 20
based extraction test was demonstrated to be significantly correlated to Pb tissue concentration in 

### 21
waterfowl exposed via in vivo studies of the same soils. 

### 22



## 7.2.8. Soil Scr eening Levels

Developed by EPA, ecological soil screening levels (Eco

- SSLs) are maximum contaminant 

### 23
concentrations in soils that are predicted to result in little or no quantifiable effect on terrestrial receptors. 

### 24
These conservative values were developed so that contaminants that could potentially present an 

### 25
unacceptable hazard to terrestrial ecological receptors are reviewed during the risk evaluation process 

### 26
while removing from consideration those that are highly unlikely to cause significant effects. Th e studies 

### 27

considered for the Eco

- SSLs for Pb and detailed consideration of the criteria for developing the Eco

- SSLs 

### 28
are provided in the 2006 Pb AQCD ( U.S. EPA, 2006 )

. Preference is given to studies using the most 

### 29

bioavailable form of Pb, to derive conservat ive values. Soil concentration protective of avian and 

### 30
mammalian diets are calculated by first converting dietary concentration to dose (mg/kg body weight per 

### 31
day) for the critical study, then using food (and soil) ingestion rates and conservatively derive d uptake 

### 32
factors to calculate soil concentration that would result in unacceptable dietary doses. This frequently 

### 33May 2011 7

- 43 DRAFT

– DO NOT C ITE OR QUOTE results in Eco

- SSL values below the average background soil concentration, as is the case with Pb for 

### 1

birds. The Pb Eco

- SSL was completed in M arch 2005 and has not been updated since. Values for 

### 2
terrestrial birds, mammals, plants, and soil invertebrates are 11, 56, 120, and 1

, 700 mg Pb/kg soil (dry 

### 3
weight), respectively. 

### 4



## 7.2.9. Characterization of Sensitivity and Vulnerability

Research has long demonst rated that Pb affects survival, reproduction, growth, metabolism, and 

### 5
development in a wide range of species. The varying severity of these effects depends in part upon 

### 6
species differences in metabolism, sequestration, and elimination rates. Dietary factor s also influence 

### 7
species sensitivity to Pb. Because of effects of soil aging and other bioavailability factors discussed above 

### 8
(Section 7.2. 2 ), in combination with differing species assemblages and biological accessibility within 

### 9
prey items, ecosystems may also differ in their sensitivity and vulnerability to Pb. The 2006 Pb AQCD 

### 10
reviewed many of these factors which are updated herein by reference to recent literature. 

### 11

7.2.9.1. Species Sensitivity

There is wide variation in sensitivity of terrestrial species to Pb exposure, even among closely 

### 12
related organisms. Langdon et al. ( 2005 ) showed a two

- fold difference in LC 50 values among three 

### 13
common earthworm species, with the standard laboratory species, E. andrei , being the least sensitive. 

### 14
Similarly, 28

- day EC 50 values derived for F. candida collembola (springtails) were between 2,060 and 

### 15
3,210 mg/kg in Pb

- spiked soil s ( Lock et al., 2006 ) , while the springtail spe cies S. curviseta exhibited no 

### 16
response to a 28

- day exposure to 3,200 mg/kg Pb

- spiked soil ( Xu et al., 2009 ) . Mammalian NOEC values 

### 17
expressed as blood P b levels were shown to vary by a factor of 8, while avian blood NOECs varied by a 

### 18

factor of 50 ( Buekers et al., 2009 ) . Age at exposure, in particular, may affect sensitivity to Pb. For 

### 19

instance, earlier instar C. elegans were more likely than older individuals to exhibit neurobehavioral 

### 20
toxicity following Pb exposure ( Xing, Guo, et al., 2009 ) , and also demonstrated more pronounced neural 

### 21
degeneration than older larvae and adults ( Xing, Rui, et al., 2009 )

. 

### 22

7.2.9.2. Nutritional Factors

Dietary factors can exert significant influence on the uptake and toxicity of Pb in many species of 

### 23
birds and mammals. The 2006 Pb AQCD describes how Ca, Zn, Fe, vitamin E, Cu, thiamin, P, Mg, fat, 

### 24

pro tein, minerals, and ascorbic acid dietary deficiencies increase Pb absorption and its toxicity. For 

### 25
example, vitamin E content was demonstrated to protect against Pb

- induced lipid peroxidation in mallard 

### 26
ducks. Generally, Pb exposure is more likely to prod uce adverse behavioral effects in conjunction with a 

### 27

nutrient

- deficient diet. As previously reported in the 2006 Pb AQCD, Ca deficiencies may increase the 

### 28
susceptibility of different terrestrial species to Pb, including plant ( Antosiewicz, 2005 ) , avian ( Dauwe et 

### 29May 2011 7

- 44 DRAFT

– DO NOT C ITE OR QUOTE al., 2006 ; Snoeijs et al., 2005 ) and invertebrate species. Antosiewicz determined that, for plants, Ca 

### 1
deficiency decreased the sequestration capacity of several species (tomato, mustard, rye, and maize), and 

### 2
that this likely resulted in an increased proportion of Pb at sites of toxic action. Because Pb ions can 

### 3
interact with plant Ca channel pores, in the presence of low Ca and high Pb concentration, a hi gher 

### 4
proportion of Pb can interact with these channels and be taken up by plants. A similar phenomenon has 

### 5
been observed in invertebrates, where the metabolic pathway of metals mimics the metabolic pathway of 

### 6
Ca (Simkiss et al. ( 1982 ) , as cited in Jordaens et al. ( 2006 ) ). Hence, in environments with 

### 7
disproportionately high Pb versus Ca concentration, accumulation of Pb may be accelerated, as in plants. 

### 8
Ca deficiency in birds was demonstrated to stimulate the production of Ca

- binding proteins in the 

### 9
intestinal tract, which extract more Ca from available diet; however, this response also e nhances the 

### 10
uptake and accumulation of Pb from diet and drinking water (Fullmer et al. ( 1997 ) , as cited in Dauwe et 

### 11

al. ( 2006 ) ). 

### 12

7.2.9.3. Soil Aging and Site

- Specific Bioavailability

Total soil Pb concentration is a poor predictor of hazards to avian or mammalian wildlife, because 

### 13

site

- specific biogeochemical and physical properties (e.g., pH, OM , metal oxide concentration) can affect 

### 14
the sequestration capacity of soils. Additionally, soil aging processes have been de monstrated to decrease 

### 15
the bioavailable Pb fraction; as such, laboratory toxicity data derived from spiked soils often overestimate 

### 16

the environmental risk of Pb. Smolders et al. ( 2009 ) compared the toxicity of freshl y Pb

- spiked soils to 

### 17
experimentally aged spiked soils and field

- collected Pb

- contaminated soils. Experimental leaching and 

### 18
aging was demonstrated to increase invertebrate Pb EC 50 values by factors of 0.4 to greater than 8; in 

### 19
approximately half the cases, the proportionality of toxicity to Pb content disappeared following 

### 20
experimental aging of freshly spiked soils through leaching. The leaching

- aging factor for Pb was 

### 21
determined to be 4.2, and represented the ratio of ED 10 values derived in aged soils to fr eshly spiked soils 

### 22
(factors greater than one indicate decreased toxicity in aged field soils relative to laboratory spiked soils). 

### 23
Consequently, the sensitivity of terrestrial vertebrates to environmental Pb exposures will be heavily 

### 24
dependent on the relat ive rate of aging and site

- specific bioavailability. 

### 25

7.2.9.4. Ecosystem Vulnerability

Relative vulnerability of different terrestrial ecosystems to effects of Pb can be inferred from the 

### 26
information discussed above on species sensitivity and how soil geochemistry influences the 

### 27
bioavailability and toxicity of Pb. Soil ecosystems with low pH, particularly those with sandy soils, are 

### 28
likely to be the most sensitive to the effects of Pb. Examples of such systems are forest soils, including 

### 29

oak, beach, and conifer fore sts. The Pine Barrens in southern New Jersey (also known as the Pinelands) is 

### 30
an example of a highly vulnerable ecosystem: it is a dense coniferous (pine) forest with acidic, sandy, 

### 31May 2011 7

- 45 DRAFT

– DO NOT C ITE OR QUOTE nutrient poor soil. As agricultural areas are taken out of production and revert to old fields and eventually 

### 1
forests, their vulnerability to Pb is likely to increase as a result of decreasing OM and acidification of soils 

### 2
(from discontinuation of fertilizing and liming). On the other hand, increasing density of native or 

### 3

invasi ve plants with associated arbuscular mycorrhizal fungi will likely act to ameliorate some of the 

### 4
effects of Pb (see previous discussion of studies by Sudova and Vostka ( 2007 ) and Wong et al. ( 2007 ) . It 

### 5
is, however, difficult to categorically state that certain plant or soil invertebrate communities are more 

### 6
vulnerable to Pb than others, as the available toxicity data have not yet been standardized fo r differences 

### 7
in bioavailability (because of use of different Pb salts, different soil properties, and different lengths of 

### 8
aging of soil prior to testing), nutritional state, or organism age, or other interacting factors. Data from 

### 9
field studies are compl icated by the co

- occurrence of other metals and alterations of pH, such as 

### 10

acidification from SO 2 in smelter emissions, that are almost universal at sites of high Pb exposure, 

### 11
especially at mine or smelter sites. However, because plants primarily sequester Pb in the roots, uptake by 

### 12
soil invertebrates is the most likely pathway for Pb exposure of higher trophic level organisms. 

### 13
Invertivores are likely at higher risk than herbivores. In fact, estimations of Pb risk at a former Pb smelter 

### 14

in northern France i ndicated that area Pb concentration presented the greatest threat to insectivorous bird 

### 15
and mammal species, but only minimal risk to soil invertebrate and herbivorous mammals ( Fritsch et al., 

### 16

2010 ) . By extension, birds and mammals in ecosystems with a richer biodiversity of soil invertebrates 

### 17
may be more vulnerable to Pb than those in ecosystems with fewer invertebrates (e.g

. , arid locations). 

### 18
Regardless, the primary determinant of terrestrial ecosystem vulnerability is soil geochemistry, notably 

### 19
pH, CEC, and amount of OM

. 

### 20



## 7.2.10. Ecosystem Services

There are no publications at this time that specifically focus on the ecosystem services affected by 

### 21
Pb in terrestrial systems

. The evidence reviewed in this ISA illustrates that Pb can cause ecological effect s 

### 22
in each of the four main categories of ecosystem services (Section 7.1.2) as defined by Hassan et al. 

### 23

( 2005 )

. These effects are sorted into ecosystem services categories and summarized here: 

### 24

 Supporting: altered nutrient cycling, decreased biodiversity, decline of productivity, food 

### 25
production for higher trophic levels 

### 26

 Provisioning: plant yields 

### 27

 Regulating: decline in soil quality, detritus production 

### 28

 Cultural: ecotourism and cultural heritage values related to ecosystem integrity and 

### 29
biodiversity, impacts to terrestrial vertebrates. 

### 30May 2011 7

- 46 DRAFT

– DO NOT C ITE OR QUOTE A few studies since the 2006 Pb AQCD consider the impact of metals in genera l on ecosystem 

### 1
services. In a review of the effects of metals on insect behavior, ecosystem services provided by insects 

### 2
such as detritus reduction and food production for higher trophic levels were evaluated by considering 

### 3
changes in ingestion behavior an d taxis ( Mogren & Trumble, 2010 ) . Pb was shown in a limited number of 

### 4
studies to affect ingestion by insects

. Crickets (Chorthippus spp) in heavily contaminated sites reduced 

### 5
their consumpt ion of leaves in the presence of increasing cadmium and Pb concentrations ( Migula & 

### 6

Binkowska, 1993 )

. Decreased feeding activity in larval and adult Col orado potato beetle ( Leptinotarsa 

### 7

decemlineata ) were observed as a result of dietary exposures of Pb and copper ( Kwartirnikov et al., 

### 8

1999 ) , while no effects were found in ingestion studies of Pb with willow leaf beetle, Lochmaea caprae 

### 9

( Rokytova et al., 2004 ) mottled water hyacinth weevil, Neochetina eichhorniae ( Kay & Haller, 1986 ) and 

### 10
hairy springtail, Orchesella ci ncta ( Van Capelleveen et al., 1986 )

. 

### 11
Soil health for agricultural production and other soil

- associated ecosystem services is dependent on 

### 12

the maintenanc e of four major functions: carbon transformations, nutrient cycles, soil structure 

### 13
maintenance, and the regulation of diseases and pests and these parameters may be altered by metal 

### 14
deposition ( Kibblewhite et al., 2008 )

. Pb impacts to terrestrial systems reviewed in the previous sections 

### 15
provide evidence for impacts to supporting, provisioning, and regulating ecosystem services provided by 

### 16
soils. For example, earthworms were shown to impact soil metal mobility and availabi lity, which in turn 

### 17
resulted in changes to microbial populations (biodiversity), pH, dissolved organic carbon, and metal 

### 18
speciation ( Sizmur & Hodson, 2009 ) , all of which may directly affect soil fertility. 

### 19

There is limited evidence of Pb impacts to plant productivity. Productivity of gray birch ( Betula 

### 20
populifolia ) was impaired in soils with elevated As

, Cr , Pb, Zn and V ( Gallagher et al., 2008 )

. Tree growth 

### 21
measured in both individuals and at the assemblage level using satelli te imagery and field spectrometry 

### 22
was significantly decreased with increasing metal load in soil. 

### 23



## 7.2.11. Summary of Effects in Terrestrial Systems

This summary of the effects of Pb on terrestrial ecosystems covers information from the 

### 24
publication of the 2006 Pb A QCD to present

. Refer to Section 7.4: Causality determinations for Pb in 

### 25
Terrestrial and Aquatic Systems for a synthesis of all evidence dating back to the 1977 AQCD considered 

### 26
to determine causality. 

### 27

7.2.11.1. Biogeochemistry and Chemical Effects

The amount of Pb dissolved in pore water determines the impact of soil Pb on terrestrial 

### 28
ecosystems to a much greater extent than the total amount present. It has long been established that the 

### 29
amount of Pb dissolved in soil solution is controlled by at least six variable s: (1) solubility equilibria; (2) 

### 30
adsorption

- desorption relationship of total Pb with inorganic compounds; (3) adsorption

- desorption 

### 31May 2011 7

- 47 DRAFT

– DO NOT C ITE OR QUOTE reactions of dissolved Pb phases on soil OM ; (4) pH; (5) CEC; and (6) aging. Since 2006, further details 

### 1

have been contribu ted to the understanding of the role of pH, CEC, OM , and aging. Smolders et al. ( 2009 ) 

### 2
demonstrated that the two most important determinants of both solubility and toxicity in soils are pH and 

### 3
CEC. However, they had previously shown that aging, primarily in the form of initial leaching following 

### 4
deposition, decreases soluble metal fraction by approximately one order of magnitude ( Smolders et al., 

### 5

2007 ) . Since 2006, OM has been confirmed as an im portant influence on Pb sequestration, leading to 

### 6
longer

- term retention in soils with higher OM content, and also creating the potential for later release of 

### 7
deposited Pb. Aging, both under natural conditions and simulated through leaching , was shown to 

### 8

s ubstantially decrease bioavailability to plants, microbes, and vertebrates. 

### 9

7.2.11.2. Bioavailability and Uptake

Plants

Studies with herbaceous species growing at various distances from smelters added to the existing 

### 10
strong evidence that atmospherically transported Pb is taken up by plants. These studies did not establish 

### 11
the relative proportion that originated from atmospheric Pb deposited in the soil, as opposed to that taken 

### 12
up directly from the atmosphere through the leaves. Multiple new studies showed that in t rees, this 

### 13
proportion is likely to be very substantial. One study attempted to quantify it, and suggested that 50% of 

### 14
the Pb contained in Scots Pine in Sweden is taken up directly from the atmosphere. Studies with 

### 15
herbaceous plants found that in most speci es tested, soil Pb taken up by the roots is not translocated into 

### 16
the stem and leaves. Studies with trees found that soil Pb is generally translocated from the roots. 

### 17

Invertebrates

Since the 2006 Pb AQCD, various species of terrestrial snails have been fo und to accumulate Pb 

### 18

from both diet and soil. New studies with earthworms have found that both internal concentration of Pb 

### 19
and mortality increase with decreasing soil pH and CEC. In addition, tissue concentration differences 

### 20
have been found in species of earthworms that burrow in different soil layers. The rate of accumulation in 

### 21
each of these species may result from layer differences in interacting factors such as pH and CEC

. 

### 22
Because earthworms often sequester Pb in granules, some authors have suggested t hat earthworm Pb is 

### 23
not bioavailable to their predators. There is some evidence that earthworm activity increases Pb 

### 24
availability in soil, but it is inconsistent. In various arthropods collected at contaminated sites, recent 

### 25
studies found gradients in accu mulated Pb that corresponded to gradients in soil with increasing distance 

### 26
from point sources. 

### 27May 2011 7

- 48 DRAFT

– DO NOT C ITE OR QUOTE Vertebrates

There were few new studies of Pb bioavailability and uptake in birds since the 2006 Pb AQCD. A 

### 1
study of two species of sea ducks in Alaska found th at 3% of the birds had tissue levels of Pb that 

### 2
indicated exposure above background. Urban pigeons in Korea were found to accumulate 1.6 to 1.9 

### 3
mg/kg wet weight Pb in the lungs, while in Wisconsin 70% of American woodcock chicks and 43 % of 

### 4

young

- of

- year h ad elevated bone Pb (9.6 to 93 mg Pb /kg dry weight in chicks, 1.5 to 220 mg Pb /kg dry 

### 5
weight in young

- of

- year). None of the locations for these studies was in proximity to point sources, and 

### 6
none was able to identify the origin of the Pb. A study at the An aconda Smelter Superfund site found 

### 7
increasing Pb accumulation in gophers with increasing soil Pb around the location of capture

. A study of 

### 8

swine fed various Pb

- contaminated soils showed that the form of Pb determined accumulation. 

### 9

Food web

New studies were able to measure Pb in the components of various food chains that included soil, 

### 10
plants, invertebrates, arthropods and vertebrates. They confirmed that trophic transfer of Pb is pervasive, 

### 11
but no consistent evidence of trophic magnification was found. 

### 12

7.2.11.3. Biological Effects

Plants

Experimental studies have added to the existing evidence of photosynthesis impairment in plants 

### 13
exposed to Pb, and have found damage to photosystem II due to alteration of chlorophyll structure, as 

### 14
well as decreases in chlorophyl l content in diverse taxa, including lichens and mosses. A substantial 

### 15
amount of evidence of oxidative stress in response to Pb exposure has also been produced. Reactive 

### 16
oxygen species were found to increase in broad bean and tomato plants exposed to incre asing 

### 17
concentrations of soil Pb, and a concomitant increase in superoxide dismutase, glutathione, peroxidases, 

### 18
and lipid peroxidation, as well as decreases in catalase were observed in the same plants. Monocot, dicot, 

### 19
and bryophytic taxa grown in Pb

- contam inated soil or in experimentally spiked soil all responded to 

### 20
increasing exposure with increased antioxidant activity

. In addition, reduced growth was observed in 

### 21
some experiments, as well as genotoxicity, decreased germination, and pollen sterility. 

### 22

Inver tebrates

Recently published studies have shown neuronal damage in nematodes exposed to low 

### 23
concentrations of Pb (2.5 μM), accompanied by behavioral abnormalities. Reproductive adverse effects 

### 24
were found at lower exposure in younger nematodes, and effects on longevity and fecundity were shown 

### 25May 2011 7

- 49 DRAFT

– DO NOT C ITE OR QUOTE to persist for several generations

. Increased mortality was found in earthworms, but was strongly 

### 1
dependent on soil characteristics including pH, CEC, and aging. Snails exposed to Pb through either 

### 2
topical application or through consumption of Pb

- exposed plants had increased antioxidant activity, 

### 3
decreased food consumption, growth, and shell thickness. Effects on arthropods exposed through soil or 

### 4
diet varied with species and exposure conditions, and included diminished growth and fecundity, 

### 5
endocrine and reproductive anomalies, and body deformities. Increasing concentration of Pb in the 

### 6
exposure medium generally resulted in increased effects within each study, but the relationship between 

### 7

concentration and effects var ie d between studies, even when the same medium, e.g., soil, was used. 

### 8
Evidence suggested that aging and pH are important modifiers. 

### 9

Vertebrates

Effects on amphibians and reptiles included decreased white blood cell counts, decreased testis 

### 10
weight, and behavi oral anomalies. However large differences in effects were observed at the same 

### 11
concentration of Pb in soil, depending on whether the soil was freshly amended, or field

- collected from 

### 12
contaminated areas. As in most studies where the comparison was made, eff ects were smaller when field



### 13

collected soils were used. In some birds, maternal elevated blood Pb level was associated in recent studies 

### 14
with decreased hatching success, smaller clutch size, high corticosteroid level, and abnormal behavior. 

### 15
Some species sh ow little or no effect of elevated blood Pb level. Effects of dietary exposure were studied 

### 16
in several mammalian species, and cognitive, endocrine, immunological, and growth effects were 

### 17

observed. 

### 18

7.2.11.4. Exposure Response

Evidence reviewed in previous sections de monstrates clearly that increased exposure to Pb is 

### 19
generally associated with increases in observed effects in terrestrial ecosystems. It also demonstrates that 

### 20
many factors, including species and various soil physiochemical properties, interact strongly w ith 

### 21
concentration to modify those effects. In these ecosystems, where soil is generally the main component of 

### 22
the exposure route, Pb aging is a particularly important factor, and one that may be difficult to reproduce 

### 23
experimentally

. Without quantitative c haracterization of those interactions, characterizations of exposure



### 24
response relationships would likely not be transferable outside of experimental settings. Since the 2006 

### 25
Pb AQCD, a few studies of exposure

- response have been conducted with earthworms, a nd results have 

### 26

been inconsistent. 

### 27

7.2.11.5. Community and Ecosystem Effects

New evidence of effects of Pb at the community and ecosystem scale include several studies of the 

### 28
ameliorative effects of mycorrhizal fungi on plant growth, attributed to decreased uptake o f Pb by plants, 

### 29May 2011 7

- 50 DRAFT

– DO NOT C ITE OR QUOTE although both mycorrhizal fungus and plant were negatively affected. The presence of both earthworms 

### 1
and mycorrhizal fungi decreased solubility and mobility of Pb in soil in one study, but the presence of 

### 2
earthworms was associated with high er uptake of Pb by plants in another. The presence of snails 

### 3
increased uptake of Pb by earthworms, but not vice

- versa. Most recently published research on 

### 4
community and ecosystem scale effects of Pb has focused on soil microbial communities, which have 

### 5

bee n shown to be impacted in both composition and activity. Many recent studies have been conducted 

### 6
using mixtures of metals, but have tried to separate the effects of individual metals when possible. One 

### 7
study compared the effects of 6 metals individually ( Akerblom et al., 2007 ) , and found that their effects 

### 8
on community composition were similar. In studi es that included only Pb, or where effects of Pb could be 

### 9
separated, soil microbial activity was generally diminished, but in some cases recovered over time. 

### 10
Species and genotype composition were consistently altered, and those changes were long

- lasting or 

### 11

permanent

. 

### 12

7.2.11.6. Critical Loads, Sensitivity and Vulnerability

Exploratory studies of critical load approaches for risk assessment for Pb have been recently 

### 13
conducted in the U. K., the Netherlands, and Italy. Their authors suggested that the main limitations of 

### 14
critical loads approaches in those countries were gaps and uncertainty in both ecotoxicological and Pb 

### 15
deposition data. The most visible indication of the need for improvement was that critical load values 

### 16
were often below background values. Smolders ( 2009 ) suggested that correcting for aging and other 

### 17
interacting factors would likely raise predicted

- no

- effect concentrations, and others proposed basing risk 

### 18
management on tissue residue in organisms, or creating extra ction methods that more closely mimic 

### 19
uptake and accumulation. 

### 20
Recent studies have addressed differences in sensitivity explicitly, and clearly demonstrated high 

### 21
variability between related species, as well as within larger taxonomic groupings. Mammalian N OEC 

### 22
values expressed as blood Pb levels were shown to vary by a factor of 8, while avian blood NOECs varied 

### 23
by a factor of 50 ( Buekers et al., 2009 ) . Protective effects of dietary Ca have been found in plants, birds, 

### 24

and invertebrates. 

### 25



# 7.3. Aquatic Ecosystem Effects

7.3.1. Introduction to Aquatic Ecosystem Effects

This section of the Pb ISA reviews the new liter ature since the 2006 Pb AQCD ( U.S. EPA, 2006 ) 

### 26
on the effects of Pb on aquatic systems

. The focus is on the effects of Pb, with particular focus on ambient 

### 27
level, to aquatic organisms including algae, aquatic plants, invertebrates, vertebrates, and other bio ta with 

### 28May 2011 7

- 51 DRAFT

– DO NOT C ITE OR QUOTE an aquatic life stage (e.g., amphibians)

. The current mean and range of Pb concentrations in surface 

### 1
waters (mean 0.66 μg Pb /L, range 0.04 to 30 μg Pb /L), sediments (mean 120 μg Pb /g dry weight, range 

### 2
0.5 to 12,000 μg Pb /g dry weight) and fish tiss ues (mean 1.03 μg Pb /g dry weight, range 0.08 to 23 μg 

### 3

Pb /g dry weight [ whole body ] ) in the U.S. based on a synthesis of National Water Quality Assessment 

### 4
(NAWQA) data was reported in the previous 2006 Pb AQCD ( U.S. EPA, 2006 ) . The 2006 Pb AQCD 

### 5
provided an overview of regulatory considerations for water and sediments in addition to consideration of 

### 6
biological effects and major environmental factors that modify the response of aquatic organisms to Pb 

### 7

exposure

. Regulatory guidelines for Pb in water and sedimen ts have not changed since the 2006 Pb 

### 8
AQCD and are summarized below with consideration of limited new information on these criteria since 

### 9

the last review

. This section is followed by new information on biogeochemistry, bioavailability and 

### 10
biological effect s of Pb since the 2006 Pb AQCD. 

### 11

T he most recent ambient water quality criteria (AWQC) for Pb were released in 1985 ( U.S. EPA, 

### 12

1985 ) by the EPA Office of Water which employed empir ical regressions between observed toxicity and 

### 13
water hardness to develop hardness

- dependent equations for acute and chronic criterion . These criteria are 

### 14
published pursuant to Section 304(a) of the Clean Water Act and provide guidance to states and tribes to 

### 15
use in adopting water quality standards for the protection of aquatic life and human health in surface 

### 16
water. The ambient water quality criteria for Pb are currently expressed as a criteria maximum 

### 17
concentration (CMC) for acute toxicity and criterion co ntinuous concentration (CCC) for chronic toxicity 

### 18

( U.S. EPA, 2010 ) . In freshwater, the CMC is 65 μ g Pb/L and the CCC is 2.5 μ g Pb/L at a hardness of 100 

### 19
mg/L. In saltwater, these values are 210 μ g Pb/L CMC and 8.1 μ g Pb/L CCC, respectively. 

### 20

A draft document intended to update the AWQC for Pb ( Great L akes Environmental Center, 2008 ) 

### 21
was recently prepared for the EPA. This draft included significantly revised values for both acute and 

### 22
chronic endpoints that were also based on a hardness equation; the revised chronic AWQC in particular is 

### 23
much higher than in previous AWQC due to a substantial reduction in the acute to chronic ratio (ACR)

. 

### 24
Recent studies have suggested that the ACRs used in the existing criteria documents were too high, 

### 25
possibly because of the age and size of fish used in those studies ( Mebane et al., 2008 )

. 

### 26
The 2006 Pb AQCD summarized two approaches for establishing sediment criteria for Pb base d on 

### 27
either bulk sediment or equilibrium partitioning (Section 7.2.1, Table 7

- 2 and Section AX7.2.1.4)

. The 

### 28
first approach is based on empirical correlations between metal concentrations in bulk sediment and 

### 29
associated biological effects to derive threshol d effect concentrations (TEC) and probable effects 

### 30
concentrations (PEC) ( MacDonald et al., 2000 )

. The TEC/PEC approach derives numeric guidelines to 

### 31
compare against bulk sediment concentrations of Pb. The other approach in the 2006 Pb AQCD was t he 

### 32
equilibrium partitioning procedure published by the EPA for developing sediment criteria for metals ( U.S. 

### 33

EPA, 2005 ) . The equilibrium partitioning approach considers bioavailability by relating sediment toxicity 

### 34

to pore water concentration of metals. The amount of simultaneously extracted metal (SEM) is compared 

### 35May 2011 7

- 52 DRAFT

– DO NOT C ITE OR QUOTE with the metals extracted via acid

- vol a tile sulfides (AVS) since metals that bind to AVS (such as Pb) 

### 1
should not be toxic in sediments where AVS occurs in greater quali ties than SEM

. 

### 2
Since the publication of the 2006 Pb AQCD both of these methods

, for estimating sediment criteria 

### 3

for metals

, have continued to be used and refined. The SEM approach was further refined in the 

### 4
development of the sediment BLM ( Di Toro et al., 2005 )

. The BLM is discussed further in Section 7.3.3. 

### 5
Comparison of empirical approaches with AVS

- SEM in metal contaminated field sediments shows that 

### 6
samples where either method predicted there should be no toxicity due to metals, no toxicity was 

### 7
observed in chronic amphipod exposures ( J. A. Besser et al., 2009 ; MacDonald et al., 2009 )

. However, 

### 8
when the relationship between invertebrate habitat (epibenthic and benthic) and environmental Pb 

### 9
bioaccumulation was investigated, De Jonge et al. ( 2 010 ) determined that different environmental 

### 10
fractions of Pb were responsible for invertebrate uptake and exposure

. Pb uptake by benthic invertebrate 

### 11
taxa was not significantly correlated to AVS Pb levels, but rather to total sediment concentrations 

### 12

( De Jonge et al., 2009 )

. Conversely , epibenthic invertebrate Pb body burdens were better correlated to 

### 13

AVS concentrations, rather than total Pb sediment concentrations ( De Jonge et al., 2010 )

. 

### 14
In the following sections, new information since the 2006 Pb AQCD on Pb in aquatic ecosystems 

### 15
will be presented. Throughout the sections, brief summaries of conclusions from the 2006 Pb AQCD are 

### 16
included where appropria te. Section 7.3 is organized to consider uptake of Pb and effects at the species 

### 17
level, followed by community and ecosystem level effects. Section 7.3.2 considers the biogeochemistry 

### 18
and chemical effects of Pb in aquatic systems. New research on the bioava ilability and uptake of Pb into 

### 19
aquatic organisms including plants, invertebrates and vertebrates is presented next (Section 7.3.3). Effects 

### 20
of Pb on the physiology of aquatic fauna and biota (Section 7.3.4) are followed with data on exposure and 

### 21

response of aquatic organisms (Section 7.3.5)

. Ecosystem

- scale responses are reviewed in Section 7.3.6 

### 22
followed by a brief consideration of critical loads in aquatic systems (Section 7.3.7), characterization of 

### 23
sensitivity and vulnerability of ecosystem components (Section 7.3.8) and the effects of Pb on ecosystem 

### 24
services associated with aquatic environments (Section 7. 3 .9). 

### 25



## 7.3.2. Biogeochemistry and Chemical Effects

Quantifying Pb speciation in aquatic environments is critical for determining the toxicity of the 

### 26
metal to aquatic organisms. As reviewed in the 2006 Pb AQCD and discussed in detail in Section 3.3. of 

### 27
this assessment (Fate and Transport), the speciation pr ocess is controlled by many environmental factors

. 

### 28
Although aerially deposited Pb largely consists of the labile Pb fraction, once the atmospherically

- derived 

### 29
Pb enters surface waters its fate and bioavailability are influenced by Ca concentration, pH, alk alinity, 

### 30
total suspended solids, and dissolved organic carbon (DOC), including humic acids. In sediments, Pb is 

### 31
further influenced by the presence of sulfides and iron and manganese oxides

. For instance, in neutral to 

### 32
acidic aquatic environments, Pb is typ ically present as PbSO 4 , PbCl 4 , Pb 2 + , cationic forms of Pb 

### 33May 2011 7

- 53 DRAFT

– DO NOT C ITE OR QUOTE hydroxide, and ordinary hydroxide [Pb(OH) 2 ], while in alkaline waters, common forms of Pb include Pb 

### 1
carbonates [Pb(CO 3 )] and hydroxides [Pb(OH) 2 ]

. In freshwater systems, Pb complexes with inorgani c 

### 2

OH - and CO 3 2− and forms weak complexes with Cl − ; conversely, Pb speciation in seawater is a function of 

### 3
chloride concentration and the primary species are PbCl 3 , PbCO 3 , PbCl 2 , and PbCl +

. In many, but not all 

### 4
aquatic organisms, Pb dissolved in water can b e the primary exposure route to gills or other biotic ligands. 

### 5
The toxicity associated with Pb in the water column or sediment pore waters is directly affected by the 

### 6
competitive binding of Pb to the anions listed above. 

### 7
Currently, national and state ambi ent water quality criteria for Pb attempt to adjust measured 

### 8
concentrations to better represent the bioavailable free ions, and express the criteria value as a function of 

### 9
the hardness (i.e., amount of calcium and magnesium ions) of the water in a specific aquatic system

. 

### 10

Models such as the BLM ( Paquin et al., 2002 ) include an aquatic speciation model (WHAM V; see 

### 11
below) combined with a model of competitive binding to gill surfaces, and provides a more 

### 12
comprehensive method for expressing Pb concentrations at specific locations in terms of the bioavailable 

### 13

metal While the BLM is not yet used in setting regulatory criteria for Pb in the U.S., its application in risk 

### 14
analysis has become widely recognized ( Fairbrother et al., 2007 ) . Sediment quality criteria have yet to be 

### 15
adopted by EPA, although an equilibrium partitioning pro cedure is now available to predict which 

### 16
sediments have metal concentrations that are not toxic to aquatic organisms ( U.S. EPA, 2005 )

. The 

### 17
approach is based on the ratio of the sum of simultaneously extracted metals and amount of AVS, adjusted 

### 18
for the fraction of organic carbon present in the sediments, and is reviewed in detail in the 2006 Pb AQCD 

### 19

( 2006 )

. It is important to note that this method cannot accurately predict which sediments are toxic or 

### 20
which metal is the primary risk driver. 

### 21

A more detailed understanding of the biogeochemistry of Pb in aquatic systems (both the water 

### 22
column and sediments) is critical to accurately predicting toxic effects of Pb to aquatic organisms

. It 

### 23
should be recognized, however, that in addition to exposure via s ediment and water, chronic exposures to 

### 24
Pb also include dietary uptake, even though the toxicokinetics of this exposure pathway are not yet well 

### 25
understood in aquatic organisms and the influence of the bioavailability factors described above is 

### 26

unknown

. Fu rthermore, changes in environmental factors that reduce the bioaccessible Pb fraction can 

### 27
result in either sequestration in sediments or subsequent release as mobile, bioaccessible forms

. This 

### 28
section provides updated information about the influence of che mical parameters that affect Pb 

### 29
bioaccessibility in the aquatic environment (in sediments and the water column). 

### 30
Several models are available for estimating the speciation of dissolved Pb. These models were 

### 31
tested by Balistrieri and Blank ( 2008 ) by comparing the speciation of dissolved Pb in aquatic systems 

### 32
affected by historical mining activities with that predicted by sev eral models, including Windermere 

### 33
humic aqueous model (WHAM VI), non

- ideal competitive absorption Donnan

- type model (NICA



### 34
Donnan), and Stockholm humic model (SHM)

. Accurate prediction of labile Pb concentrations was 

### 35
achieved only with SHM, although other m etal concentrations were better described by the WHAM 

### 36May 2011 7

- 54 DRAFT

– DO NOT C ITE OR QUOTE model

. Whereas both WHAM VI and NICA

- Donnan predicted that the bulk of Pb contamination would 

### 1
be complexed with iron, SHM predicted Pb speciation predominantly characterized by both iron and 

### 2
inorganic P b complexes

. Predicted dynamic Pb concentrations developed with the WHAM VI and NICA



### 3
Donnan methods overestimated Pb concentrations measured using diffusive gradients in thin

- films in 

### 4

Lake Greifen (Switzerland) , but underestimated concentrations in Furbach stream (both in the Coeur 

### 5

D ’ Alene River Basin in Idaho ), indicating that such models may not be able to accurately describe metal 

### 6
speciation under all environmental conditions ( Balistrieri & Blank, 2008 )

. 

### 7
Quantification of different sediment metal

- binding phases, including sulfide, organic C, Fe, and Mn 

### 8
phases, is important to fully un derstand the bioaccessible fraction of Pb and the toxicity to benthic 

### 9
organisms ( Simpson & Batley, 2007 )

. However, physical disturbance, pH change, and even the biota 

### 10
themselves also alter sediment binding or release of Pb

. Atkinson et al. ( 2007 ) studied the effe cts of pH on 

### 11
sequestration or release of Pb from sediments

. Although high and circumneutral water pH (8.1 and 7.2) 

### 12
did not affect the release of sequestered Pb from sediments, lowering the pH to 6 increased the 

### 13
concentration of Pb in overlying waters from less than 100 μg Pb /L to 200

- 300 μg Pb /L

. Physical 

### 14

sediment disturbance also increased the amount of sediment

- bound Pb released into the aqueous phase

. 

### 15

When Pb

- contaminated sediment was physically disturbed, the dissolved oxygen content of the overlying 

### 16

wa ter was observed to significantly impact Pb mobilization, with greater Pb mobilization at lower 

### 17
dissolved oxygen levels (3 to 9 mg/L O 2 ) ( Atkinson et al., 2007 )

. In addition, although Pb concentrations 

### 18

in the sediments of a mine

- impacted wetland in Hezhang, China, were determined to be strongly 

### 19

associated wi th organic/sulfide and residual fractions (e.g., 34 to 82% of total Pb), the presence of aquatic 

### 20
macrophytes altered the Pb speciation, increasing the fraction of Pb bound to Fe

- Mn oxides (42% to 47% 

### 21
of total Pb) ( Bi et al., 2007 ) . This phenomenon was investigated in greater depth by Sundby et al. ( 2005 )

, 

### 22
who determined that release of oxygen from macrophyte roots resulted in the oxidation of sediment



### 23
bound Pb, leading to the release of bioaccessible Pb fractions ( Sundby et al., 2005 )

. 

### 24

7.3.2.1. Other Metals

Komjarova and Blust ( 2008 ) looked at the effect of the presence of Cd 2+ on the uptake of Pb by the 

### 25

freshwater c ladoceran Daphnia magna . While Pb uptake rates were not affected by Cu, Ni or Zn, 

### 26
enhanced Pb accumulation was observed in the presence of 0.2 μM Cd. The highest Pb concentrations 

### 27
(0.25 μM) in turn facilitated Cu uptake. Area

- specific and whole organism Pb transport rates were 

### 28
greatest in the mid

- intestine. It was concluded that Pb

- induced disruptions of ion homeostasis and metal 

### 29
absorption processes might be a possible explanation of stimulated Pb uptake in the presence of Cd, as 

### 30

well as the increase in Cu uptake rates provoked by presence of Pb at its highest studied concentration. 

### 31
Komjarova and Blust ( 2009b ) then considered the effect of Na, Ca and pH on simultaneous uptake of Cd, 

### 32

Cu, Ni, Pb and Zn. Cd and Pb showed increase d uptake rates at high Na concentration. It was thought that 

### 33May 2011 7

- 55 DRAFT

– DO NOT C ITE OR QUOTE increased Na uptake rates promoted Pb entrance to the cell. With respect to the effect of pH, reduced 

### 1
proton competition begins to influence Pb uptake in waters with high pH . A clear suppression of Cd, Ni, 

### 2
Pb and Zn uptake was observed in the presence of Ca (2.5 mM). Ca has been reported to have a protective 

### 3
effect in other studies (involving other organisms). The presence of other metals may also affect the 

### 4
uptake of Pb by f ish

. At low concentrations, Cd in a Pb

- Cd mixture out

- competed Pb at gill tissue binding 

### 5
sites in rainbow trout ( Oncorhynchus mykiss ), resulting in a less

- than additive toxicity when fish were 

### 6
exposed to both metals in tandem ( Birceanu et al., 2008 )

. 

### 7

7.3.2.2. Biofilm

Farag et al. ( 2007 ) measured Pb concentrations in various media (water, colloids, sediment, 

### 8
biofilm) as well as invertebrates and fish collected within the Boulder River watershed

. They concluded 

### 9

that the fra ction of Pb associated with Fe

- oxides was most frequently transferred to biofilms and the other 

### 10
biological components of the sampled systems ( Farag et al., 2007 )

. Consequently, an increase in the Pb 

### 11

Fe

- oxide fraction could signify a potential increase in the bioaccessible pool of Pb

. The authors also noted 

### 12
that this fraction may promote downstream transport of Pb contamination

. Anci on et al. ( 2010 ) 

### 13
investigated whether urban runoff metal contaminants could modify biofilm bacterial community 

### 14
structure and diversity and therefor e potentially alter the function of biofilms in stream ecosystems. They 

### 15
found that accumulation rates for metals in biofilm were maximal during the first day of exposure and 

### 16
then decreased with time. Equilibrium between metal concentrations in the water an d in the biofilm was 

### 17

reached for all metals after 7

- 14 days of exposure. The affinity of the biofilm for Pb was, however, much 

### 18
greater than for Cu and Zn. With respect to recovery, the release of metals was slow and after 14 days in 

### 19

clean water 35% of Pb r emained in the biofilm. By retaining and releasing such metal pollutants, biofilms 

### 20
may play a key role in determining both the concentration of the dissolved metals in the water column and 

### 21
the transfer of the metals to invertebrates and fish grazing on the m. An enrichment factor of 6

, 000:1 for 

### 22
Pb between the biofilm and the water was measured after 21 days exposure to synthetic urban runoff. The 

### 23
relatively slow release of such metal may greatly influence the transfer of Pb to organisms feeding on the 

### 24

biofil ms. This may be of particular importance during storm events when large amounts of Pb are present 

### 25
in the urban runoff. It was suggested that biofilms constitute an integrative indicator of metal exposure 

### 26
over a period of days to weeks. 

### 27

7.3.2.3. Carbonate

An investi gation of heavy metal concentrations in an industrially impacted French canal (Deule 

### 28
canal) indicated that total extractable Pb in sediments ranged from 27 to 10,079 mg Pb /kg, with 52.3% 

### 29
present in Fe

- Mn oxide fractions, 26.9% as organic sulfide fraction, 10.7% in carbonates, and 10.1% in 

### 30

the residual fraction ( Boughriet et al., 2007 )

. The relatively high fraction of Pb associated with carbonates 

### 31May 2011 7

- 56 DRAFT

– DO NOT C ITE OR QUOTE was not observed at other sites, as sediments in these areas contained low proportions of carbonates

. 

### 1

Hence, addition of carbona tes (either from anthropogenic or natural sources) can significantly impact Pb 

### 2
speciation in sediments, and potential bioavailability to resident organisms. In addition, increased surface 

### 3

water carbonate concentrations also reduced the bioaccessible Pb fra ction as measured by chronic Pb 

### 4
accumulation in the fathead minnow, ( Pimephales promelas ) ( Mager et al., 2010 ) , and by Pb toxicity to 

### 5

fathead minnow and the cladoceran Ceriodaphnia dubia ( Mager et al., 2011 )

. 

### 6

7.3.2.4. Dissolved Organic Matter (DOM)

Uptake of Pb by water

- column organisms is affected by the concentration of DOM ( Mager et al., 

### 7

2010 )

. The specific composition of DOM has been shown to affec t the bioaccessibility of environmental 

### 8

Pb

. Humic acid

- rich DOM resulted in decreased free Pb ion concentration when compared to systems 

### 9
containing DOM with high concentrations of polysaccharides ( Lamelas & Slaveykova, 2008 )

. When the 

### 10
sequestering abilities of various components of DOM were compared, humic acid again was shown to be 

### 11
most efficient at reducing the Pb free ion concentration, followed by fulvic acid, alginic acid, 

### 12
polygalacturoni c acid, succinoglycan, and xanthan ( Lamelas et al., 2005 ) . Lamelas et al. ( 2009 ) 

### 13
considered the effect of humic acid on Pb(II) uptake by freshwater algae taking account of kinetics and 

### 14
cell wall speciation. The uptake flux was described by a Michaelis

- Men ten type equation. Comparison of 

### 15
Cu(II), Cd(II) and Pb(II) uptake by green freshwater algae, C. Kesslerii, in the presence of either citric 

### 16
acid or humic acid was made. The uptake fluxes, percentage adsorbed and percentage internali z ed for Cu 

### 17

and Cd were i dentical in the presence of either citric or humic acid. In contrast, however, there was a ten



### 18
fold increase in the respective values for Pb. The increase in adsorbed Pb was attributed to the increase in 

### 19
adsorption sites from the adsorbed humic acid on the surface of the algae. Two hypotheses were 

### 20
considered to explain the increase in internalized Pb and the internalization flux: ( 1 ) direct interaction of 

### 21

Pb

- humic acid complexes with the internali z ation sites

, and ( 2 ) uptake of Pb(II) after dissociation fro m 

### 22

the Pb

- humic acid complex. The authors favor the former hypothesis but no evidence is presented for the 

### 23
proposed ternary Pb

- humic acid

- internali z ed site complexes, nor is there an explanation as to why this 

### 24

behavior is not observed for Cd or Cu. 

### 25

There is evidence, however, that DOC/DOM does not have the same effect on free Pb ion 

### 26
concentration in marine systems as in freshwater systems. No correlation was observed between DOM 

### 27
concentration or composition and Pb toxicity when examined using the marine inve rtebrate Paracentrotus 

### 28

lividus embryo

- larval bioassay ( Sanchez

- Marin, Santos

- Echeandia, et al., 2010 ) . For marine invertebrates, 

### 29
the presence of humic acid increased both the uptake and toxicity of Pb, despite the fact that a larger 

### 30
fraction of Pb is complexed with humic acid (25 to 75%). Alt hough the authors could not provide a 

### 31
precise explanation for this, they theorized that in marine environments, addition of humic acid could 

### 32
induce and enhance uptake of Pb via membrane Ca 2+ channels ( Sanchez

- Marin, Slaveykova, et al., 2010 )

. 

### 33May 2011 7

- 57 DRAFT

– DO NOT C ITE OR QUOTE This mechanism was observed in the marine diatom Thalassiosira weissflogii , in that humic acids 

### 1
absorbed to cell surfaces increased metal uptake; however, water column Pb

- humic acid associations did 

### 2
appear to reduce free Pb ion concentrations ( Sanchez

- Marin, Slaveykova, et al., 2010 )

. Formation of a 

### 3
ternary complex that is better absorbed by biological membranes was another proposed mechanism that 

### 4
could describe the increased bioaccessibility to marine invertebrates of Pb bound to humic acid ( Sánchez



### 5

Marín et al., 2007 )

. 

### 6
As little as 1 μmol of humic acid introduced into surface waters was sufficient to reduce Pb uptake 

### 7
by perennial ryegrass, Lolium perenne , grown in nutrient solution. This resulted from a decrease in the 

### 8
concentration of the free Pb fraction by several orders of magnitude following complexation with the OM. 

### 9

Pb content on the root surf ace was reduced to 8 μmol/g from 20 μmol/g following humic acid addition, 

### 10
and relative Pb absorption (absorption in the presence of humic acid divided by absorption in the absence 

### 11
of humic acid) was determined to be approximately 0.2 ( Kalis et al., 2006 )

. Conversely, humic acid may 

### 12
increase the bioaccessible Pb fraction for green algae through formation o f a ternary complex that 

### 13
promotes algal uptake of the metal

. Lamelas and Slaveykova ( 2007 ) found that aqueous Pb formed 

### 14
complexes with humic acid, which in turn would become a d sorbed to Chlorella kesslerii algal surfaces, 

### 15
and that the presence of Pb sorbed to humic acid did not interfere with humic acid

- algae complexation

. 

### 16
The authors concluded that humic acids bound to algae acted as additional binding sites for Pb, thus 

### 17
increasing t he concentrations associated with the algal fraction ( Lamelas & Slaveykova, 2007 )

. 

### 18

Based on the above, the recent literature indicates the existence of a number of deviations from 

### 19
current models used to predict bioaccess i bility of Pb

. In marine aquatic systems, for instance, surface 

### 20
water DOM was found to increase (rather than decrease) uptake of Pb by fish gill structures, potentially 

### 21
through the alt eration of membrane Ca

- channel permeability

. This phenomenon would not be accurately 

### 22
predicted by a BLM developed using data from freshwater organisms

. Further, in both freshwater and 

### 23
marine environments, algal biosorption of labile Pb fraction was also in creased by humic acid and DOM, 

### 24
likely through the formation of ternary complexes that increase Pb binding sites on the algal surface

. 

### 25
Although it is unclear whether Pb in this form is available for toxic action on algae, it is likely to 

### 26
comprise a signific ant source of dietary Pb for primary consumers

. Moreover, the attempted field 

### 27
verification of freshwater bioaccessibility models was conducted at sites with distinct point

- sources of Pb 

### 28
contamination, and only one model (SHM) was found to adequately predic t Pb bioaccessibility. 

### 29



## 7.3.3. Bioavailability in Aquatic Systems

Bioavailability was defined in the 2006 Pb AQCD as “ the proportion of a toxin that passes a 

### 30
physiological membrane (the plasma membrane in plants or the gut wall in animals) and reaches a target 

### 31
receptor (cytosol or blood). ” In 2007, EPA took cases of bioactive adsorption into consideration and 

### 32
revised the definition of bioavailability as “ the extent to which bioaccessible metals absorb onto, or into, 

### 33May 2011 7

- 58 DRAFT

– DO NOT C ITE OR QUOTE and across biological membranes of organisms, expressed as a fraction of the total amount of metal the 

### 1
organism is proximately exposed to (at the sorption surface) during a given time and under defined 

### 2

conditions ” ( Fairbrother et al., 2007 )

. In brief, trace metals, and their complexes, must first diffuse from 

### 3
the external medium to the surface of the organism (mass transport). Metal complexes may dissociate and 

### 4

re

- associat e in the time that it takes to diffuse to the biological surface. To have an effect on the organism, 

### 5
metals must then react with a sensitive site on the biological membrane (adsorption/desorption processes), 

### 6
often but not necessarily followed by biological transport (internalization). Any of these processes may be 

### 7
the rate limiting step for the overall biouptake process. Internalization is, however, the key step in the 

### 8
overall biouptake process. Although the transport sites often have a high affinity for re quired metals they 

### 9
do not always have high selectivity and so a toxic metal may bind to the site of an essential metal with a 

### 10

similar ionic radius or co

- ordination geometry, e.g., Pb 2+ , Cd 2+ and Zn 2+ are similar to Ca 2+ . At the 

### 11

molecular level, there are t hree major classes of transition metal transporter: P

- type ATPases, zinc 

### 12
regulated transporter/iron

- regulated transporter, and natural resistance associated macrophage proteins 

### 13

( Worms et al., 2006 ) . Of these, natural resistance associated macrophage proteins have been shown to 

### 14
promote the uptake of various metals including Pb. This type of trace metal transport can be described by 

### 15

Michaelis

- Menten uptake kinetics and equilibrium considerations. 

### 16
According to the 2006 Pb AQCD, Pb adsorption, complexation, chelation, etc., are processes that 

### 17

a lter its bioavailability to different aquatic species, and it was suggested that multiple exposure routes 

### 18
may be important in determining overall bioavailability of Pb

. Given its low solubility in water, 

### 19
bioaccumulation of Pb by aquatic organisms may preferentially occur via exposure routes other than 

### 20
direct absorption from the water column, including ingestion of contaminated fo od and water, uptake 

### 21
from sediment pore waters, or incidental ingestion of sediment

. If uptake and accumulation are 

### 22
sufficiently faster than depuration and excretion, Pb tissue levels may become sufficiently high to result in 

### 23

adverse effects ( Luoma & Rainbow, 2005 ) . Pb accumulation rates are controlled, in part, by metabolic 

### 24
rate. Other factors that influence bioavailability of Pb to organisms in aquatic systems are reviewed in 

### 25

Secti on 7.3.2

. As summarized in the 2006 Pb AQCD, organisms exhibit three Pb accumulation strategies : 

### 26
(1) accumulation of significant Pb concentrations with low rate of loss resulting in substantial 

### 27

accumulation ; (2) balance between excretion and bioava ilable m etal in the environment; and (3) very low 

### 28
metal uptake rate without significant excretion, resulting in weak net accumulation ( Rainbow, 1996 )

. 

### 29
Uptake experiments with aquatic plants, invertebrates and vertebrates reviewed in the 2006 Pb AQCD 

### 30
showed increases in Pb uptake with incr easing Pb in solution

. The 2006 Pb AQCD findings included 

### 31
consideration of bioaccumulation in different trophic levels. Pb concentrations were found to be typically 

### 32
higher in algae and benthic organisms and lower in higher trophic

- level consumers. 

### 33

In this section, recent information on bioavailability and uptake in algae, plants, invertebrates and 

### 34
vertebrates from marine and freshwater systems are reviewed with summary material from the 2006 Pb 

### 35
AQCD where appropriate

. An overview of the BLM is presented as the most widely used method for 

### 36May 2011 7

- 59 DRAFT

– DO NOT C ITE OR QUOTE predicting both the bioaccessible and bioavailable fractions of Pb in the aquatic environment

. This is 

### 1
followed by a discussion of bioavailability in algae, plants, invertebrates and vertebrates

. As reviewed by 

### 2
Wang and Rai nbow ( 2008 )

, aquatic organisms exhibit distinct patterns of metal bioaccumulation

. The 

### 3
authors suggest that the observed differences in accumulation, body burden, and elimination between 

### 4
species are due to metal biogeochemistry and physiological and biological respons es of the organism

. The 

### 5
studies presented below generally support the observations of Wang and Rainbow ( 2008 ) that closely 

### 6
related species can vary greatly in bioaccumulation of Pb and other non

- essential metals

. 

### 7
The bioaccumulation and toxicity of Pb to aquatic organisms are closely linked to the 

### 8
environmental fate of the metal under variable environmental conditions (Section 3.3) as they are highly 

### 9
dependent upon the relative proportion of free metal ions in the water column

. However, information is 

### 10
lacking on the uptake of Pb through ingestion of Pb

- sorbed particles or dietary exposure to biologically



### 11
incorporated Pb. Such routes of exposure are not included in models such as the BLM that predict toxicity 

### 12

as a function of Pb concentration in the water column

. This uncertaint y may be greater for Pb than for 

### 13
other more soluble metals (such as Cu) as a greater proportion of the total mass of Pb in an aquatic 

### 14
ecosystem is likely to be bound to particulate matter

. Therefore, estimating chronic toxicity of Pb to 

### 15
aquatic receptors m ay have greater uncertainty than predicting acute effects. 

### 16
In addition to the biogeochemical effects that govern the environmental pool of accessible Pb, 

### 17
reactions of Pb with biological surfaces and membranes determines the bioavailability and uptake of th e 

### 18
metal by aquatic organisms

. The BLM predicts both the bioaccessible and bioavailable fraction of Pb in 

### 19
the aquatic environment, and can be used to estimate the importance of environmental variables such as 

### 20
DOC in limiting uptake by aquatic organisms ( Alonso

- Castro et al., 2009 )

. The BLM integrates the 

### 21
binding affinities of various natural ligands in surface waters and the biological uptake rates of aquatic 

### 22
organisms to de termine the site

- specific toxicity of the bioavailable fraction. In the 2006 Pb AQCD, 

### 23
limitations of the use of BLM in developing air quality criteria were recognized including the focus of 

### 24
this model on acute endpoints and the absence of consideration of dietary uptake as a route of exposure. 

### 25
Atmospheric deposition of Pb to aquatic systems and subsequent effects on ecosystem receptors is likely 

### 26
characterized as a chronic, cumulative exposure rather than an acute exposure. Recommendations from 

### 27

the 2006 Pb A QCD included developing both chronic toxicity BLMs and BLMs that consider the dietary 

### 28
route of Pb uptake. This section reviews the literature from the past 5 years on applications of the BLM to 

### 29
predicting bioavailability of Pb to aquatic organisms. However , the primary focus of initial BLMs has 

### 30
been acute toxicity endpoints for fish and invertebrates following gill or cuticular uptake of metals

. 

### 31

Di Toro et al. ( 2005 ) constructed BLMs for metals exposure in sediments, surface water, and 

### 32

sediment p ore water to determine how to most accurately predict the toxicity of metals

- contaminated 

### 33

sediments

. Results from models were compared with literature

- derived acute toxicity values for benthic 

### 34
and epibenthic invertebrates to establish the accuracy of the d eveloped models

. Although the models 

### 35
tended to overestimate the toxicity of aqueous and sediment

- bound Pb in freshwater environments, it was 

### 36May 2011 7

- 60 DRAFT

– DO NOT C ITE OR QUOTE determined that the model significantly underestimated Pb toxicity to marine invertebrates ( Di Toro et al., 

### 1

2005 )

. This may be because pore water metal concentrations were not modeled

. Consequently, these 

### 2
results may suggest that either 1) mobilization of Pb concentrations from sediments into pore water is 

### 3
greater in marine environment s, or 2) marine invertebrates are significantly more susceptible to Pb 

### 4
exposures than are freshwater species. 

### 5
A number of deviations from results predicted by Pb exposure models (such as the BLM) were 

### 6
documented by Ahlf et al. ( 2009 )

. They highlighted that uptake of metals by sediment

- dwelling bivalves 

### 7
was significantly greater than predicted, because bivalves accumulate Pb from multip le sources not 

### 8
included in the model, such as ingestion of algae, bacteria, and colloidal matter

. Species

- specific dietary 

### 9
assimilation of ingested particulate

- bound metals is also likely to play a role in the toxicity of Pb to 

### 10
aquatic organisms, yet insuf ficient data are available to permit modeling of this additional factor ( Ahlf et 

### 11

al., 2009 )

. The authors outlined the need for additional data in developing bioavailability models for 

### 12
chronic metal exposures. 

### 13
Similarly, although the presence of humic acid is considered to reduce the bioavailable fraction of 

### 14
metals in surface water, green algae uptake and biosorption o f metals, including Pb, was actually 

### 15
increased by humic acid

. The authors determined that humic acid bound to algal surfaces served to 

### 16
increase the total number of metal binding sites over those afforded solely by the algal surface ( Lamelas 

### 17
& Slaveykova, 2007 )

. This highlights the complexity of modeling chronic metals bioavailability through 

### 18
multiple exposure routes, as humic acid would decrease gill or cuticular uptake of metals from the water 

### 19
column, but could potentially enhance dietary exposure by increasing algal metal content

. Slaveykova and 

### 20

Wilkinson ( 2005 ) also noted that humic acid is likely to interact with other biological membranes and 

### 21
alter their pe rmeability to metals, especially in acidic environments

. Further, they observed that increased 

### 22
surface water temperatures can not only increase membrane permeability but also change metabolic rates, 

### 23
both of which can enhance metals uptake and assimilation; however, this factor is not included in 

### 24
bioavailability models such as the BLM ( Slaveykova & Wilkinson, 2005 ) . Despite this, the authors noted 

### 25
that, in most cases, the BLM could predict acute metals toxicity with a reasonable degree of accuracy. 

### 26

Veltman et al. ( 2010 ) proposed an integration of BLM and bioaccumulation models in order to 

### 27
more accurately predict metal uptake by fish and invertebrates

. Although Pb was not the specific focus of 

### 28
the paper, calculated metal absorption efficiencies for marine fish species from both BLM and 

### 29
bioaccumulation models were determined to be highly comparable for Ag, Cd, Cu, and Zn

. Authors also 

### 30
noted that affinity constants for Ca, Cd, Cu, Na, and Zn were highly similar across different aquatic 

### 31
species, including fish and invertebrates ( Veltman et al., 2010 )

. These findings suggest that the BLM can 

### 32
be integrated with bioaccumulation kinetics to account for both environmental chemic al speciation and 

### 33
biological and physiological factors. 

### 34May 2011 7

- 61 DRAFT

– DO NOT C ITE OR QUOTE 

### 7.3.3.1. Plants and Algae

Aquatic macrophytes and algae can accumulate Pb from either the water column or sediments, 

### 1
based on their specific microhabitats

. For instance, rooted macrophytes may be more likely to accumulate 

### 2
Pb from sediment sources, while floating macrophytes or algae will take up Pb suspended or dissolved in 

### 3

the water column

. However, significant species

- dependent differences in bioaccumulation rates, as well as 

### 4
concentrations of sequestered me tals within different parts of the plants (shoots versus roots), have also 

### 5
been observed and some authors have concluded that the plant species is a more important determinant of 

### 6
Pb uptake than is habitat type. Uptake and translocation studies of Pb in pla nts and algae reviewed in the 

### 7
2006 Pb AQCD indicated that all plants tend to sequester larger amounts of Pb in their roots than in their 

### 8

shoots

. Recent studies on bioavailability of Pb to plants support the findings of the 2006 Pb AQCD and 

### 9
provide addition al evidence for species

- dependent differences in responses to Pb in water and sediments. 

### 10
The microalgae Spirulina platensis was demonstrated to accumulate Pb from the water column, 

### 11
with 2.7, 6.9, 19, 45 and 145 μg Pb /mg accumulated at aqueous Pb concentrat ions of 5, 10, 30, 50, and 

### 12

100 μg Pb /L, following a 10

- day incubation period ( Arunakumara et al., 2008 )

. Pb concentrations 

### 13
accumulated by algae appeared to decrease when culture time increased from 2 to 10 days

. This may have 

### 14
occurred as a result of a gradual recovery of growth and an addition of biomass that would have reduced 

### 15
the concentration of Pb in algal tissue (known as “ biodilution ” )

. An aquatic moss, Fontinalis antipyretica

, 

### 16
accumulated up to an average of 3 μmol Pb/g dry weight over a 7

- day exposure to 100 μmol Pb, despite 

### 17
saturation of intracellular Pb concentrations after 5 days of exposure ( Rau et al., 2007 )

. Interestingly, 

### 18

e xperimentation with concurrent Cu and Pb exposure indicated that the presence of Cu increased the 

### 19
uptake of Pb by the green algae Chlamydomonas reinhardtii ( Z. Z. Chen et al., 2010 )

. The authors noted 

### 20

that, in the case of Cu

- Pb binary exposures, uptake rates of Pb exhibited complex non

- linear dynamics in 

### 21
other aquatic organisms as well. 

### 22
Pb bioaccumulation studies conducted with five species of ma rine algae, ( Tetraselmis chuii

, 

### 23

Rhodomonas salina , Chaetoceros sp., Isochrysis galbana and Nannochloropsis gaditana ) demonstrated 

### 24
that bioaccumulation rates varied with species. I. galbana accumulated the lowest concentrations of Pb 

### 25
(0.01 and 0.6 pg Pb /cel l at water concentrations of 51 and 6,348 μg Pb /L), while Chaetoceros sp. was 

### 26
observed to be the most efficient Pb bioaccumulator, adsorbing 0.04 and 54 pg Pb /cell at 1.4 and 6,348 μg 

### 27

Pb /L ( Debelius et al., 2009 )

. 

### 28
When exposed to water concentrations of up to 100 μmol Pb, floating (non

- rooted) coontail plants 

### 29

( Ceratophyllum demersum ) accumulated an average Pb concentration of 1,748 mg Pb/kg after 7 days, 

### 30
although this was not significantly higher than levels accumulated in the first day of exposure ( S. Mishra 

### 31

et al., 2006 )

. Induction of the antioxidant system improved the tolerance of the aquatic plant Najas indica 

### 32

for bioaccumulated Pb, allow ing for increased biomass and the potential to accumulate additional Pb 

### 33

mass

. High Pb accumulation (3,554 mg Pb/kg dry weight tissue following a 7

- day exposure to 100 μmol 

### 34May 2011 7

- 62 DRAFT

– DO NOT C ITE OR QUOTE Pb) was considered to be a function of plant morphology; as a submerged, floating pl ant, N. indica 

### 1
provides a large surface area for the absorption of Pb ( Singh et al., 2010 )

. 

### 2
Given that atmospherically

- derived Pb is likely to become sequestered in sediments, uptake by 

### 3
aquatic macrophytes is a significant route of Pb removal from sediments, and a potential route for Pb 

### 4
mobilization into the aquatic fo od web

. The rooted aquatic macrophyte Eleocharis acicularis was 

### 5
determined to be a hyperaccumulator of Pb in an 11

- month bioaccumulation experiment with mine 

### 6
tailings

. When grown in sediments containing 1,930 mg Pb /kg, the maximum concentration of Pb in E. 

### 7

acicularis was determined to be 1,120 mg Pb /kg dry weight

. However, calculated BCF ’ s for Pb were all 

### 8
less than one, indicating that Pb uptake, although high, was less efficient than for other metals present ( Ha 

### 9

et al., 2009 )

. 

### 10
Aquatic plants inhabiting a wetlands containing an average sediment Pb concentration of 99 

### 11

mg Pb /kg exhibited variable Pb tissue concentr ations, but these do not appear to be related to macrophyte 

### 12
type (e.g., submerged, floating, emergent, etc.)

. Consequently, the authors concluded that uptake of Pb by 

### 13
aquatic plants appears to be dependent on species, at the exclusion of habitat or type

. F or instance, among 

### 14
the submerged plant species, Ceratophyllum demersum accumulated the greatest amount of Pb (22 μg/g 

### 15
dry weight), while Potamogeton malainus tissue contained the least amount of Pb, 2.4 μg/g dry weight 

### 16

( Bi et al., 2007 )

. Tissues of the floating plants Azolla imbricata and Spirogyra communis were found to 

### 17

contain 12 and 20 mg Pb/ k g dry weight, respectively, while emergent macrophytes Scirpus triqueter and 

### 18
Alternanthera philoxeroides accumulated 1.4 and 10 m g Pb/ k g dry weight

. Fritioff and Greger ( 2006 ) 

### 19
determined that anywhere from 24

– 59% of the total Pb taken up by Potamogeton natans aquatic plants 

### 20
was sequestered in the cell wall fraction, depending on plant tissue an d environmental Pb concentration

. 

### 21
More importantly, no translocation of Pb was observed when plant tissues (leaf, stem, root) were exposed 

### 22
to Pb solutions separately ( Fritioff & Greger, 2006 )

. 

### 23

Dwivedi et al. ( 2008 ) reared nine different species of aquatic plants in a fly

- ash contaminated 

### 24
medium containing approximately 7 m g Pb/ k g dry weight. Not only did species exhibit different Pb 

### 25
accumulation efficiencies but they also compartmentalized sequestered Pb differently. The submerged 

### 26

macrop hyte Hydrilla verticillata accumulated the greatest amount of Pb (approximately 180 m g Pb / k g 

### 27
dry weight tissue), but Pb was sequestered solely in the shoot tissue

. In contrast, other plant species 

### 28

accumulated between 15 and 100 m g Pb/ k g dry weight ( Ranuncu lus scloralus and Marsilia quadrifolia ) 

### 29
with the majority compartmentalizing the metal in root tissue, except for C. demersum and M. 

### 30
quadrifolia , which also utilized shoot tissue for Pb storage ( Dwivedi et al., 2008 )

. 

### 31

Pb concentrat ions in the root, leaf, and stem tissues of three aquatic plant species were found to 

### 32
correlate most closely with the concentration of the exchangeable Pb fraction (e.g., the fraction of Pb that 

### 33
is easily and freely leachable from the sediment). Authors no ted that seasonal variations can alter the 

### 34
amount of Pb present in the exchangeable fraction, and that Pb was more likely than Cd or Cu to remain 

### 35May 2011 7

- 63 DRAFT

– DO NOT C ITE OR QUOTE tightly bound to sediments, and therefore the relationship between total sediment Pb and Pb in aquatic 

### 1
plant t issues was weaker ( Ebrahimpour & Mushrifah, 2009 )

. 

### 2
Lemna sp., a rooted macrophyte, incubated in a water extract of waste ash containing 19 μg Pb /L 

### 3

accumulated 3.5 m g Pb / k g dry weight over 7 days of exposure. Slight toxic effects, including suppression 

### 4
of growth, were observed over this exposure period, but this may have been a result of exposures to 

### 5
multiple metals in the water extract, including Cr, Mn, Cu, and Zn ( Horvat et al., 2007 )

. Lemna sp. was 

### 6
also demonstrated to be effective in the biosorption of Pb f rom solution, even in the presence of sediments 

### 7
(1 g per 700 mL water)

. Over 7 days of exposure to 5 and 10 mg Pb/L, plant biomass was found to 

### 8
contain an average of 2.9 and 6.6 mg Pb, respectively, versus 0.2 and 0.3 mg in sediment ( Hurd & 

### 9
Sternberg, 2008 )

. 

### 10
Young Typha latifolia , another rooted macrophyte, were grown in 5 and 7.5 mg/L Pb

- spiked 

### 11
sediment for 10 days to determine their value as metal accumulators

. Within the exposure period, plants 

### 12
exposed to the lower concentration were able to remove 89% of Pb, while 84% of the Pb present in the 

### 13
higher treatment was taken up by T. latifolia

. Pb concentrations measured in root and leaf tissue ranged 

### 14

from 1,365 to 4,867 m g Pb /kg and 272 to 927 mg Pb /kg, respectively, and were higher at the greater 

### 15
environmental Pb exposure ( Alonso

- Castro et al., 2009 )

. 

### 16
Common reeds ( Phragmites australis ) grown in metal

- impacted aquatic environments in Sicily, 

### 17
Italy, preferentially accumulated Pb in root and rhizome tissues ( Bonanno & Lo Giudice, 2010 )

. 

### 18

Environmental Pb concentratio ns in water and sediment averaged 0.4 μg Pb /L and 2.7 mg Pb /kg

. These 

### 19
levels yielded root and rhizome concentrations of 17 and 15 mg Pb/kg, respectively, whereas stem and 

### 20
leaf Pb concentrations were lower (9.9 and 13 mg Pb /kg)

. These tissue concentrations were significantly 

### 21

correlated to both water and sediment concentrations ( Bonanno & Lo Giudice, 2010 )

. The roots of two 

### 22

salt marsh species, Sacorconia fructicosa and Spartina maritima significantly accumulated Pb, to 

### 23
maximum concentrations of 2,870 mg Pb /kg and 1,755 mg Pb /kg, respectively ( Caetano et al., 2007 )

. 

### 24
Roots had similar isotopic signature to sediments in vegetated zones indicating that Pb uptake by plants 

### 25
reflects the input in sediments. Conversely, the semi

- aquatic plant Ammania baccifera , grown in mine 

### 26
tailings containing 35 to 78 mg Pb /kg, d id not accumulate analytically detectable levels of Pb in either 

### 27
root or shoot tissues, despite the fact that other metals (Cu, Ni, Zn) were bioaccumulated ( Das & Maiti, 

### 28

2007 )

. This would indicate that at low/moderate environmental Pb concentrations, some plant species 

### 29
may not bioaccumulate significant (or measurable) levels of Pb. 

### 30
The average concentration of Pb in the tissues of rooted aquatic macrophytes ( Callitriche verna

, P. 

### 31

natans
, C. demersum

, Polygonum amphibium

, Veronica beccabunga ) collected from two metals

- polluted 

### 32
streams in Poland (average sediment concentration 38 to 58 mg Pb/kg) was less than 30 mg Pb /kg

. Pb 

### 33
bioaccumulation in plants was signi ficantly correlated with sediment Pb concentrations ( Samecka



### 34
Cymerman & Kempers, 2007 )

. A similar significant correlation was established between reed sweet grass 

### 35May 2011 7

- 64 DRAFT

– DO NOT C ITE OR QUOTE root Pb concentration and sediment Pb concentrations, yielding BCFs ranging from 0.5 to 1.5, wit h an 

### 1
average BCF of 0.9 ( Skorbiowicz, 2006 )

. 

### 2

Pb tissue concentrations of aquatic plants P. australis and Ludwigia prostrata collected from 

### 3
wetlands containing an average of 52 mg Pb /kg in surficial sediments were predominantly in root tissues, 

### 4
indicating poor translocation of Pb from roots

. In the former, Pb decreased from an average of 37 mg 

### 5

Pb /kg in roots to 17, 14, and 12 mg Pb /kg in rhizome, stem and leaf tissues, respectively, while L. 

### 6

prostrata Pb tissue concentrations decreased from 77 mg Pb /kg in fibrous root to 7 and 43 mg Pb /kg in 

### 7

stem and leaf tissu es ( H. J. Yang et al., 2008 )

. The authors proposed that this diminished transfer ability 

### 8
explained the relatively low BCF ’ s for Pb uptake in t hese two species, when compared with those of other 

### 9
metals (Table 7

- 3 ). 

### 10
Despite no significant seasonal effect on surface water Pb concentrations, shining pondweed 

### 11

( Potamogeton lucens ), a rooted aquatic macrophyte grown in an urbanized metal

- contaminated lake in 

### 12
Turkey, exhibited seasonal alterations in Pb tissue concentrations

. Average measured water Pb 

### 13
concentrations were 28 μg Pb /L in spring, 27 μg Pb /L in summer, and 30 μg Pb /L in autumn

. Over this 

### 14
same time period, root tissue Pb concentrations signif icantly increased from 6 m g Pb / kg dry weight in 

### 15
spring, to 9 m g Pb / k g dry weight in summer, and to 10 m g/ k g dry weight in autumn ( Duman et al., 2006 )

. 

### 16

No differences were detected in stem Pb concentrations betwe en spring and summer (approximately 4 m g 

### 17

Pb / k g dry weight), but stem Pb concentrations were found to be significantly higher in autumn (6 m g 

### 18

Pb / k g dry weight)

. In the same system, P. australis plants accumulated the most Pb during win ter: 103, 

### 19

23, and 21 m g Pb / k g dry weight in root, rhizome, and shoot tissue, respectively, in sediments containing 

### 20

13 m g Pb/ k g dry weight

. By contrast, Schoenoplectus lacustris accumulated maximum rhizome and stem 

### 21

Pb concentrations of 5.1 and 7.3 m g Pb/ k g dry weight in winter, but sequestered the greatest amount of Pb 

### 22
in root tissues during the spring (30 m g Pb/ k g dry weight) at a comparable sediment concentration, 18 m g 

### 23

Pb/ k g dry weight ( Duman et al., 2 007 )

. The authors suggest that this indicated that metal uptake was 

### 24
regulated differently between species. 

### 25
Tree species that inhabit semi

- aquatic environments have also been shown to absorb Pb from Pb



### 26

contaminated sediments

. Bald

- cypress trees ( Taxodium distichum ) growing in sediments of a refinery



### 27
impacted bayou in Louisiana accumulated significantly greater amounts of Pb than did trees of the same 

### 28
species growing in bankside soil, despite the lower Pb concentrations of sediments

. Bankside soils 

### 29

contain ed greater than 2,700 mg Pb /kg versus concentrations of 10 to 424 mg Pb/kg in sediments, yet Pb 

### 30
concentrations in trees averaged 4.5 and 7.8 mg Pb/kg tissue, respectively ( Devall et al., 2006 )

. The 

### 31
authors theorized that Pb was more readily released from sediments and that soil dispersion to the swamp 

### 32
sediments provides additional, if periodic, loads of Pb into the system. 

### 33
BCFs for Pb in root tissue from mangrove tree species range between 0.09 and 2.9, depending on 

### 34
the species and the habitat, with an average BCF of 0.84

. The average BCF for mangrove species leaf 

### 35
tissue was considerably less (0.11), as these species are poor translocators of Pb ( MacFarlane et al., 2007 )

. 

### 36May 2011 7

- 65 DRAFT

– DO NOT C ITE OR QUOTE In contrast, willow seedlings planted in Pb

- contaminated sediment were more effective at removing Pb 

### 1
from the media than a diffusive gradient in thin film technique predicted ( Jakl et al., 2009 )

. T he authors 

### 2
proposed that the plant ’ s active mobilization of nutrients from soil during growth also resulted in 

### 3
increased Pb uptake and sequestration. 

### 4
Given that sediments are a significant sink for Pb entering aquatic systems, it is not surprising that 

### 5

roo ted macrophytes bioaccumulate significant quantities of the metal

. Although there are some 

### 6
similarities to Pb accumulation observed in terrestrial plants (e.g., preferential sequestration of the metal 

### 7
in root tissue), Pb appears to be more bioavailable in sediment than it is in soil

. This may be a result of 

### 8
differences in plant physiology between aquatic and terrestrial plants (e.g., more rapid growth or more 

### 9

efficient assimilation of nutrients and ions from a water

- saturated medium)

. While rooted macrophyt es are 

### 10
likely to be chronic accumulators of Pb sequestered in sediments, aerial deposition of Pb into aquatic 

### 11
systems may result in pulsed inputs of labile Pb that would be available for uptake by floating 

### 12
macrophytes and algae

. 

### 13
Reported values for BCF ’ s in aquatic plants from the 2006 Pb AQCD range from 840 to 20,000 (

, 

### 14

Table AX7

- 2.3.1U.S. EPA, 2006 )

. Duckweed ( Lemna minor ) had BCF values ranging from 840 to 3,560 

### 15
depending on the method of measurement

. A dditional BCF ’ s established for aquatic plants since the 2006 

### 16
Pb AQCD are summarized in Table 7

- 3 and include data on field

- collected plants as well as BCF ’ s 

### 17
obtained from laboratory exposures. 

### 18

Table 7

- 3. Bioconcentration factors for Pb in aquatic plants

Species BCF Test conditions Reference
Typha latifolia 649 10 days, Pb nitrate

- spiked water Alonso

- Castro et al. ( 2009 ) Spirulina platensis 1500 10 days, Pb nitrate

- spiked water Arunakumara et al. ( 2008 ) Ceratophyllum demersum 0.2 Field

- collected plants Bi et al. ( 2007 ) Spirogyra communis 0.2 Field

- collected plants Bi et al. ( 2007 ) Phragmites australis 6.4 Field

- collected plants Bonanno and Lo Giudice ( 2010 )
Taxodium distichum 0.02 Field

- collected tissue Devall et al. ( 2006 ) Hydrilla verticillata 26 Field

- collected plants Dwivedi et al. ( 2008 )
Eleocharis acicularis 0.8 11 mo , field

- collected sediment Ha et al. ( 2009 ) Lemna sp. 0.01 7 days, ash water extract Hor vat et al. ( 2007 ) Mangrove species 0.8 Field

- collected tissue MacFarlane et al. ( 2007 ) Glyceria aquatica 0.9 Field

- collected tissue (roots) Skorbiowicz ( 2006 ) Phragmites australis 0.7 Field

- collected plants Yang et al. ( 2008 ) Ludwigia prostrata 1.5 Field

- collected plants Yang et al. ( 2008 )



### 7.3.3.2. Invertebrates

Uptake and subsequent bioaccumulation of Pb in marine and freshwater invertebrates varies greatly 

### 19
between species and across taxa as previously characterized in the 2006 Pb AQCD

. This section expands 

### 20
on the findings from the 2006 Pb AQCD on bioaccumulation and sequestration of Pb in aquatic 

### 21May 2011 7

- 66 DRAFT

– DO NOT C ITE OR QUOTE invertebrates

. In the case of invertebrates, Pb can be bioaccumulated from multiple sources, including the 

### 1

water column, sedi ment, and dietary exposures, and factors such as proportion of bioavailable Pb, life 

### 2
stage, age, and metabolism can alter the accumulation rate

. In this section, new information on Pb uptake 

### 3
from sediments by invertebrates will be considered, followed by a discussion on dietary and water routes 

### 4
of exposure and factors that influence species

- specific Pb tissue concentrations such as invertebrate 

### 5
habitat and functional feeding group. 

### 6
The 2006 Pb AQCD summarized studies of uptake of Pb from sediment by aquati c invertebrates 

### 7

and noted that sediment pore water, rather than bulk sediment, is the primary route of exposure

. However, 

### 8
a recent study suggests that in the midge, Chironomus riparius , total metal concentrations in bulk 

### 9
sediment are better predictors of m etal accumulation than dissolved metal concentrations in sediment pore 

### 10
water based on bioaccumulation studies using contaminated sediments from six different sites ( Roulier et 

### 11

al., 2008 )

. Vink ( 2009 ) studied six river systems and found that, for a range of metals, uptake b y benthic 

### 12
organisms (the oligochaete, Limnodrilus (Family Tubificidae) and the midge, C. riparius ) from the 

### 13
sediment pore water (as compared with surface water) was observed only occasionally, and solely for Pb. 

### 14
The physiological mechanisms of Pb uptake ar e still unclear but it is suggested that uptake and 

### 15
elimination of Pb obey different mechanisms than for other heavy metals. Additionally, Metian et al. 

### 16

( 2009 ) showed that king scallop ( Pecten maximus ) exhibited low bioaccumulation efficiency of Pb from 

### 17
spiked sediment. 

### 18
The 2006 Pb AQCD recognized the potential importance of the diet ary uptake pathway as a source 

### 19
of Pb exposure for invertebrates

. Specifically, in a study with the freshwater amphipod Hyalella azteca

, 

### 20
dietary exposure was found to contribute to the chronic toxicity of Pb, while acute toxicity was unaffected 

### 21

( J. M. Besser et al., 2004 )

. Since the 2006 Pb AQCD, additional studies have considered the relative 

### 22
importance of water and dietary uptake of Pb in aquatic invertebrates

. A stable isotope technique was used 

### 23
to simultaneously measure uptake of environmentally relevant concentrations of Pb (0.05 μmol Pb in the 

### 24
water column) by the freshwater cladoceran D. magna directly from water and through food, the green 

### 25
algae Pseudokirchneriella subcapitata

. ( Komjarova & Blust, 2009a )

. D. magna accumulated the metal 

### 26
from both sources, but the relative proportion of uptake from each source changed over the exposure 

### 27

pe riod

. After the first day of exposure, 12% of accumulated Pb was determined to have been absorbed 

### 28
from dietary (algal) sources, but this percentage decreased by day four of exposure to 4%

. Pb absorbed 

### 29
from water exposure only resulted in Daphnia body burde ns of approximately 300 μmol Pb /kg dry 

### 30
weight, and was similar to the amount absorbed by algae ( Komjarova & Blust, 2009a )

. 

### 31
Stable isotope analysis was to used measure uptake and elimination simultaneously in netspinning 

### 32
caddisfly larvae (Hydropsyche sp.) exposed to aqueous Pb concen trations of 0.2 to 0.6 μg Pb /L ( Evans et 

### 33

al., 2006 )

. The measured uptake constant for Pb in this study was 7.8 g/dry weight

· day and the 

### 34

elimination rate constant of 0.15 d - 1 for Pb

- exposed larvae was similar in both presence and absence of 

### 35

the metal in the water

. Caddisflies accumulated significant amounts of the metal over 18 days of 

### 36May 2011 7

- 67 DRAFT

– DO NOT C ITE OR QUOTE exposure. Measured tissue concentrations ranged from approximately 15 to 35 μg Pb/g

. Hydropsychid Pb 

### 1

BCF ’ s ranged from 41 to 65, and averaged 54, indicating a rela tively high accumulation rate when 

### 2
compared to other metals tested (average BCF of 17 for Cd, 7.7 for Cu, and 6.3 for Zn) ( Evans et al., 

### 3

2006 )

. In larvae of the mosquito, Culex quinquefasciatus , exposed to 100 μg Pb /L for seven days the BCF 

### 4

was 62 ( Kitvatanachai et al., 2005 )

. 

### 5
In a comparison of dietary and waterborne exposure as sources of Pb to aquatic invertebrates, no 

### 6
correlation between Pb uptake and dietary exposure was observed in the amphipod H. azteca ( Borgmann 

### 7

et al., 2007 )

. Metian et al. ( 2009 ) investigated the uptake and bioaccumulation of 210 Pb in Chlamys varia 

### 8
(variegated scallop) and king scallop to determine the major accumulation route (seawater or food) and 

### 9

then asse ss subsequent tissue distribution

. Dietary Pb from phytoplankton in the diet was poorly 

### 10

assim i lated (<20%) while more than 70% of Pb in seawater was retained in the tissues

. In seawater, 210 Pb 

### 11
was accumulated more rapidly in C. varia than P. maximus and so ft tissue distribution patterns differed 

### 12
between the species. C. varia accumulated Pb preferentially in the digestive gland (50%) while in P. 

### 13

maximus , Pb was equally distributed in the digestive gland, kidneys, gills, gonad, mantle, intestine

, and 

### 14

adductor muscle with each tissue representing 12

- 30% of 210 Pb body load

. An additional test with Pb



### 15
spiked sediment with P. maximus showed low bioaccumulation efficiency of Pb from sediment. 

### 16
With the exception of the above

- mentioned study with scallops ( Metian et al., 2009 ) recent reports 

### 17
on Pb distribution generally su pport s the findings of the 2006 Pb AQCD that Pb is primarily sequestered 

### 18
in the gills, hepatopancreas

, and muscle

. Uptake of Pb by the crayfish ( Cherax destructor ) exposed to 

### 19
5,000 μg Pb /L for 21 days resulted in accumulation at the highest concentration i n gill, followed by 

### 20

exoskeleton < mid

- gut gland < muscle < hemolymph ( Morris et al., 2005 )

. The gills were the main sites 

### 21

of Pb accumulation in Pinctada fucata (pearl oyster) followed by mantle, in 72

- hour exposures to 103.5 

### 22

μg Pb /L ( Jing et al., 2007 )

. Following a 10 day exposure to 2,500 μg Pb /L as Pb nitrate, accumulation of 

### 23
Pb was higher in gill than digestive gland of Mytilus edulis : after a 10 day depuration, Pb content was 

### 24
decreased in the gills and digestive gland of these mussels ( Einsporn et al., 2009 )

. In blue crabs, 

### 25

Calli nectes sapidus , collected from a contaminated and a clean estuary in New Jersey, U.S., the 

### 26
hepatopancreas was found to be the primary organ for Pb uptake ( Reichmuth et al., 2010 )

. Body burden 

### 27
analysis following 96 hour exposure to 50, 100 and 500 μg Pb /L in the freshwater snail Biomphalaria 

### 28
glabrata indicated that bioaccumulation increased with increasing concentrations of Pb and the highest 

### 29

l evels were detected in the digestive gland ( Ansaldo et al., 2006 )

. 

### 30

There is more information now on the cel lular and subcellular distribution of Pb in invertebrates 

### 31
than there was at the time of writing the 2006 Pb AQCD. Specifically, localization of Pb at the 

### 32
ultrastructural level has been assessed in the marine mussel ( M. edulis ) through an antibody

- based 

### 33

det ection method ( Einsporn et al., 2009 ; Einsporn & Koehler, 2008 ) . Dissolved Pb was detected mainly 

### 34
within specific lysosomal structures in gill epithelial cells and digestive gland cells and was also localized 

### 35
in nuclei and mitochondria. Transport of Pb is thought to be via lysosomal granules associated with 

### 36May 2011 7

- 68 DRAFT

– DO NOT C ITE OR QUOTE hemocytes ( Einsporn et al., 2009 ) . In the digestive gland of the vari e gated scallop, Pb was also mainly 

### 1
bound to organelles, i.e., 66% of the total metal burden ( Bustamant e & Miramand, 2005 )

. In the digestive 

### 2
gland of the cephalopod Sephia officinalis , (cuttlefish) most of the Pb was found in the organelles (62%) 

### 3

( Bustamante et al., 2006 ) . In contrast, only 7% of Pb in the digestive gland of the octopus ( Octopus 

### 4
vulgaris ) was associated with the fraction containing nuclei, mitochondria, lysosome and microsomes: the 

### 5
majority of Pb in this species was found in cytosolic proteins ( Raimundo et al., 2008 )

. 

### 6

Since the publication of the 2006 Pb AQCD

, additional factors have been consider ed that may 

### 7
affect Pb uptake in aquatic organisms

. Pb tissue concentrations fluctuated seasonally in mussels ( Mytilus 

### 8
galloprovincialis ) harvested near Istanbul, Turkey ( Ozden, 2008 ) . Tissue Pb concentrations were lowest 

### 9
during the summer months (average of 0.9 mg Pb/kg), followed by spring, autumn and winter (1. 3, 1.4, 

### 10
and 1.6 mg Pb/kg, respectively). The authors speculated that the slight seasonal differences indicate that 

### 11
bioavailability of the metal may be related to seasonal changes in surface water or sediment chemistry

. 

### 12
Additionally, alterations in growth o ver the year, as well as different rates of Ca uptake may have 

### 13
impacted Pb bioaccumulation rates. When the relationship between invertebrate habitat (epibenthic and 

### 14
benthic) and environmental Pb bioaccumulation was investigated, De Jonge et al. ( 2010 ) determined that 

### 15
different environmental fractions of Pb were responsible for invertebrate uptake and exposure

. Pb uptake 

### 16
by benthic invertebrate taxa w as not significantly correlated to AVS Pb levels, but rather to total sediment 

### 17

concentrations ( De Jonge et al., 2009 ) . Conversely, epibenthic invertebrate Pb body burdens were better 

### 18

correlated to AVS concentrations, rather than total Pb sediment concentrations ( De Jonge et al., 2010 )

. 

### 19
Reported BAF values for Pb in aquatic invertebrates from the 2006 Pb AQCD range d from 499 to 

### 20

3,670 [See Table AX7

- 2.3.2 ( U.S. EPA, 2006 ) ]. Since the publication of the 2006 Pb AQCD, additional 

### 21

BAF value s have been established for invertebrates in field studies which tend to be higher than BCF 

### 22

value s calculated in laboratory exposures ( Casas et al., 2008 ; Gagnon & Fisher, 1997 ) (Table 7

- 4 ). A 

### 23
complicating factor in establishing BAF value s is that laboratory studies usually assess uptake in water



### 24
only or sediment only exposures while field studies take into account dieta ry sources of Pb as well as 

### 25
waterborne Pb resulting in BAF value s that are frequently 100

- 1,000 times larger than BCF value s for the 

### 26
same metal and species ( DeForest et al., 2007 ) . Mean Pb levels in both predatory and grazing 

### 27
zooplanktonic species in El Niagra reservoir, (in Aguascalientes, Mexico ) were used to calculate BAF 

### 28

values ( Rubio

- Franchini et al., 2008 ) to assess biomagnification of Pb. The BAF of the predatory rotifer 

### 29
Asplanchna brig thwellii (BAF 49,300) was up to four times higher than the grazing cladocerans Daphnia 

### 30

similis (BAF 9,022) and Moina micrura (BAF 8,046)

. Limpet (Patella sp

. ) from the Lebanese Coast had 

### 31

Pb BAF values ranging from 2,500 to 6,000 and in the same field study mussel ( Brachidontes variabilis ) 

### 32

Pb BAF values ranged from 7,500

- 8,000 ( Nakhle et al., 2006 )

. 

### 33May 2011 7

- 69 DRAFT

– DO NOT C ITE OR QUOTE Table 7

- 4. Bioaccumulation factors for Pb in aquatic invertebrates

Species BAF Test conditions Reference
Hydropsyche sp

. ( caddisfly larvae ) 54 18 days, Pb laboratory exposure Evans et al. ( 2006 )

Culex Quinquefasciatus (mosquito) 62 7 days, Pb laboratory exposure Kitvatanachai et al. ( 2005 )

Daphnia similis (zooplankton) 9,022 Field

- collected Rubio

- Franchini et al. ( 2008 )
Moina micrura (zooplankton) 8,046 Field

- collected Rubio

- Franchini et al. ( 2008 ) Asplanchna brig th welli i (rotifer) 49,344 Field

- collected Rubio

- Franchini et al. ( 2008 ) Patella sp

. (limpet) 6,000 Field

- collected Nakhle et al. ( 2006 )
Brachidontes variabilis (mussel) 8,000 Field

- collected Nakhle et al. ( 2006 )

Recently, several studies have attempted to establish biodynamic exposure assessments for various 

### 1

contaminants

. In an in situ metal kinetics field study with the mussel M. galloprovincialis , simultaneous 

### 2
measurements of metal concentrations in water and suspended particles with mussel biometrics and 

### 3
physiological indices were conducted to establish uptake and excretion rates in the natural environment 

### 4

( Casas et al., 2008 )

. A mean log of 4.3 of the metal concentration in mussels (ng Pb /kg wet flesh 

### 5
weight)/metal concentration in water (ng Pb /L) was determined for Pb for M. galloprovincialis in this 

### 6
study based on the rate constants of uptake and efflux in a series of transplantation experiments between 

### 7

contaminated and clean environments

. Equil i brium concentrations of Pb in mussels leveled out at 

### 8
approximately 30 days with a concentration of 6.7 mg Pb /kg. 

### 9

7.3.3.3. Vertebrates

Uptake of Pb by vertebrates considered here includes data from fish species as well as a limited 

### 10
amount of new information on amphibians and aquatic mammals. In fish, Pb is taken up from water via 

### 11
the gills and from food via ingestion

. Amphibians and aquatic mammals are exposed to waterborne Pb 

### 12
primarily through dietary sources

. In the 2006 Pb AQCD, dietary Pb was recognized as a potentiall y 

### 13
significant source of exposure to all vertebrates since Pb adsorbed to food, particulate matter and 

### 14
sediment can be taken up by aquatic organisms. 

### 15
Since the 2006 Pb AQCD, tissue accumulation of Pb via gill and dietary uptake has been further 

### 16

characteriz ed in vertebrates

, and new techniques such as the use of stable isotopes have been applied to 

### 17

further elucidate bioaccumulation of Pb

. For example, patterns of uptake and subsequent excretion of Pb 

### 18
in fish as measured by isotopic ratios of Pb in each tissu e can determine whether exposure was due to 

### 19
relatively long term sources (which favor accumulation in bone) or short term sources (which favors 

### 20
accumulation in liver) ( Miller et al., 2005 )

. New information since the 2006 Pb AQCD on uptake of Pb by 

### 21

fish from water is reviewed below, followed by studies on dietary uptake as a route of Pb exposure

. Next, 

### 22
tissue accumulation patterns in fish species are reported with special consideration of the anterior intestine 

### 23
as a newly identified target of Pb from dietary e xposures

. New data on uptake studies in marine fishes are 

### 24May 2011 7

- 70 DRAFT

– DO NOT C ITE OR QUOTE presented followed by new evidence for additional Pb detoxification mechanisms in fish. Finally, new 

### 1
data on uptake and tissue distribution of Pb in amphibians and aquatic mammals are presented. 

### 2

Fr eshwater Fish

Pb uptake in freshwater fish is accomplished largely via direct uptake of dissolved Pb from the 

### 3
water column through gill surfaces and by ingestion of Pb

- contaminated diets

. According to the data 

### 4
presented in the 2006 Pb AQCD, accumulation ra tes of Pb are influenced by both environmental factors, 

### 5
such as water pH, DOC, and Ca concentrations, and by species

- dependent factors, such as metabolism, 

### 6
sequestration, and elimination capacities

. The effects of these variables on Pb bioaccumulation in f ish are 

### 7
largely identical to the effects observed for invertebrates (discussed above)

. 

### 8

Since the publication of the 2006 Pb AQCD, multiple studies on uptake of Pb from water by 

### 9
fathead minnow have been conducted. Spokas et al. ( 2006 ) showed that Pb accumulates to the highest 

### 10
concentration in gill when compared to other tissues ove r a 24 day exposure

. This pattern was also 

### 11
observed in larval fathead minnows exposed to 26 μg Pb /L for 10

- 30 days, where gill exhibited the 

### 12
highest Pb concentration compared to carcass, intestine, muscle and liver ( Grosell et al., 2006a )

. In the 

### 13
larval minnows, Pb concentration in the intestine exhibited the highest initial accumulation of all tissues 

### 14
on day 3 but then decreased for the remainder of the experiment while con centrations in the other organs 

### 15

continued to increase

. By day 30, gill tissue exhibited the highest Pb concentration (approximately 120 μg 

### 16

Pb /g), followed by whole fish and carcass (whole fish minus gill, liver, muscle and intestine) Pb 

### 17
concentrations (app roximately 70 to 80 μg Pb /g)

. However, in considering overall internal Pb body 

### 18
burden, nearly 80% was largely concentrated in the bone tissue, while gill contributed <5%. 

### 19
In another study with fathead minnow, chronic (300 day) exposure to 120 μg Pb /L resulted in 

### 20
accumulation of approximately 200 nmol Pb/g tissue, although this number was decreased from initial 

### 21
body burdens of greater than 500 nmol Pb/g at test initiation ( Mager et al., 2010 )

. Tissue distribution at 

### 22
300 days was consistent with Grosell et al. ( 2006a ) with highest concentration in gill, followed by kidney, 

### 23

anterior intestine, an d carcass. Addition of humic acid and carbonate both independently reduced uptake 

### 24
of Pb in these fish over the exposure time period

. Interestingly, fathead minnow eggs collected daily 

### 25
during 21 day breeding assays that followed the chronic exposure describ ed above accumulated similar 

### 26
levels of Pb from the test solutions regardless of Pb concentration or water chemistry (e.g., addition of 

### 27
humic acid and carbonate) ( Mager et al., 2010 ) . Direct acute exposure from water rather than parental 

### 28
transfer accounted for the majority of the Pb accumulation in eggs. Similarly, exposure of fish to 157 nM 

### 29
Pb in base water for 150 days resulted in fathead minnow whole body c oncentrations of approximately 

### 30
150 nmol Pb/g tissue, with the most rapid accumulation rate occurring within the first 10 days of 

### 31
exposure, followed by an extended period of equilibrium ( Mager et al., 2008 )

. In this same study, fish 

### 32
were tested in two additional treatments: 177 nM Pb in hard water (Ca 2+ 500 μM) or 187 nM Pb in humic 

### 33May 2011 7

- 71 DRAFT

– DO NOT C ITE OR QUOTE acid supplemented water (4 mg/L). While the addition of humic acid significantly reduced Pb 

### 1
bioaccumulation in minnows (to approximately 50 nmol Pb/g on a whole body basis), Ca sulfate did not 

### 2
alter uptake. Despite the fact that Ca

- mediated Pb toxicity occurred in larval fathead minnow, there wa s 

### 3
no concurrent effect on whole body Pb accumulation

. 

### 4
Uptake studies in other teleosts of Pb from freshwater have generally followed the pattern of 

### 5
uptake described above for fathead minnow

. In the cichlid, Nile tilapia ( Oreochromis niloticus ) Pb 

### 6

accumula ted significantly in gill (45.9 +34.4 μg/g dry weight at 10 μ M, 57.4 +26.1 μg/g dry weight at 20 

### 7

μ M) and liver (14.3 μg/g dry weight at 10 μM and 10.2 μg/g dry weight at 20 μM) during a 14

- day 

### 8

exposure ( Atli & Canli, 2008 )

. In rainbow trout exposed to 100 μg Pb /L for 72 hours, the accumulation in 

### 9
tissues was gill>kidney>liver and this same pattern was observed in all concentrations tested (100

- 10,000 

### 10

μg Pb /L) ( Suicmez et al., 2006 ) . Sloman et al. ( 2005 ) investigated the u ptake of Pb in dominant



### 11
subordinate pairings of rainbow trout exposed to 46 μg/L or 325 μg/L Pb

- nitrate for 48 hours

. Significant 

### 12
Pb accumulation in gill, liver and kidney was only observed in the highest concentration

. Pb accumulated 

### 13
preferentially in liv er of subordinate trout when compared to dominant trout

. Brown trout ( Salmo trutta ) 

### 14
exposed to aqueous Pb concentrations ranging from 15 to 46 μg Pb /L for 24 days accumulated 6 μg Pb/g 

### 15
dry weight in gill tissue and Pb concentrations in liver tissue reached 14 μg Pb /g dry weight

. Interestingly, 

### 16
Pb in gill tissue peaked on day 11 and decreased thereafter, while liver Pb concentrations increased 

### 17
steadily over the exposure period, which may indicate translocation of Pb in brown trout from gill to liver 

### 18

( Heier et al., 2009 )

. 

### 19
Zebrafish ( Danio rerio ) Pb uptake rates from media containing 0.025 μmol Pb was significantly 

### 20
increased by neutral pH (versus a pH of 6 or 8) and by Ca concentrations of 0. 5 mmol; uptake rate of Pb 

### 21
was increased from 10 L/kg

· h to 35 L/kg

· h by increasing pH from 6 to 7, and from 20 L/kg

· h to 35 L/kg

· h 

### 22
by increasing Ca concentration from 0.1 mmol to 0.5 mmol ( Komjarova & Blust, 2009c ) . This study also 

### 23
demonstrated that zebrafish gill tissue is the main uptake site for the metal, as Pb concentrations in these 

### 24
tissues were up to eight time s as high as that in other tissues

. 

### 25
The Eurasian silver crucian carp ( Carrasius auratus ) collected from a pond containing an average 

### 26
of 1,600 mg Pb /kg in the sediments exhibited increased Pb body burdens ranging from 12 to 68 mg Pb /kg 

### 27
dry weight ( Khozhina & Sherriff, 2008 )

. Pb was primarily sequestered in skin, gill, and bone tissues, but 

### 28
was also detected at elevated levels in muscle and liver tissues, as well as in eggs

. Two fish species 

### 29

( Labeo rohita and Ctenopharyngodon idella ) collected from the Upper Lake of Bhopal, India with 

### 30
average Pb concentration of 0.03 mg Pb/L in the water column contained elevated Pb tissue 

### 31

concentrations ( Malik et al., 2010 )

. However, while liver and kidney Pb concentrations were similar 

### 32
between the two species (1.5 and 1.1 μg Pb /g tissue and 1.3 and 1.0 μg Pb /g tissue for C. idella and L. 

### 33

rohita , respectively), they accumulated significantly different amounts of Pb in gill and muscle tissues

. C. 

### 34

idella accumulated more than twice the Pb in these tissues (1.6 and 1.3 μg Pb /g) than did L. rohita (0. 5 

### 35
and 0.4 μg Pb /g). 

### 36May 2011 7

- 72 DRAFT

– DO NOT C ITE OR QUOTE The studies reviewed above generally support the conclusions of the 2006 Pb AQCD that the gill is 

### 1
a major site of Pb uptake in fish and that there are species

- dependent differences in the rate and pattern of 

### 2

Pb accumulation. As indicat ed in the 2006 Pb AQCD, exposure duration can be a factor in Pb uptake from 

### 3
water. In a 30 day exposure study, Nile tilapia fingerlings had a three

- fold increase in Pb uptake at the gill 

### 4
on day 30 compared to Pb concentration in gill at day 10 and 20 ( Kamaruzzaman et al., 2010 ) . In addition 

### 5
to uptake at the gill, a tim e

- dependent uptake of Pb into kidney in rainbow trout exposed to 570 μg Pb /L 

### 6

for 96 hours ( Patel et al., 2006 ) was observed. Pb was accumulated preferentia lly in the posterior kidney 

### 7
compared to the anterior kidney

. A similar pattern was observed by Alves and Wood ( 2006 ) in a dietary 

### 8
exposure. In catla ( Catla catla ) fingerlings, the accumulation pattern of Pb was k idney > liver > gill > 

### 9
brain > muscle in both 14 day and 60 day Pb exposures ( Palaniappan et al., 2009 ) . In multiple studies 

### 10

with fat head minnow at different exposure durations, tissue uptake patterns were similar at 30 days 

### 11

( Grosell et al., 2006a ) and 300 days ( Mager et al., 2010 ) . In the larval minnows, Pb concentration in the 

### 12
intestine exhibited the highest initial accumulation of all tissues on day 3 but then decreased for the 

### 13
remainder of the experimen t while concentrations in the other organs continued to increase ( Grosell et al., 

### 14

2006a ) . By day 30, gill tissue exhibited the highest Pb concentration followed by whole fi sh and carcass 

### 15
(whole fish minus gill, liver, muscle and intestine)

. The most rapid rate of Pb accumulation in this species 

### 16
occurs within the first 10 days of exposure ( Mager et al., 2008 )

. African catfish ( Clarias gariepinus ) 

### 17
exposed to aqueous Pb concentrations of 50 to 1,000 μg Pb /L (as Pb nitrate) for 4 weeks accumulated 

### 18
significant amounts of Pb in heart (520

- 600 mg Pb /kg), liver (150

- 242 mg Pb /kg), and brain (120

- 230 mg 

### 19

Pb /kg) tissues ( Kudirat, 2008 ) . Doublin g the exposure time to 8 weeks increased sequestration of Pb in 

### 20
these tissues as well as in skin (125

- 137.5 mg Pb /kg) and ovaries (30

- 60 mg Pb /kg). 

### 21

Since the publication of the 2006 Pb AQCD, several studies have focused on dietary uptake of Pb 

### 22

in teleosts

. Alves et al. ( 2006 ) administered a diet of three concentrations of Pb (7, 77 and 520 μg Pb/g 

### 23
dry weight) to rainbow trout for 21 days. Doses were calculated to be 0.02 μg Pb /day (control), 3.7 

### 24

μ g Pb /day (low concentration), 39.6 μg Pb /day (intermediate concentration) and 221 .5 μg Pb /day (high 

### 25
concentration). Concentrations in the study were selected to represent environmentally relevant 

### 26
concentrations in prey. After 21 days exposure to the highest concentration, Pb accumulation was greatest 

### 27
in the intestine, followed by carca ss, kidney and liver leading the authors to hypothesize that the intestine 

### 28
is the primary site of exposure in dietary uptake of Pb. All tissues, (gill, liver, kidney, intestine, carcass) 

### 29
sequestered Pb in a dose

- dependent manner. The gills had the greatest concentration of Pb on day 7(8.0 

### 30
μg Pb/g tissue wet weight) and this accumulation decreased to 2.2 μg Pb/g tissue wet weight by the end of 

### 31
the experiment suggesting that the Pb was excreted or redistributed ( Alves et al., 2006 ) . Furthermore, 

### 32
with increasing dietary concentrations, the percentage of Pb retained in the fish decreased

. Additionally, 

### 33
in this study red blood cells were identified as a reservoir for dietary Pb. Plasma did not accumulate 

### 34
significant Pb (0.012 μg Pb g wet weight in the high dose), however, Pb was elevated in blood cells (1.5 

### 35
μg Pb g wet w eight in the high dose) ( Alves et al., 2006 )

. 

### 36May 2011 7

- 73 DRAFT

– DO NOT C ITE OR QUOTE Additional st udies have supported the anterior intestine as a target for Pb in fish

. Nile tilapia 

### 1
exposed to dietary Pb for 60 days (100, 400, and 800 μ g Pb /g dry weight) accumulated the greatest 

### 2
concentration of Pb in the intestine, followed by the stomach and then th e liver ( Dai, D u, et al., 2009 )

. 

### 3
The amount of Pb in tissue increased with increasing dietary Pb concentration. In a 42 day chronic study 

### 4
of dietary uptake in rainbow trout, fish fed 50 or 500 μg Pb/g, accumulated Pb preferentially in anterior 

### 5

intestine ( Alves & Wood, 2006 ) . Pb accumulation in the gut was followed by bone, kidney, liver, spleen, 

### 6
gill, carcass, brain and white muscle ( Alve s & Wood, 2006 ) . Ojo and Wood ( 2007 ) investigated the 

### 7
bioavailability of ingested Pb within different compartments of the rainbow trout gut using an in vitro gut 

### 8
sac technique

. Although a significant incre ase in Pb uptake was observed in the mid

- intestines, this was 

### 9
determined to be much lower than Pb uptake rates via gill surfaces

. However, given that intestinal uptake 

### 10
rate for Pb did not significantly differ from those derived for essential metals (e.g., Cu, Zn, and Ni), this 

### 11
uptake route is likely to be significant when aqueous Pb concentrations are low and absorption via gill 

### 12
surfaces is negligible ( Ojo & Wood, 2007 )

. 

### 13
Following a chronic 63

- day dietary exposure to Pb, male zebrafish had significantly increased Pb 

### 14
body burdens, but did not exhibit any significant impairment when compared with controls. Fish were fed 

### 15
diets consisting of field

- collected Nereis diversicolor oligochaetes that contained 1.7 or 33 mg Pb /kg dry 

### 16
weight

. This resulted in a daily Pb dose of either 0.1 or 0.4 mg Pb /kg ( Boyle et al., 2010 ) . At the end of 

### 17
the exposure period, tissue from male fish reared on the high

- Pb diet contained approximately 0.6 mg 

### 18
Pb/kg wet weight, as compared with approximately 0.48 mg Pb/kg wet weight in the low

- Pb dietary 

### 19

exp osure group. Pb level was elevated in female fish fed the high

- Pb diet, but not significantly so. 

### 20

Ciardullo et al. ( 2008 ) examined bioaccumulation of Pb in rainbow trout tissues following a 3

- year 

### 21

c hronic dietary exposure to the metal

. Diet was determined to contain 0.19 μg Pb/g wet weight

. Fish skin 

### 22
accumulated the greatest Pb concentrations (0.02 to 0.05 μg Pb /g wet weight), followed by kidney, gills, 

### 23

liver, and muscle

. Pb accumulation in muscles ( 5 ng Pb /g) remained constant over all sampled growth 

### 24

stages ( Ciardullo et al., 2008 )

. The authors concluded that dietary Pb was poorly absorbed by rainbow 

### 25

trout

. Compariso n of dietary and water

- borne exposures suggest that although accumulation of Pb can 

### 26
occur internally from dietary sources, toxicity does not correlate with dietary exposure, but does correlate 

### 27
with gill accumulation from waterborne exposure ( Alves et al., 2006 )

. Comparison of uptake rates across 

### 28
the gut and gill h ave shown that transporter pathways in the gill have a much higher affinity for Pb than 

### 29
do similar pathways in the gut ( Ojo & Wood, 2007 )

. 

### 30
Since the 2006 Pb AQCD, several field studies hav e considered Pb uptake and bioaccumulation in 

### 31
fish as a tool for environmental assessment. Pb tissue concentrations were elevated in several species of 

### 32
fish exposed in the field to Pb from historical mining waste, and blood Pb concentrations were highly 

### 33

co rrelated with elevated tissue concentrations, suggesting that blood sampling may be a useful and 

### 34
potentially non

- lethal monitoring technique ( Brumbaugh et al., 2005 ) . The Western Airborne 

### 35
Contaminants Assessment Projec t assessed concentrations of semi

- volatile organic compounds and metals 

### 36May 2011 7

- 74 DRAFT

– DO NOT C ITE OR QUOTE in up to seven ecosystem components (air, snow, water, sediment, lichen, conifer needles and fish) in 

### 1
watersheds of eight core national parks during a multi

- year project co n ducted from 2002

- 2007 ( Landers et 

### 2

al., 2008 ) . The goals of t he study were to assess where these contaminants were accumulating in remote 

### 3
ecosystems in the Western U

. S

. , identify ecological receptors for the pollutants, and to determine the 

### 4
source of the air masses most likely to have transported the contaminants to the parks. Results from this 

### 5
study are considered in in Chapter 3 of this ISA

. 

### 6

Marine Fish

In comparison to freshwater fish, fewer studies have been conducted on Pb uptake in marine fish

. 

### 7

Since marine fish drink seawater to maintain osmotic homeostasis, Pb can be taken up via gills and 

### 8

intestine ( W. X. Wang & Rainbow, 2008 )

. Pb was significantly accumulated in gill, liver, plasma, kidney, 

### 9
rectal gland, intestine, skin, muscle of a marine shark species, spotted dogfish ( Scyliorhinus canicula ) 

### 10

exp osed to 2

, 072 μg Pb /L for one week ( De Boeck et al., 2010 ) . Egg cases of th e spotted dogfish exposed 

### 11

to 210 Pb in seawater for 21 days, accumulated radiolabeled Pb rapidly and the metal was subsequently 

### 12
detected in embryos indicating the permeability of shark eggs to Pb in coastal environments ( Jeffree et al., 

### 13

2008 )

. 

### 14

The 20 06 Pb AQCD considered detoxification mechanisms in fish including mucus production and 

### 15
Pb removal by scales through chelation with keratin

. Since the 2006 review, additional Pb detoxification 

### 16

mechanisms in marine fish have been further elucidated. Mummicho g ( Fundulus heteroclitus ) 

### 17
populations in metal

- polluted salt marshes in New York exhibited different patterns of intracellular 

### 18
partitioning of Pb although body burden between sites was not significantly different ( Goto & Wallace, 

### 19

2010 ) . Mummichogs at more polluted sites stored a higher amount of Pb in metal rich granules as 

### 20
compared to other detoxifying cellular components such as heat

- stable proteins, heat

- denaturable proteins 

### 21
and organelles. 

### 22
A study of Pb bioaccumulation in five marine fish species ( Chloroscombrus chrysurus

, Sardinella 

### 23

aurita

, Ilisha africana

, Galeoides decadactylus

, Caranx latus ) found that C. chrysurus was an especially 

### 24
strong bioaccumulator, yielding Pb concentrations of 6 to 10 mg Pb /kg ( Gnandi et al., 2006 ) . However, C

. 

### 25
chrysurus metal content was not correlated to the Pb concentrations along the mine tailings gradient from 

### 26
which they were collected (8.5 and 9 .0 μg Pb /L for minimum and maximum tissue con centrations, 

### 27
respectively). This lack of correlation was also observed for fish species that were considered to be 

### 28
weaker Pb bioaccumulators, indicating that diffuse, non

- quantified sources of Pb (e.g., in sediments or in 

### 29
dietary sources) may be contributi ng to Pb uptake by marine fish. 

### 30
This review of the recent literature indicates that the primary and most efficient mode of Pb 

### 31
absorption for freshwater fish is assimilation of labile Pb via gill surfaces; recent research indicates that 

### 32
chronic dietary Pb e xposure may result in some Pb bioaccumulation although it is not the predominant 

### 33May 2011 7

- 75 DRAFT

– DO NOT C ITE OR QUOTE route of exposure. Nevertheless, if benthic invertebrates comprise a large portion of fish diets in 

### 1
chronically contaminated systems, assimilated Pb loads may be significant

. This was demonstrated by 

### 2
Boyle et al. ( 2010 ) , who showed that laboratory diets consisting of less than one third field

- collected Pb



### 3

contaminated invertebrates were sufficient to raise fish tissue Pb levels

. However, data from field sites 

### 4
suggest that fish accumulation of Pb from dietary sources is highly variable and may be strongly 

### 5
dependent on the physiology of individua l species and absorption capacities. 

### 6
Reported BCF ’ s in fish species from the 2006 Pb AQCD were 42 for brook trout ( Salvelinus 

### 7
fontinalis ) and 45 for bluegill ( Lepomis macrochirus ). Since the 2006 Pb AQCD, additional BAF ’ s have 

### 8

been established in water

- onl y exposures, from dietary exposures and from field

- collected fish (Table 7

- 

### 9

5 )

. 

### 10

Table 7

- 5. Bioaccumulation factors for Pb in fish

Species BAF Test conditions Reference
Clarias gariepinus 800 56 days, Pb nitrate Kudirat ( 2008 ) Ctenopharyngodon idella 44 Field

- collected Malik et al. ( 2010 ) Pimephales promelas 100 to 100,000 30 days, Pb nitrate Grosell et al. ( 2006a )
Carassius auratu s 0.04 Field

- collected Khozhina and Sherriff ( 2008 )
Danio rerio 1.4 63 days, dietary exposure Boyle et al. ( 2010 )

Amphibians

Since the 2006 Pb AQCD, there are a few new studies that consider uptake of Pb in amphibians. In 

### 11
a chronic study with tadpoles of the Northern Leopard frog ( Rana pipiens ), Pb tissue concentrations were 

### 12
evaluated following exposures to 3, 10, and 100 μg Pb /L from embryo to metamorphosis

. The tadpole 

### 13
tissue concentrations ranged from 0.1 to 224.5 mg Pb /kg dry mass and were positively correlated to Pb 

### 14

concentrations in the water ( T. H. Chen et al., 2006 )

. Dose

- dependent bioaccumulation of Pb was 

### 15
observed in the livers of tadpoles of the African clawed frog ( Xenopus laevis ) exposed to concentrations 

### 16
ranging from 0.001 to 30 mg Pb /L (2.91 to 114.5 Pb/g wet weight) for 12 days ( Mouchet et al., 2007 )

. Pb 

### 17

concentrati ons were measured in livers, bodies without liver and whole bodies in Southern leopard frog 

### 18

( Rana spenocephala ) tadpoles exposed to Pb in sediment (45 to 7,580 mg Pb /kg dry weight) with 

### 19
corresponding pore water concentrations of 123 to 24,427 μg Pb /L from embryonic stage to 

### 20
metamorphosis ( Sparling et al., 2006 )

. There was 100% mortality at 3

, 940 mg Pb /kg and higher. In all 

### 21
body residues analyzed there was a significant positive correlation between Pb in sediment and Pb in 

### 22

sediment pore water

. Concentrations of Pb in liver were similar to results with whole body and bodies 

### 23
without liver indicating that Pb is not preferentially sequestered in liver. 

### 24May 2011 7

- 76 DRAFT

– DO NOT C ITE OR QUOTE Mammals

Studies that consider uptake of Pb in aquatic mammals are limited. Kannan et al. ( 2006 ) in a 

### 1
comparison of trace element concentrations in livers of free

- ranging sea otters ( Enhydra lutris nereis ) 

### 2
found dead along the California coast, detected Pb in all individuals sampled (N=80) in a rang e of 0.019 

### 3
to 1.06 μg Pb /g

. The otters were classified by cause of death (infectious causes, non

- infectious causes, 

### 4
those that died in an emaciated condition) and trace element patterns of tissue distribution were compared. 

### 5

Livers from emaciated otters had significantly elevated levels of Pb compared to non

- diseased 

### 6

individuals. 

### 7

7.3.3.4. Food Web

At the time of the publication of the 2006 Pb AQCD, trophic transfer of Pb through aquatic food 

### 8
chains was considered to be negligible ( U.S. EPA, 2006 ) . Measured concentra tions of Pb in the tissues of 

### 9
aquatic organisms were found to be generally higher in algae and benthic organisms and lower in higher 

### 10
trophic

- level consumers indicating that Pb was bioconcentrated but not biomagnified ( Eisler, 2000 ; U.S. 

### 11

EPA, 2006 ) . New literature since the 2006 Pb AQCD provides evi dence of the potential for Pb to be 

### 12
transferred in aquatic food webs while other studies indicate Pb is decreased with increasing trophic level. 

### 13
This section incorporates recent literature on transfer of Pb through aquatic food chains including the 

### 14
applica tion of stable isotope techniques to trace the accumulation and dilution of metals through 

### 15
producers and consumers. 

### 16
Pb was transferred through at least one trophic level in El Niagra reservoir, Aguascalientes, 

### 17
Mexico, an ecosystem that lacks fishes ( Rubio

- Franchini et al., 2008 )

. Pb was measured in sediment, 

### 18
water, and zooplankton sampl es of this freshwater system. BCF ’ s were calculated for predatory and 

### 19
grazing zooplanktonic species

. The BCF of the rotifer A. brigthwellii (BCF 49,300) was up to four times 

### 20
higher than the grazing cladocerans D. similis (BCF 9,022) and M. micrura (BCF 8,0 46)

. According to 

### 21

the authors, since M. micrura are prey for A. brigthwellii this may explain the biomagnifications of Pb 

### 22
observed in the predatory rotifer and provides evidence that Pb biomagnifies at intermediate trophic 

### 23

levels

. Partial evidence for biom agnification was observed in a subtropical lagoon in Mexico with 

### 24

increases of Pb concentration oc c urring in 14 of the 31 (45.2%) of trophic interactions considered 

### 25

( Ruelas

- Inzunza & Páez

- Osuna, 2008 ) . The highest rate of transference of Pb as measured in muscle 

### 26
tissue occurred between the prey species white shrimp ( Litopenaeus vannimei ) and mullet ( Mugil 

### 27
cephalus ) to pelican ( Pelecanus occidentalis ). 

### 28
The relative contribution of water and food as source of trace metals including Pb was investigated 

### 29

in the larva e of the alderfly Sialis velata ( Croisetiere et al., 2006 )

. Its prey , the midge ( C. riparius ) was 

### 30
reared in the laboratory and then exposed to trace elements in a metal

- contaminated lake for one week 

### 31
prior to being fed to S. velata

. During the one

- week exposure period of C. riparius to the contaminated 

### 32May 2011 7

- 77 DRAFT

– DO NOT C ITE OR QUOTE water, five of six t race elements, including Pb, reached steady state within C. riparius

. Alderfly larvae 

### 1

were held in the lab in uncontaminated lake water and feed one of the treated C. riparius per day for up to 

### 2
six days to measure Pb uptake via prey. A separate group of al derfly larvae were exposed directly to the 

### 3
contaminated lake water for six days and fed uncontaminated C. riparius while a third group was exposed 

### 4
to Pb via prey and water. Trace metal concentrations in S. velata that consumed contaminated C. riparius 

### 5

incr eased significantly compared to S. velata in water

- only exposures. Food was concluded to be the 

### 6
primary source of Pb (94%) to these organisms, not Pb in the water. 

### 7
The trophic transfer of Pb from the sediment dwelling polychaete worm N. diversicolor to the 

### 8
invertebrate polychaete predator Neris virens provides additional evidence for assimilation of Pb by a 

### 9
predator and the potential for further transport up the food chain ( Rainbow et al., 2006 )

. N. virens 

### 10
significantly accumulated Pb from a diet of N. diversicolor and there was a significant inverse linear 

### 11

relation ship between the trophic transfer coefficient and prey Pb concentration

. In the same study, another 

### 12
predator, the decapod Palaemonetes varians , did not significantly accumulate Pb from N. diversicolor 

### 13
indicating that trophic transfer is dependent on specie s

- specific differences in metal assimilation 

### 14
efficiencies and accumulation patterns. 

### 15
In a recent dietary metal study, field

- collected invertebrates representing ecologically relevant 

### 16
sources of Pb were fed to zebrafish, to assess bioavailability of this m etal via food

. The polychaete worm 

### 17

N. diversicolor was collected from two sites; an estuary contaminated with Pb and a reference site with 

### 18

low metal concentrations ( Boyle et al., 2010 )

. Male zebrafish fed Pb

- enriched N. diversicolor had 

### 19
significant increases in whole

- body Pb burden when compared to zebrafish fed prey from the reference 

### 20
site, brine shrimp or flake food diets. There was a trend toward increased Pb levels in females under the 

### 21
same dietary regimen

. In this study, deposit feeding invertebrates were shown to mobilize sediment



### 22

bound metals in the fo od chain since zebrafish were exposed only to biologically incorporated metal. 

### 23
The concentration of Pb in the tissues of various aquatic organisms was measured during the 

### 24
biomonitoring of mining

- impacted stream systems in Missouri, U.S. Generally, Pb concentrations 

### 25
decreased with increasing trophic level: detritus contained 20 to 60 μg Pb /g dry weight, while periphyton 

### 26
and algae contained 1 to 30 μg Pb /g dry weight ; i nvertebrates and fish collected from the same areas 

### 27

exhibited Pb tissue concentrations of 0.1 to 8 μg Pb /g dry weight ( J. M. Besser et a l., 2007 )

. In addition, 

### 28
Pb concentrations in invertebrates (snails, crayfish, and other benthos) were negatively correlated with Pb 

### 29
concentrations in detritus, periphyton, and algae

. Fish tissue concentrations, however, were consistently 

### 30
correlated only with detritus Pb concentrations ( J. M. Besser et al., 2007 )

. 

### 31
Other studies have traced Pb in aquatic food webs and have found no evidence of biomagnification 

### 32
of Pb with increasing trophic level

. Pb exposure at the base of the food web did not biomagnify in a 

### 33
simplified

- four level marine food chain fro m Tetraselmis suecica (phytoplankton) to Artemia franciscana 

### 34
(crustacean, brine shrimp) then L. vannamei (crustacean white shrimp) and finally to Haemulon scudderi 

### 35
(fish, grunt) ( Soto

- Jiménez et al. ) . In the southeastern Gulf of California, Mexico, Pb was not positively 

### 36May 2011 7

- 78 DRAFT

– DO NOT C ITE OR QUOTE transferred (biomagnification factor <1) through primary producers (seston, detritus) and 14 consumer 

### 1
species in a lagoon food web ( Jara

- Marini et al., 2009 )

. No biomagnification of Pb was detected from 

### 2
mesozooplankton to macrozooplankton in Bahia Blanca estuary, Argentina ( Fernández S everini et al. ) . In 

### 3
a Brazilian coastal lagoon food chain, Pb was significantly higher in invertebrates than in fishes ( Pereira 

### 4

et al., 2010 ) . Watanabe et al. ( 2008 ) also observed decreasing Pb concentrations through a stream 

### 5
macroinvertebrate food web in Japan from producers to primary and secondar y consumers. 

### 6
Introduction of exotic species into an aquatic food web may alter Pb concentrations at higher tropic 

### 7
levels. In Lake Erie, the invasive round goby ( Neogobius melanastomus ) and the introduced zebra mussel 

### 8

( Dreissena polymorpha ) have created a n ew benthic pathway for transfer of Pb and other metals 

### 9

( Southward Hogan et al., 2007 ) . The goby is a predator of the benthic zebra mussel, while the endemic 

### 10
smallmouth bass ( Micropterus dolomieui ) feed on goby

. Since the introduction of goby into the lake, total 

### 11

Pb concentrations have decreased in bass

. The authors attribute t his decrease of Pb in bass to changes in 

### 12
food web structure, changes in prey contaminant burden or declines in sediment Pb concentrations. 

### 13



## 7.3.4. Biological Effects

This section focuses on the studies of biological effects of Pb on aquatic biota including algae, 

### 14
aquatic plants, invertebrates, fish and other biota with an aquatic lifestage (e.g., amphibians) published 

### 15
since the 2006 Pb AQCD

. Waterborne Pb is highly toxic to aquatic organisms with toxicity varying 

### 16
depending upon the species and lifestage tested, du ration of exposure, the form of Pb tested, and water 

### 17
quality characteristics

. The 2006 Pb AQCD noted that the physiological effects of Pb in aquatic organisms 

### 18
can occur at the biochemical, cellular, and tissue levels of organization and include inhibition of heme 

### 19
formation, adverse effects to blood chemistry, and decreases in enzyme levels

. Functional growth 

### 20
responses resulting from Pb exposure include changes in growth patterns, gill binding affinities, and 

### 21
absorption rates

. A review of the more recent lit erature corroborated these findings, and added 

### 22
information about induction of oxidative stress by Pb, alterations in chlorophyll, and changes in 

### 23
production and storage of carbohydrates and proteins. Since this document focuses on atmospheric 

### 24

sources of Pb to ecosystem receptors, areas of research not addressed here include literature related to 

### 25
exposure to Pb from shot or pellets. Biological effects of Pb on algae and plants are considered below, 

### 26
followed by information on effects on aquatic invertebrates a nd vertebrates

. 

### 27

7.3.4.1. Plants and Algae

Effects of Pb on algae reported in the 2006 Pb AQCD included decreased growth, deformation and 

### 28
disintegration of algae cells, and blocking of the pathways that lead to pigment synthesis, thus affecting 

### 29
photosynthesis

. Obse rvations in addi ti onal algal species since the 2006 Pb AQCD support these findings

. 

### 30
Pb exposure in microalgae species has been linked to several adverse effects, including disruption of 

### 31May 2011 7

- 79 DRAFT

– DO NOT C ITE OR QUOTE thylakoid structure and inhibition of growth in both Scenedesmus quadr icauda and Anabeana flos

- aquae 

### 1

( Arunakumara & Zhang, 2008 )

. Arunakumara et al. ( 2008 ) determined the effect of aqueous Pb on the 

### 2
algal species S. platensis using solutions of Pb

- nitrate

. While low Pb exposures (5 μg Pb /mL) stimulated 

### 3

10

- day algal grow th, growth was inhibited at higher concentrations of 10, 30, 50, and 100 μg Pb /mL by 5, 

### 4
40, 49, and 78%, respectively

. In addition to growth inhibition, algal chlorophyll a and b content were 

### 5
significantly diminished at the three highest Pb exposures ( Arunakumara et al., 2008 )

. Although no 

### 6
specific morphological abnormalities were linked to Pb exposure, filamen t breakage was observed in S. 

### 7
platensis at Pb concentrations > 50 μg Pb /mL. The effect of Pb exposure on the structure and function of 

### 8
plant photosystem II was studied in giant duckweed, S. polyrrhiza ( Ling & Hong, 2009 ) . The Pb 

### 9
concentration of extracted photosystem II particles was found to increase with increasing environmental 

### 10
Pb concentration, and increased Pb concentration was shown to decrease emission peak intensity at 340 

### 11
nm, amino acid excitation peaks at 230 nm, tyrosine residues, and absorption intensities. This results in 

### 12
decreased efficiency of visible light absorption by affected plants. The authors theorized that Pb 2+ may 

### 13
replace either Mg 2+ or Ca 2+ in chlorophy ll or the oxygen

- evolving center, inhibiting photosystem II 

### 14
function through an alteration of chlorophyll structure. 

### 15
An increase in levels of antioxidant enzymes is commonly observed in aquatic plant, algae, and 

### 16
moss species exposed to Pb

. An aquatic moss, F. antipyretica , exhibited increased SOD and ascorbate 

### 17
levels following a 2

- day exposure to Pb

- chloride solutions of concentrations of 1, 10, 100, and 1000 

### 18
μmol. When exposure duration was increased to 7 days, only SOD activity remained significantly 

### 19

incr eased by Pb exposure ( Dazy et al., 2009 )

. Bell

- shaped concentration

- response curves were commonly 

### 20
observed for the induction of antioxidant enzymes in F. antipyretica . The chlorophyll, carotenoid, and 

### 21
protein contents of the aquatic macrophyte Elodea canadensis were significantly reduced following Pb 

### 22
accumulation at exposures of 1, 10, and 100 mg Pb /L ( Dogan et al., 2009 )

. This, along with the induction 

### 23
of some antioxidant systems and the reduction of growth at the highest two exposures, indicated that 

### 24
exposure to the met al caused significant stress, and that toxicity increased with exposure. In addition, 

### 25

native Myriophyllum quitense exhibited elevated antioxidant enzyme activity (glutathione

- S

- transferase, 

### 26
glutathione reductase, peroxidase) following transplantation in an thropogenically polluted areas 

### 27
containing elevated Pb concentrations. These were correlated with sediment Pb concentrations in the 

### 28
range of 5 to 23 mg Pb /g dry weight ( Nimptsch et al., 2005 )

. 

### 29
Toxicity and oxidative stress were also observed in coontail ( C. demersum ) rooted aquatic 

### 30
macrophytes following 7

- day exposures to aqueous Pb (1 to 100 μmol), with increasing effects observed 

### 31
with greater exposure concentrations and times. Chlorosis and leaf fragmentation were evident following 

### 32

a 7

- day exposure to the highest concentration, while induction of antioxidant enzymes (glutathione, 

### 33
superoxide dismutase, p eroxidases, and catalase) was observed at lower exposure concentrations and 

### 34
times. However, as the duration and concentration of Pb exposure was increased, activities of these 

### 35
antioxidant enzymes decreased ( S. Mishra et al., 2006 )

. 

### 36May 2011 7

- 80 DRAFT

– DO NOT C ITE OR QUOTE Sobrino et al. ( 2010 ) observed reductions in soluble starch stores and proteins with subsequent 

### 1
increases in free sugars and amino acids in Lemna gibba plants expose d to Pb (50 to 300 mg Pb /L); total 

### 2
phenols also increased with increasing Pb exposure. Authors noted that this species exhibited similar 

### 3
responses under extreme temperatures, drought, and disease ( Sobrino et al., 2010 )

. According to Odjegba 

### 4

and Fasidi ( 2006 ) , exposure to 0.3 mmol of Pb for 21 days was sufficient to induce a gradual reduction of 

### 5
both chlorophyl l and protein content in the macrophyte Eichhornia crassipes

. Decreased proteins were 

### 6
theorized to be related to inefficient protein formation following disruption of nitrogen metabolism after 

### 7
Pb exposure ( Odjegba & Fasidi, 2006 ) . Foliar proline (which is thought to act as an antioxidant) 

### 8

concentrations were found to increase in a concentration

- dependent manner as Pb concentrations increase 

### 9

from 0.1 to 5.0 mmol. 

### 10
Following 72

- hour aqueous exposure to 41 μmol Pb

- nitrate, phytochelatin and glutathione 

### 11
concentrations in the algae Scenedesmus vacuolatus were significantly increased over that of non

- exposed 

### 12
algal cultures {F, 2006, 358857} . The 72

- hour Pb exposure also significantly reduced S. vacuolatus 

### 13
growth, and of all the metals tested ( Cu, Zn, Ni, Pb, Ag, As, and Sb), Pb was determined to be the most 

### 14
toxic to the algae species. 

### 15
Pb exposure (as Pb

- nitrate) caused oxidative damage, growth inhibition, and decreased biochemical 

### 16
parameters, including photosynthetic pigments, proteins, and mo nosaccharides, in Wolffia arrhiza plants. 

### 17
Fresh weight of plants was reduced following both 7

- and 14

- day exposures to Pb concentrations greater 

### 18
than 10 mmol, while chlorophyll a content was decreased at concentrations greater than 1 mmol Pb 

### 19

( Piotrowska et al., 2010 )

. 

### 20
Root elongation was significantly reduced in a number of wetland plant species ( Beckmannia 

### 21
syzigachne

, Juncus effusus

, Oenanthe javanica

, Cyperus flabelliformis

, Cyperus malaccensis , and 

### 22

Ney raudia reynaudiana ) following Pb exposures of 20 mg Pb /L ( Deng et al., 2009 )

. Further, while both 

### 23

Zn and Fe exposures exerted some selective p ressure on plants, the authors did not observe the same with 

### 24
Pb, leading them to theorize that concentrations of bioavailable Pb were not present in high enough 

### 25
quantities to have such an effect. However, while Lemna sp. aquatic plants were determined to e ffectively 

### 26
sequester aqueous Pb, the plant growth rate was not significantly different from zero following exposures 

### 27
of 5 and 10 mg Pb/L, while exposure to 15 mg Pb /L was associated with notable plant mortality ( Hurd & 

### 28
Sternberg, 2008 ) . In fact, Paczkowska et al. ( 2007 ) observed that low Pb exposures (0.1 to 1.0 mmol for 9 

### 29
days) stimulated the growth of Lemna minor cultures, although there was concurrent evidence of 

### 30
chlorosis and induction of antioxidant enzymes

. Additionally, Cd was found to be more toxic than Pb, 

### 31
although the authors determined that this resulted from poor uptake of Pb by L. minor ( Paczkowska et al., 

### 32

2007 ) 

### 33May 2011 7

- 81 DRAFT

– DO NOT C ITE OR QUOTE 

### 7.3.4.2. Invertebrates

Effects of Pb on aquatic invertebrates recognized in the 2006 Pb AQCD include adverse impacts on 

### 1
reproduction, growth, survival and metabolism

. Pb was recognized to be more toxic in longer

- term 

### 2

exposures than shorter

- term exposures with chronic toxicity thresholds for reproduction in water fleas ( D. 

### 3

magna ) ranging as low as 30 μg Pb /L. As observed in terrestrial invertebra tes, the antioxidant system, 

### 4
survival, growth and reproduction are affected by Pb in aquatic organisms

. In aquatic invertebrates, Pb 

### 5
has also been shown to affect stress responses and osmoregulation. New evidence that supports previous 

### 6
findings of Pb on re production and growth in invertebrates are reviewed here as well as limited studies on 

### 7

behavioral effects. 

### 8
Recent literature strengthens the evidence indicating that Pb affects enzymes and antioxidant 

### 9
activity in aquatic invertebrates. Increased SOD activ ity was observed in mantles of pearl oyster but 

### 10
decreased with time although always remaining higher than in the control animals during 72

- hour 

### 11
exposures to 0.5 μM Pb ( Jing et al., 2007 ) . In contrast, activity of Se

- dependent glutathione peroxidase 

### 12
was not changed with Pb exposure. SOD, catalase, and glutathione peroxi dase were significantly reduced 

### 13
at environmentally relevant concentrations of Pb (2 μg Pb /L as measured in Bohai Bay, China) in the 

### 14
digestive gland of the bivalve Chlamys farreri ( Y. Zhang et al., 2010 ) . In contrast, Einsporn et al

. ( 2009 ) 

### 15
observed no change in catalase activity in the digestive gland and gill of bl ue mussel M. edulis following 

### 16
exposures to 2,500 μg Pb /L as Pb nitrate for 10 days and measured again following a 10 day depuration 

### 17
period. However, in this same species, glutathione

- S

- transferase activity was elevated in the gills after Pb 

### 18
exposure and re mained active during depuration while no changes to glutathione

- S

- transferase activity 

### 19
were observed in the digestive gland. In black mussel ( M. galloprovincialis ) exposed 10 days to sublethal 

### 20
concentrations of Pb, fluctuations in SOD activity were observe d over the length of the exposure 

### 21

( Vlahogianni & Valavanidis, 2007 ) . Catalase activity was decreased in the mantle of these mussels but 

### 22
fluctuated in their gills, as compared with the control group. In the bivalve C. farreri exposed to Pb, there 

### 23
was induction of lipid peroxidation measured as MDA of 24% and a 37% reduction in 7

- ethoxyresorufin



### 24

o

- deethylase (EROD) activity when compared to controls ( Y. Zhang et al., 2010 ) . In black mussel 

### 25
exposed for 10 days to sublethal concentrations of Pb, MDA levels were increased in mantle and gill 

### 26

( Vlahogianni & Valavanidis, 2007 )

. 

### 27

Aminolevulinic acid dehydratase (ALAD) is a recognized biomarker of exposure across a wide 

### 28
range of taxa including bacteria ( Korcan et al., 2007 ) , invertebrates and vertebrates. Since the 2006 Pb 

### 29
AQCD, there are additional studies measuring changes in ALAD activity in field

- collected bivalves and 

### 30

crustaceans. In the bivalve Chamelea gallina colle cted from the coast of Spain, ALAD inhibition was 

### 31
greater with higher concentrations of Pb measured in whole tissue ( Kalman et al., 2008 ) . In another study 

### 32
from Spain, ALAD activity was negatively correlated with total Pb concentration in seven marine 

### 33
bivalves ( C. gallina

, Mactra corallina

, Donax trunculus

, Cerastoderma edule

, M. galloprovinci alis

, 

### 34May 2011 7

- 82 DRAFT

– DO NOT C ITE OR QUOTE Scrobicularia plana and Crassotrea angulata ), however, the authors of this study indicated the need to 

### 1
consider species

- dependent responses to Pb ( Company et al., 2011 )

. Pb content varied significantly 

### 2
among species and was related to habitat (sediment versus substrate) and feeding behavior. In red fingered 

### 3

marsh crab, Parasesar ma erythodactyla , collected from sites along an estuarine lake in New South Wales, 

### 4
Australia, elevated glutathione peroxidase activity was correlated with individuals with higher metal body 

### 5

burdens ( MacFarlane et al., 2006 )

. 

### 6
Studies of stress responses to Pb in invertebrates

, conducted since the 2006 Pb AQCD

, include 

### 7
induction of heat shock proteins and depletion of glycogen reserves. Induction of heat shock proteins in 

### 8
zebra mussel exposed to 500 μg Pb /L for 10 weeks exhibited a 12

- fold higher induction rate as compared 

### 9

to co ntrol groups ( Singer et al., 2005 ) . Energetic reserves in the freshwater snail B. glabrata in the form 

### 10
of glycogen levels were significantly decreased by 20%, 57% and 78% in gonads compared to control 

### 11
animals following 96

- hour exposures to 50, 100 and 500 μg Pb /L, respectively ( Ansaldo et al., 2006 )

. 

### 12
Decreases in glycogen levels were also observed in the pulmonary and digestive gland region at 50 and 

### 13
100 μg Pb /L treatment levels. Pb did not exacerbate the effects of sustained hypoxia in the crayfish ( C. 

### 14

destructor ) exposed to 5,000 μg Pb /L for 14 days while being subjected to decreasing oxygen levels in 

### 15

water ( Morris et al., 2005 )

. The crayfish appeared to cope with Pb by lowering metabolic rates in the 

### 16
presence of the metal. Activity of enzymes associated with the immune defense system in the mantle of 

### 17
pearl oyster were measured at 0, 24, 48 and 72 ho ur exposure to 104 μg Pb /L ( Jing et al., 2007 ) . Activity 

### 18

of AcPa se, a lysosomal marker enzyme, was detected at 24 hours and decreased at subsequent time 

### 19
points. Phenoloxidase activity was depressed compared with controls and remained significantly lower 

### 20
than control at 72 hours of exposure to Pb. 

### 21

The effect of Pb on t he osmoregulatory response has been studied after the 2006 Pb AQCD. The 

### 22
combined effects of Pb and hyperosmotic stress on cell volume regulation was analyzed in vivo and in 

### 23

vitro in the freshwater red crab, Dilocarcinus pagei ( Amado et al., 2006 )

. Crabs held in either freshwater 

### 24
or brackish water lost 10% of their body weight after one day when exposed to 2

, 700 μg Pb 2+ /L. This 

### 25
weight loss was transient and was not observed during days 2

- 10 of the exposure

. In vitro, muscle from 

### 26
red crabs exposed to hyperosmotic saline solution had increased ninhydrin

- positive substances and 

### 27
muscle weight decreased in isosmotic conditions upon exposure to Pb indicating that this metal affects 

### 28
tissue volume regulation in crabs although the exact mechanism is unknown. 

### 29
Additional evidence of reproductive and developmental effects of Pb on aquatic invertebrates is 

### 30
available since the 2006 Pb AQCD. Sublethal concentrations of Pb negatively affected the total number of 

### 31
eggs, hatching success and embryonic survival of the freshwater snail B. glabrata exposed to 50, 100, or 

### 32
500 μg Pb /L ( Ansaldo et al., 2009 ) . Following exposure of adult snails for 96 hours, adults were removed 

### 33
and the eggs were left in the Pb solutions. The total num ber of eggs was significantly reduced at the 

### 34
highest concentration tested (500 μg Pb /L). Time to hatching was doubled and embryonic survival was 

### 35
significantly decreased at 50 and 100 μg Pb /L

, while no embryos survived in the highest concentration. 

### 36May 2011 7

- 83 DRAFT

– DO NOT C ITE OR QUOTE Formatio n of tentacles and eyes was significantly impaired in embryos of the freshwater ramshorn snail 

### 1

Marisa cornuarietis at 15,000 μg Pb /L ( Sawasdee & Köhler, 2010 ) . Theegala et al. ( 2007 ) observed that 

### 2
the rate of reproduction was significantly impaired in Daphnia pulex at >500 μg Pb /L in 21 day 

### 3

exposures

. Reproductive variables includi ng average lifespan, rate of reproduction, generation time and 

### 4
rate of population increase were adversely affected in the rotifer Brachionus patulus under conditions of 

### 5
increasing turbidity and Pb concentration ( Garcia

- Garcia et al., 2007 )

. 

### 6
In larvae of the mosquito, C. quinquefasciatus , exposed to 50 μg Pb /L , 100 μg Pb /L or 200 μg 

### 7

Pb /L, Pb

- nitrate exposure was found to significantly redu ce hatching rate and egg

- production at all 

### 8
concentrations and larval emergence rate at 200 μg Pb /L ( Kitvatanachai et al., 2005 ) . Larval emergence 

### 9
rates of 78% (F0), 86% (F1) and 86% (F2) were observed in the control group while emergence rates 

### 10
decreased in each generation 46% (F0), 26% (F1) and 58% (F2) in mosquito e s reared in a concentration 

### 11
of 200 μg Pb /L

. The time to first emergence also increased slightly to 10 days in the Pb

- exposed group as 

### 12
compared to the control group where emerge nce was first observed on day 9. In the F2 generation of 

### 13
parents exposed to 200 μg/L

, the ratio of female to male offspring was 3.6:1 .0 . No effects were observed 

### 14
on oviposition preference of adult females, larval weight or larval deformation. 

### 15

Since the pu blication of the 2006 Pb AQCD, limited studies on marine invertebrates have indicated 

### 16
adverse effects of Pb on reproduction in saltwater environments

. In a long term (approx imately 60 days) 

### 17
sediment bioassay with the marine amphipod Elasmopus laevi , onset to reproduction was significantly 

### 18
delayed at 118 μg Pb /g compared to controls

. In the higher concentrations, start of offspring production 

### 19
was delayed further; 4 days in 234 μg Pb /g and 8 days in 424 μg Pb /g ( Ringenary et al., 2007 )

. Fecundity 

### 20
was also reduced with increasing Pb c oncentration in sediment. Exposure of gametes to Pb prior to 

### 21
fertilization resulted in a decrease of the fertilization rates of the marine polychaete Hydroides elegans 

### 22

( Gopalakrishnan et al., 2008 ) . In sperm pretreated in 1 00 μg Pb /L filtered seawater for 20 minutes, 

### 23
fertilization rate decreased by approximately 70% compared to controls

. In a separate experiment, eggs 

### 24
were pretreated with Pb prior to addition of an untreated sperm suspension. The fertilization rate of eggs 

### 25
pretreated in 50 μg Pb/L filtered seawater decreased to 20% of the control. In another test with H. elegans 

### 26
in which gametes were not pre

- treated, but instead added directly to v arying concentrations of Pb for 

### 27

fertilization

, there appears to be a protective effect following fertilization due to the formation of the 

### 28
fertilization membrane during the first cell division that may prevent Pb from entering the oocytes 

### 29

( Gopalakrishnan et al., 2007 )

. 

### 30

T he protective barrier against Pb toxicity formed by the egg structure in some invertebrates ( e.g., 

### 31
Daphnia) was recognized in the 2006 Pb AQCD. Consideration of toxicity of Pb to embryos that develop 

### 32
surrounded by a protective egg shell has been expanded s ince the 2006 Pb AQCD. In a study with 

### 33
cuttlefish ( S. officinalis ) eggs, radioisotopes were used to assess the permeability of the egg to Pb at low 

### 34
exposure concentrations ( 210 Pb activity concentration corresponding to 512 μg/L Pb) ( Lacou e

- Labarthe et 

### 35

al., 2009 ) . Retention and diffusion properties of the cuttlefish egg change throughout the development of 

### 36May 2011 7

- 84 DRAFT

– DO NOT C ITE OR QUOTE the embryo and since the eggs are fixed on substrata in shallow coastal waters they may be subject to 

### 1
acute and chronic Pb exposures

. In the radiotracer experiments, 210 Pb was never detected in the internal 

### 2
compartments of the egg during the embryonic development stage, however concentrations associated 

### 3
with the eggshell increased throughout the 48 day exposure. These results are consis tent with cuttlefish 

### 4
eggs collected from the field in which Pb was only detected in the eggshell and indicate the protective 

### 5
barrier provided by cuttlefish egg to Pb toxicity ( Miramand et al., 2006 )

. 

### 6
As noted in the 2006 Pb AQCD, Pb exposure negatively affects the growth of aquatic invertebrates. 

### 7

Some stu dies reviewed in the previous document suggested that juveniles do not discriminate between the 

### 8
uptake of essential and non

- essential metals ( Arai et al., 2002 ) . In new literature, the freshwater 

### 9
pulomonate snail Lymnaea stagnalis has been identified as a species that is extremely sensitive to Pb 

### 10

exposure

. Growth of juveniles was inhibited at EC 20 <4 μg Pb/L. ( Grosell & Brix, 2009 ; Grosell et al., 

### 11

2006b )

. In L. stagnalis exposed to 18.9μg/L Pb for 21 days, Ca 2+ influx was significantly inhibited and 

### 12
model estimates indicated 83% reduction in growth of newly hatched snails after 30 days at this exposure 

### 13

concentration ( Grosell & Brix, 2009 ) . The authors speculate that the high Ca 2+ demand of juvenile L. 

### 14
stagnalis for shell formation and interference of the Ca 2+ uptake pathway by Pb result in the susceptibility 

### 15
of this species.Wang et al., {, 2009, 533439} observed growth of embryos of the Asian Clam ( Meretrix 

### 16

meretrix ) was significantly reduced by Pb with an EC 50 of 197 μg/L. In juvenile Catarina scallop, 

### 17
Argopecten ventricosus , exposed to Pb for 30 days, the EC 50 for growth was 4,210 μg/L ( A. S. Sobrino



### 18
Figueroa et al., 2007 ) . Rate of growth of the deposit feeding polychaete Capitella sp. decreased 

### 19
significantly with increasing concentrations of Pb associated with sediment ( Horng et al., 2009 )

. 

### 20
Aquatic invertebrate strategies for detoxifying Pb were reviewed in the 2006 Pb AQCD and include 

### 21
sequestration of Pb in lysosomal

- vacuolar systems, excretion of Pb by some organisms, and deposition of 

### 22
Pb to molted exoskeleton. Molting of the exoskeleton ca n result in depuration of Pb from the body (see 

### 23

Knowlton et al., ( 1983 ) and Anderson et al., ( 1997 ) as cited in the 2006 Pb AQCD). New research has 

### 24
provided further evidence of depuration of Pb via molting in invertebrates

. Mohapatra et al. ( 2009 ) 

### 25
observed that Pb concentrations in body tissues were lower in the newly molted mud crabs ( Scylla 

### 26

serrata ) than in the pre

- molt, hard

- shelled crabs. Additionally, the c arapace of hard shelled crabs have 

### 27
lower concentrations of Pb than the exuvium of the soft shell crabs, leading the authors to speculate that 

### 28
some of the metal might be excreted during the molting process

. Bergey and Weis ( 2007 ) showed that 

### 29
differences in the proportion of Pb stored in exoskeleton and soft tissues changed during intermolt and 

### 30
immediate postmolt in two populations of fiddler crabs ( Uca pugnax ) collected from New Jersey. One 

### 31
population from a relative ly clean estuary eliminated an average of 56% of Pb total body burden during 

### 32
molting while individuals from a site contaminated by metals eliminated an average of 76% of total Pb 

### 33
body burden via this route

. Pb distribution within the body of crabs from the clean site shifted from 

### 34
exoskeleton to soft tissues prior to molting

. The authors observed the opposite pattern of Pb distribution 

### 35
in fiddlers from the contaminated site where larger amounts of Pb were depurated in the exoskeleton. 

### 36May 2011 7

- 85 DRAFT

– DO NOT C ITE OR QUOTE Behavioral responses of aquatic invertebrates to Pb reviewed in the 2006 Pb AQCD included 

### 1
avoidance. A limited number of new studies have considered additional behavioral endpoints. Valve 

### 2
closing speed was used as a measure of physiological alterations due to Pb exposure in the Catarina 

### 3
scallop ( A. Sobrino

- Figueroa & Caceres

- Martinez, 2009 )

. The average valve closing time increased from 

### 4
under one second in the control group to 3 to 12 seconds in juvenile scallops exposed to Pb (40 μg/L to 

### 5

400 μg/L) for 20 days

. Damage to sensory cilia of the mantle was observed following microscopic 

### 6

examination of Pb

- exposed individuals

. Feeding rate of the blackworm L. variegatus was signif icantly 

### 7
suppressed by day 6 of a 10 day sublethal test in Pb

- spiked sediments ( Penttinen et al., 2008 ) as compared 

### 8
to feeding rates at the start of the experiment

. However, this decrease of approximately 50% of the initial 

### 9
feeding rate was also observed in the controls; therefo re it is likely caused by some other factor other than 

### 10
Pb exposure

. 

### 11
Although Pb is known to cause mortality when invertebrates are exposed at sufficiently high 

### 12
concentrations, species that are tolerant of Pb may not exhibit significant mortality even at h igh 

### 13

concentrations of Pb. In a 10

- day Pb

- spiked sediment exposure (1,000 mg Pb /kg), 100% of individuals of 

### 14

the Australian estuarine bivalve Tellina deltoidalis survived ( King et al., 2010 ) . In the deposit feeding 

### 15
polychaete Capitella sp ., exposure to varying concentrations of Pb associated with sediment up to 0.41 

### 16
μmol/g had no effect on survival ( Horng et al., 2009 )

. In freshwater habitats, odonates are highly tolerant 

### 17
of Pb with no significant differences in survival time of dragonfly larvae ( Pachydiplax longipennis and 

### 18
Erythemis simplicicollis ) exposed to concentrations as high as 185 mg Pb /L Pb (185,000 μg Pb /L) ( Tollett 

### 19

et al., 2009 ) . Other species are more susceptible to Pb in the environment and these responses are 

### 20

reviewed in Section 7.3.5. 

### 21

7.3.4.3. Vertebrates

Biological effects of Pb on fish that have been studied since the 2006 Pb AQCD report are 

### 22

r eviewed here, and limited new evidence of Pb effects on amphibians, and marine mammals are 

### 23

considered

. As noted in the 2006 Pb AQCD, commonly observed effects of Pb on fish included inhibition 

### 24
of heme formation, alterations in brain receptors in fish, adve rse effects on blood chemistry, and decreases 

### 25
in some enzyme activities

. ( U.S. EPA, 2006 )

. Functional responses resulting from Pb exposure included 

### 26
increased production of mucus, changes in growth patterns, and gill binding affinities. According to Eisler 

### 27

( 2000 ) and reviewed in the 2006 Pb AQCD, the general symptoms of Pb toxicity in fish include 

### 28
production of excess mucus, lordosis, anemia, darkening of the dorsal tail region, degeneration of the 

### 29
caudal fin, destruction of spinal neurons, ALAD inhibi tion, growth inhibition, renal pathology, 

### 30
reproductive effects, growth inhibition and mortality. More recent experimental data presented here 

### 31
expand and support these observations. As in terrestrial vertebrates, Pb has been shown to affect 

### 32

antioxidant and enzymatic activity in aquatic vertebrates and new evidence of this since the 2006 Pb 

### 33May 2011 7

- 86 DRAFT

– DO NOT C ITE OR QUOTE AQCD is reviewed in this section

. This section also presents the limited new information available on the 

### 1

mechanism of Pb as a neurotoxicant in fish and effects of this me tal on blood chemistry. Additional 

### 2
mechanisms of Pb toxicity have been elucidated in the gill and the renal system of fish since the 2006 Pb 

### 3

AQCD

. Further supporting evidence of reproductive and growth effects of Pb on fish is discussed along 

### 4

with limited new information on behavioral effects of Pb

. Finally, limited new information since the 2006 

### 5
Pb AQCD on physiological effects of Pb on amphibians and marine mammals is presented

. 

### 6

Fish

In environmental assessments of metal

- impacted habitats, ALAD is a reco gnized biomarker of Pb 

### 7

exposure ( U.S. EPA, 2006 ) . For example, lower ALAD activity has been significantly correlated with 

### 8
elevated blood Pb concentrations in wild caught fish from Pb

- Zn mining areas although there are 

### 9
differences in species sensitivity ( Schmitt et al., 2005 ; Schmitt et al., 2007 ) . Sup pression of ALAD 

### 10
activity in brown trout transplanted to a metal contaminated stream was linked to Pb accumulation on 

### 11
gills and in liver in a 23 day exposure ( Heier et al., 2009 ) . Costa et al. ( 2007 ) observed inhibition of 

### 12
ALAD in hepatocytes of the neotropical traira ( Hoplias malabaricus ) following dietary dosing of 21 μg 

### 13
Pb/g every 5 days for 70 days. Cytoskeletal and cytoplasmic disorganization were observed in 

### 14
histopat hological examination of affected hepatocytes

. In fathead minnow exposed to Pb in either control 

### 15
water (33 μg Pb /L), CaSO 4 (37μg Pb /L) or (39 μg Pb /L) humic acid

- supplemented water and 

### 16
subsequently analyzed by quantitative PCR analysis there were no signif icant changes in ALAD mRNA 

### 17
gene response leading the authors to speculate that water chemistry alone does not influence this gene 

### 18

response ( Mager et al., 2008 )

. 

### 19
Pb was shown to inhibit hepatic cytochrome P450 in carp ( Cyprinus carpio ), silver carp 

### 20

( Hypothalmichtys molitrix ) and wels catfish ( Silurus glanis ) in a concentration

- dependent manner from 0

- 

### 21
4.0 μg/mL (Pb 2 + ) ( Henczova et al., 2008 )

. The concentrations of Pb that resulted in 50% inhibition of 

### 22

EROD and 7

- ethoxycoumarin

- o

- deethylase (ECOD) isoenzymes varied with the fish species. Silver carp 

### 23
was the least sensitive to the inhibitory effects of Pb (EROD 1.21, ECOD 1.52 μ g Pb /L) while carp 

### 24
EROD activity was inhibited at 0.76 μg Pb /L. Interaction of Pb with cytochrome P450 was verified b y 

### 25
spectral changes using Fo u rier Transform Infrared (FTIR) spectroscopy. Liver damage to Afri can catfish 

### 26
exposed to Pb (50

- 1,000 μg Pb /L) for 4 or 8 weeks included hepatic vacuolar degeneration followed by 

### 27
necrosis of hepatocytes ( Adeyemo, 2008b ) . The severity of observed histopathological effects in the liver 

### 28
was proportional to the duration of exposure and concentration of Pb. 

### 29
Upregulation of antioxidant enzymes in fish is a well

- recognized response to Pb exposure. Since 

### 30
the last review, additional studies demonstrating antioxidant activity as well as evidence for production of 

### 31
reactive oxygen species following Pb exposure are available. Silver crucian carp ( Caras sius auratus 

### 32
gibelio ) injected with 10, 20 or 30 mg Pb /kg wet weight Pb

- chloride showed a significant increase in the 

### 33May 2011 7

- 87 DRAFT

– DO NOT C ITE OR QUOTE rate of production of superoxide ion and hydrogen peroxide in liver ( Ling & Hong, 2010 ) . In the same 

### 1

fish, activities of liver SOD, catalase, asco r bate peroxidase, and glutathione peroxidase were significantly 

### 2
inhibited. Both glutathione and ascorbic acid levels decreased and malondialdehyde content increas ed 

### 3
with increasing Pb dosage, suggesting that lipid peroxidation was occurring and the liver was depleting 

### 4

antioxidants. In fathead minnow

, three genes, glucose

- 6

- phosphate dehydrogenase, glutathione

- S



### 5
transferase and ferritin were upregulated

, in microarr ay analysi s , during 30 day exposures to Pb in base 

### 6
water (33μg Pb /L)

, or (37μg Pb /L [hard]

- water supplemented with 500 μM Ca 2+ ) or (39 μg Pb /L [DOC]

- 

### 7
water supplemented with 4 mg/L humic acid)

. However, no changes in whole body ion concentrations 

### 8

were observed ( Mager et al., 2008 ) . In the freshwater fish Nile t ilapia, liver catalase, liver alkaline 

### 9
phosphatase, sodium and potassium

- ATPase (NA, K

- ATPase) and muscle Ca

- ATPase activities were 

### 10
quantified in various tissues following a 14 day exposure to 5, 10, and 20 μM concentrations of Pb nitrate 

### 11

( Atli & Canli, 2007 )

. Liver catalase activity significantly increased in the 5 and 20 μM concentrations 

### 12
while liver alkaline phosphatase activity was significantly increased only at the 20 μM concentration. No 

### 13
significant change in alkaline phosphatase activity was observed in intestine or serum. Ca

- ATPase activity 

### 14
was significantly decreased in muscle. Na, K

- ATPase was elevated in gill in the highest concentration of 

### 15
Pb while all concentrations resulted in significant decreases of this enzyme in intestine

. In another study 

### 16

w ith O. niloticus , Pb had no effect on glutathione measured in liver, gill, intestine, muscle and blood and 

### 17
liver metallothionein levels following a 14 day exposure to 5, 10, and 20 μM concentrations of Pb nitrate 

### 18

( Atli & Canli, 2008 )

. 

### 19
Metabolic enzyme activity in teleosts has also been measured following dietary exposures

. Alves 

### 20

and Wood ( 2006 ) in a 42 day chronic dietary Pb study with 50 to 500 μg Pb/g found that gill Na, K



### 21
ATPase activity was not a ffected in rainbow trout while increased Na, K

- ATPase was observed in the 

### 22

anterior intestine. Metabolic activit ies measured in liver and kidney of Nile tilapia following 60 day 

### 23
dietary administration of 100, 400, and 800 μg Pb /g indicated that alanine transaminase, aspirate 

### 24
transaminase, and lactate dehydrogenase activities significantly decreased in kidney in a co ncentration



### 25
dependent manner ( Dai, Fu, et al., 2009 ) and increased in liver with increasing concentration of dietary 

### 26
Pb. In a subsequent study using the same exposure paradigm, the digestive enzymes amylase, trypsin and 

### 27
lipase in tilapia were inhibited by dietary Pb in a concentration

- dependent manner ( Dai, Du, et al., 20 09 )

. 

### 28
Lesions were also evident in histological sections from livers of Pb

- exposed fish from this study and 

### 29
included irregular hepatocytes, cell hypertrophy, and vacuolation although no quantification of lesions by 

### 30

dose

- group was presented. 

### 31

Data on the physiological effects of Pb on marine elasmobra n chs are limited. De Boeck et al. 

### 32

( 2010 ) exposed the s potted dogfish to 2,072 μg Pb /L for one week and measured metallothionein 

### 33
induction, and the electrolytes Na, K, Ca and Cl. No effects were observed in Pb

- exposed fish in any of 

### 34
the physiological parameters measured in this study, however Pb was measured i n all organs ( De Boeck et 

### 35

al., 2010 )

. 

### 36May 2011 7

- 88 DRAFT

– DO NOT C ITE OR QUOTE Additional evidence of the neurotoxic effects of Pb on teleosts has become available since the 2006 

### 1
Pb AQCD. The mitogen

- activated protein kinases (MAPK), extracellular signal

- regulated kinase 

### 2
(ERK)1/2 and p38 MAPK were identified for the first time as possible molecular targets for Pb neuroto xicity 

### 3

in a teleost ( Leal et al., 2006 ) . The phosphorylation of ERK1/2 an d p38 MAPK by Pb was determined in 

### 4
vitro and in vivo in the catfish ( Rhamdia quelen ). R. quelen exposed to 1 ,000 μg Pb /L acetate for two 

### 5
days showed a significant increase in phosphorylation of ERK1/2 and p38 MAPK in the nervous system. 

### 6

Incubation of cerebel lar slices for 3 hours in 5 and 10 μ M Pb acetate also showed significant 

### 7
phosphorylation of MAPKs. The observed effects of Pb on the MAPK family of signaling proteins have 

### 8
implications for control of brain development, apoptosis and stress response

. In the neotropical fish traira 

### 9

( Hoplias malabaricus ) muscle cholinesterase was significantly inhibited after 14 dietary doses of 21 μg 

### 10

Pb /g wet weight ( Rabitt o et al., 2005 ) . Histopathological observations of brains of African catfish exposed 

### 11
to 500 μg Pb /L or 1

, 000 μg Pb /L Pb for 4 weeks included perivascular edema, focal areas of malacia

, and 

### 12
diffuse areas of neuronal degeneration ( Adeyemo, 2008b )

. 

### 13
Adverse effects of Pb on blood chemistry of fish were noted in the 2006 Pb AQCD and limited new 

### 14

literature since the last Pb review has considered effects on blood. In the African catfish, packed cell 

### 15
volume decreased with increasing concentration of Pb (25,000 to 200,000 μg Pb /L as Pb

- nitrate) and 

### 16
platelet counts increased in a 96

- h our exposure ( Adeyemo, 2007 ) . Red blood cell counts also decreased in 

### 17
some of the treatments when compa red to controls, although the response was not dose

- dependent and so 

### 18
may not have been caused by Pb exposure. 

### 19
The gill has long been recognized as a target of Pb in teleosts. Acute Pb toxicity at the fish gill 

### 20
primarily involves disruption of Ca homeostas is as previously characterized in the 2006 Pb AQCD 

### 21

( Rogers & Wood, 2004 ; Rogers & Wood, 2003 ) . In addition to this mechanism, Pb was found to induce 

### 22
ionoregulatory toxicity at the gill of rainbow trout throug h a binding of Pb with Na

- K, ATPase and rapid 

### 23
inhibition of carbonic anhydrase activity thus enabling noncompetitive inhibition of Na + and Cl - influx 

### 24

( Roger s et al., 2005 ) . Alves et al. ( 2006 ) administered a diet of three concentrat ions of Pb (7, 77 and 520 

### 25
μg Pb/g dry weight) to rainbow trout for 21 days, and measured physiological parameters including Na + 

### 26

and Ca + influx rate from water. Dietary Pb had no effect on brachial Na + and Ca + rates except on day 8 

### 27

where Na + influx rates were significantly elevated

. These studies suggest that Pb is intermediate between 

### 28
purely Ca antagonists such as Zn and Cd and disruptors of Na and Cl balance such as Ag and Cu. This 

### 29
finding has implications for BLM modeling since it suggests that both Ca and Na need to be considered 

### 30
as protective cations for Pb toxicity. Indeed, protection from Pb toxicity by both Na and Ca have been 

### 31

documented in freshwater fish ( Komjarova & Blust, 2009b )

. 

### 32
Long

- term exposures of Pb can adversely impact gill structure and function. Histopathological 

### 33
observations of gill tissue in the catfish ( C. gariepinus ) following an 8

- week aqueous exposure to Pb 

### 34

nitrate revealed focal ar eas of epithe l ial hyperplasia and necrosis at the lower exposure concentrations (50 

### 35

μg Pb /L and 100 μg Pb /L) ( Adeyemo, 2008a ) . Hyperplasia of mucous cells and epithelial cells were 

### 36May 2011 7

- 89 DRAFT

– DO NOT C ITE OR QUOTE apparent in the tissue from fish exposed the highest concentrations of Pb in the s tudy (500 μg Pb /L and 

### 1

1

, 000 μg Pb /L). In vitro incubation of gill tissue from fathead minnow with Pb concentrations of 2.5, 12.5 

### 2
and 25 mg Pb /L decreased the ratio of reduced glutathione to oxidized glutathione, indicating that lipid 

### 3
peroxidation at the gi ll likely contributes to Pb toxicity at low water hardness ( Spokas et al., 2006 )

. 

### 4
In addition to recent evidence of Pb interruption of Na + and Cl - at the gill ( Rogers et al., 2005 ) , Pb 

### 5

can interfere with the ionoregulation of Na + and Cl and reabsorption of Ca + , Mg 2+ , glucose, and water in 

### 6
the teleost kidney ( Patel et al., 2006 ) . Renal parameters includi ng urine flow rate, glomerular filtration 

### 7
rate, urine pH

, and ammonia excretion were monitored in a 96

- hour exposure of rainbow trout to 1.2 mg 

### 8

Pb /L as Pb nitrate. Rates of Na + and Cl - excretion decreased by 30% by 48 hours while Mg excretion 

### 9

increased two

- to

- three fold by 96 hours. Urine flow rate was not altered by Pb exposure, although urinary 

### 10
Pb excretion rate was significantly increased. After 24 hours of Pb exposure, the urine excretion rate of 

### 11

Ca + increased significantly by approximately 43% and rema ined elevated above the excretion rate in the 

### 12
control group for the duration of the exposure. Glomerular filtration rate significantly decreased only 

### 13
during the last 12 hours of the exposure

. Ammonia excretion rate increased significantly at 48 hours as 

### 14

ur ine pH correspondingly decreased

. At the end of the experiment glucose excretion was significantly 

### 15
greater in Pb

- exposed fish

. Although the exposures in this study approached the 96

- hour LC 50 , 

### 16
nephrotoxic effects of Pb indicate the need to consider additio nal binding sites for this metal in the 

### 17
development of biotic ligand modeling ( Patel et al., 2006 )

. 

### 18
Limited new studies on reproductive effects of Pb in f ish from oocyte formation to spawning are 

### 19
available. Decreased oocyte diameter and density in the toadfish ( Tetractenos glaber ) were associated 

### 20
with elevated levels of Pb in the gonad ( Alquezar et al., 2006 ) . The authors state this is suggestive of a 

### 21
reduction in egg size which ultimately may lead to a decline in female reproductive output. The effects of 

### 22
metals on embryonic stage of fish development in Cyprinus carpio and other species were reviewed in 

### 23

Jezierska et al. ( 2009 ) and included developmental abnormalities during organogenesis as well as 

### 24
embryonic and larval malformations. The authors concluded that the initial period of embryonic 

### 25
development, just after fertilization, and the period of hatching are the times at which developing embryos 

### 26
are most sensitive to metals. Reproductive performance of zebrafish as measured by incidence of 

### 27
spawning, numbers of eggs per breeding pair or hatch rate of embryos was unaffected following a 63 day 

### 28

diet of field

- collected Pb

- contaminated polych ae tes that were representative of a daily dose of 0.3

- 0.48 g 

### 29

Pb /kg

· day (dry we ight diet/wet weight fish) through food ( Boyle et al., 2010 )

. Mager et al. ( 2010 ) 

### 30
conducted 21 day breeding exposures at the end of chronic 300 day toxicity testing with fathead minnow. 

### 31

Non

- exposed breeders were switched to water containing Pb and Pb

- exposed breeders were mov ed to 

### 32
control tanks and effects on egg hatchability and embryo Pb accumulation were assessed. Fish in the high 

### 33
Pb concentration (120 μg Pb /L) reduced total reproductive output

, while a significant increase in average 

### 34
egg mass was observed in the high Pb HC O 3 - and DOC treatments as compared to egg mass size in 

### 35May 2011 7

- 9 0 DRAFT

– DO NOT C ITE OR QUOTE controls and in low HCO 3 - and DOC treatments with Pb. No significant differences were present between 

### 1
treatments in egg hatchability

. 

### 2
Reproductive effects of Pb have also been observed at the cellular level, including alterations in 

### 3
gonadal tissue and hormone secretions that are associated with Pb

- exposure. Histopathological 

### 4
observations of ovarian tissue in the African catfish following an 8

- week aqueous exposure to Pb nitrate 

### 5

indicated necrosis of ov arian follicles at the lowest concentration tested (50 μg Pb /L) ( Adeyemo, 2008a )

. 

### 6
Severe degeneration of ovarian follicles was observed in the highest concentrations of 500 μg Pb /L and 

### 7

1,000 μg Pb /L. Chaube et al. ( 2010 ) considered the effects of Pb on steroid levels through 12 and 24 hour 

### 8
in vitro exposures of post

- vitellogenic ovaries from the catfish ( Heteropneustes fossilis ) to Pb

- nitrate (0, 

### 9
001, 0.1, 1, 3, and 10 μg Pb /mL). Progesteron e, 17

- hydroxyprogesterone, 17, 20 beta



### 10
dihydroxyprogesterone, corticosterone, 21

- deoxycortisol and deoxycorticosterone were inhibited in a 

### 11

dose

- dependent manner

. Pb was stimulatory on the steroids estradiol

- 17

- β, testosterone and cortisol at low 

### 12

concentrat ions, and inhibitory at higher concentrations. The disruption of steroid production and altered 

### 13
hormone secretion patterns observed at the low concentrations of Pb in this study are suggestive of the 

### 14
potential for impacts to fish reproduction ( Chaube et al., 2010 )

. 

### 15
Reduction of growth was noted as an adverse effect of Pb on fish in the 2006 Pb AQCD

. No new 

### 16
evidence of growth effects in fish have been reported wi th the exception of Grosell et al. ( 2006a )

. In a 

### 17
series of exposures in which Ca +2 , DOC and pH were varied to assess effects on Pb toxicity to fathead 

### 18

minnows

, Grosell et al. ( 2006a ) observed a significant increase in growth in some groups exposed to 

### 19
higher concentrations, however, the increase in body mass was noted to have occurred in tanks with high 

### 20
mortality earlier in the exposur e ( Grosell et al., 2006a )

. No effects on growth rates were observed in 

### 21
rainbow trout administered a diet containing three concentrations of Pb (7, 77 and 520 μg Pb/g dry 

### 22

we ight) for 21 days ( Alves et al., 2006 ) or in Nile tilapia f ed diets with 100, 400, or 800 μg/g Pb dry 

### 23
weight for 60 days ( Dai, Du, et al., 2009 )

. Growth and survival were not adversely affected in juvenile 

### 24
rainbow trout, fathead minnow and channel catfish ( Ictalurus punctatus ) fed a live diet of L. vari e gatus 

### 25

contaminated wit h Pb (850

- 1

, 000 μg Pb /L

· g dry mass for 30 days. ( Erickson et al., 2010 )

. In 30 day 

### 26
chronic tests in which a range of pH values (6.4, 7.5 and 8.3) were tested with low (25

- 32 μg Pb /L), 

### 27

intermediate (82

- 156 μg Pb /L) and high (297

- 453 μ g Pb/L ) concentrations of Pb, Mager et al. ( 2011 ) did 

### 28
not observe growth impairment in fathead minnows at environmentally relevant c oncentrations of Pb. 

### 29

Since the publication of the 2006 Pb AQCD, several studies integrating behavioral and 

### 30
physiological measures of Pb toxicity have been conducted on fish

. The ornate wrasse ( Thalassoma pavo ) 

### 31
was exposed to sublethal (400 μg Pb /L) or a m aximum acceptable toxicant concentration (1,600 μg Pb /L) 

### 32
dissolved in seawater for one week to assess the effects of Pb on feeding and motor activities ( Giusi et al., 

### 33

2008 )

. In the sublethal concentration group

, hyperactivity was elevated 36% over controls

. In the h igh 

### 34
concentration, a 70% increase in hyperactivity was observed and hyperventilation occurred in 56% of 

### 35

behavioral observations

. Elevated expression of heat shock protein 70/90 orthologs was detected in the 

### 36May 2011 7

- 91 DRAFT

– DO NOT C ITE OR QUOTE hypothalamus and mesencephalic areas of the brain s of Pb

- treated fish

. No changes in feeding activity 

### 1

were noted between non

- treated and treated fish. 

### 2

Sloman et al. ( 2005 ) investigated the effect of Pb on hierarchical social interactions and the 

### 3
corresponding monoaminergic profile s in rainbow trout. Trout were allowed to establish dominant



### 4
subordinate relationships for 24 h ours, then were exposed to 46 μg Pb /L or 325 μg Pb /L ( Pb

- nitrate ) for 

### 5

48 ho urs to assess effects on behavior and brain momoamines. In non

- exposed fish, subordinate 

### 6
individuals had higher concentrations of circulating plasma cortisol and telencephalic 

### 7

5

- hydroxyindoleacetic acid/5

- hydroxytryptamine (serotonin) (5

- HIAA/5

- HT) ratios

. In the high 

### 8
concentration of Pb, there was significant uptake of Pb into gill, kidney and liver when compared with the 

### 9
control group and dominant fish appeared to have elevated hypothalamic 5

- HIAA/5HT ratios

. Uptake of 

### 10
Pb into the liver was higher in subo rdinate fish when compared to the dominant fish

. No significant 

### 11
differences were observed in cortisol levels or behavior after metal exposure

. 

### 12
Mager et al. ( 2010 ) conducted prey capture assays with 10 day old fathead minnow larvae born 

### 13
from adult fish exposed to either 35 or 120 μg Pb /L for 300 days, then subsequently tested in a breeding 

### 14
assay for 21 days. The time interval between 1st and 5th ingestion of 10 prey items ( Artemia nauplii ) was 

### 15
used as a measure of behavior and motor function of offspring of Pb

- exposed fish

. Larvae were offered 10 

### 16
Artemia and the number ingested within 5 minutes was scored. The number of larvae ingesting 5 Artemia 

### 17

decreased within the time period in offspring of Pb

- exposed fish as compared to the control group, 

### 18
leading the authors to suggest this behavior is indicative of motor/behavioral impairment. 

### 19

Amphibians

Amphibians move between terrestrial and aquatic habitats and can therefore be exposed to Pb both 

### 20
on land and in water. The studies reviewed here are all aquatic or sediment exposures. Biological effects 

### 21
of Pb on amphibians in terrestrial exposure scenarios are reviewed in Section s 7 .2. 2.3 and 7.2. 4. 3. 

### 22
Amphibians lay their eggs i n or around water making them susceptible to water

- borne Pb during 

### 23
swimming, breeding and development. In the 2006 Pb AQCD amphibians were considered to be 

### 24
relatively tolerant to Pb. Observed responses to Pb exposure included decreased enzyme activity ( e.g ., 

### 25
ALAD reduction) and changes in behavior summarized in Table AX7

- 2.4.3 ( U.S. EPA, 2006 ) . Since the 

### 26
2006 Pb AQCD, studies conducted at environmentally relevant concentrations of Pb have indicated 

### 27
sublethal effects on tadpole endpoints including growth, def ormity, and swimming ability. Genotoxic and 

### 28
enzymatic effects of Pb following chronic exposures have been assessed in laboratory bioassays. 

### 29
Various sublethal endpoints (growth, deformity, swimming ability, metamorphosis) were evaluated 

### 30
in northern leopard frog ( R. pipiens ) tadpoles exposed to nominal concentrations of 3, 10, and 100 μg 

### 31

Pb /L as Pb nitrate from embryonic stage to metamorphosis ( T. H. Ch en et al., 2006 )

. In this chronic study, 

### 32
the concentrations represent the range of Pb found in surface freshwaters across the U.S. The lowest 

### 33May 2011 7

- 92 DRAFT

– DO NOT C ITE OR QUOTE concentration of 3 μg Pb /L approaches the EPA chronic criterion for Pb of 2.5 μg Pb /L at a hardness of 

### 1
100 mg/L or 4.5 μg Pb /L at a hardness of 170 mg/L ( U. S. EPA, 2002 ) . No effects were observed in the 

### 2
lowest concentration. In the 100 μg Pb /L treatment, tadpole growth rate was slower (Gosner stages 25

- 

### 3
30), 92% of tadpoles had lateral spinal curvature (compared with 6% in the control) and maximum 

### 4
swimming speed was significantly slower than the other treatment groups. In this study, Pb concentrations 

### 5
in the tissues of tadpoles were quantified and the authors reported that they were within the range of 

### 6
reported tissue concentrations from wild

- caught populati ons

. 

### 7

The effects of Pb

- contaminated sediment on early growth and development were assessed in the 

### 8
southern leopard frog ( Sparling et al., 2006 ) . Tadpoles exposed to Pb in sediment (45, 75, 180, 540, 2,360, 

### 9
3,940, 5,520, and 7,580 mg Pb /kg dry weight) with corresponding sediment pore water co ncentrations of 

### 10
123, 227, 589, 1,833, 8,121, 13,579, 19,038 and 24,427 μg Pb /L from embryonic stage to metamorphosis 

### 11
exhibited sublethal responses to Pb in sediment at levels below 3,940 m g Pb /kg

. There was 100% 

### 12
mortality in the 3,940, 5,520 and 7,580 mg P b /kg exposures by day 5. The authors noted that the most 

### 13
profound effects of Pb on the tadpoles were on skeletal development. At 75 mg Pb /kg

, subtle effects on 

### 14
skeletal formation such as clinomely and brachydactyly were observed

. Skeletal malformations inc reased 

### 15
in severity at 540 mg Pb /kg and included clinodactyly, brachymely and spinal curvature and these effects 

### 16
persisted after metamorphosis

. At the highest concentration with surviving tadpoles (2,360 mg Pb /kg) all 

### 17
individuals displayed severe skeletal m alformations that impacted mobility

. Other sublethal effects of Pb 

### 18
observed in this study were reduced rates of early growth of tadpoles at concentrations ≤ 540 mg Pb /kg 

### 19
and increased time to metamorphosis in the 2,360 mg Pb /kg (8

, 121 μg Pb /L sediment pore water) 

### 20
treatment. Conversely, no effects were observed on organogenesis in X. laevis embryos exposed to a 

### 21
range of Pb concentrations from 8,600 to 220,500 μg Pb /L using the Frog Embryo Teratogenesis Assay 

### 22

( Gungordu et al., 2010 )

. 

### 23
Endpoints of oxidative damage were measured in testes of the black

- spotted frog ( Rana 

### 24
nigromaculata ) treated with 100 μg Pb /L, 200 μg Pb /L, 400 μg Pb /L, 800 μg Pb /L or 1,600 μg Pb /L Pb



### 25
nitrate by epidermal adsorption for 30 days ( M. Z. Wang & Jia, 2009 )

. All doses significantly increased 

### 26
MDA, a product of o xidative stress, and glutathione levels were elevated in all but the lowest treatment 

### 27
group. In the same study, damage to DNA assessed by DNA tail length showed effects at >200 μg Pb /L 

### 28

and DNA tail mov e ment showed effects at >400 μg Pb /L

. The authors concl uded that the effects on 

### 29
endpoints of oxidative stress and DNA damage detected in testes indicated a possible reproductive effect 

### 30

of Pb to black

- spotted frogs. 

### 31
The genotoxic potential of Pb to larvae of the toad ( X. laevis ) was assessed by determining the 

### 32
number of micronucleated erythrocytes per thousand (MNE) following a 12 day exposure ( Mouchet et al., 

### 33

2007 )

. The lowest Pb concentrations with X. laevis (10 and 100 μg Pb /L) did not exhibit genotoxic effects 

### 34
while both 1,000 and 10,000 μg Pb /L significantly increased MNE to 14 and 202, respectively compared 

### 35
to the control (6 MNE)

. In a nother chronic genotoxic study, eryth r ocytic micronuclei and er y throcytic 

### 36May 2011 7

- 93 DRAFT

– DO NOT C ITE OR QUOTE nuclear abnormalities were significantly increased with increasing Pb concentrations (700 μg Pb /L , 1,400 

### 1

μg Pb /L , 14,000 μg Pb /L, 70,000 μg Pb /L) during 45, 60, and 75 day exposure s of tadpoles Bufo raddei 

### 2

( Y. M. Zhang et al., 2007 )

. The authors noted that the eryth r ocytic micronuclei and er y throcytic nuclear 

### 3
abnormalities frequencies generally decreased with increasing exposure time and that this may be 

### 4
indicative of regulation of genotoxic factors by tadpoles

. 

### 5
In a study with 4

- day

- old X. laevis tadpoles exposed to a range of concentrations of Pb from 25,500 

### 6
to 137,000 μg Pb /L for 24 hours, acetylcholin esterase was significantly inhibited in all treatments 

### 7

( Gungordu et al., 20 10 )

. The authors suggest that the 35

- 60% inhibition of acetylcholinesterase is 

### 8
indicative of a neurotoxic effect. In the same study, glutathione

- s

- transferase activity significantly 

### 9

increased in a concentration

- dependent manner

. Alanine aminotransferase and aspartate aminotra n sferase 

### 10
activities were decreased, however, the degree of inhibition did not reflect Pb concentration. The 

### 11
concentrations used in this study were selected based on the LC 50 of Frog Embryo Teratogenesis Assay 

### 12

results with X. laevis a nd are much higher than ambient levels of Pb. 

### 13

Birds

In addition to effects on amphibians, consideration of toxicity of Pb to vertebrate embryos that 

### 14
develop surrounded by a protective egg shell has been expanded since the 2006 Pb AQCD

. Pb treatment 

### 15

of mall ard duck ( Anas platyrhynchos ), eggs by immersion in 100 μg Pb / L for 30 minutes on day 0 of 

### 16
development did not increase malformations or mortality of embryos ( Kertész & Fáncsi, 2003 )

. However, 

### 17
immersion of eggs in 2

, 900 μg Pb /L under the same experimental conditions resulted in increased rate of 

### 18
mortality and significant malformations including hemorrhages of the body, stunted growth, and absence 

### 19
of yolk sac circulatory system ( Kertesz et al., 2006 )

. The second study was conducted to emulate 

### 20
environmental levels of Pb following a dam failure in Hungary

. 

### 21

Mammals

Although Pb co ntinues to be detected in tissues of marine mammals in U.S. coastal waters ( Bryan 

### 22

et al., 2007 ; Kannan et al., 2006 ; Stavros et al., 2007 ) few studies exist that consider biological effects 

### 23

associated with Pb

- exposure

. Pb effects on immune parameters including cell viability, apoptosis, 

### 24
lymphocyte proliferation, and phagocytosis were tested in vitro on phagocytes and lymphocytes isolated 

### 25
from the peripheral blood of bottlenose dolphin ( Tursiops truncates ) ( Cámara Pellissó et al., 2008 )

. No 

### 26

effect s on viability of immune cells, apoptosis, or phagocytosis were observed in 72 hour exposure to 

### 27
concentrations of 1, 10, 20 and 50 mg Pb /L. Proliferative response of bottlenose dolphin leukocytes was 

### 28
significantly reduced at 50 mg Pb /L, albeit by only 10% in comparison to the control. 

### 29May 2011 7

- 94 DRAFT

– DO NOT C ITE OR QUOTE 

## 7.3.5. Exposure and Response of Aquatic Species

To support the development of air quality criteria standards that are protective of aquatic 

### 1
ecosystems, threshold levels for Pb effects on aquatic populations must be evaluated. The Ann ex of the 

### 2
2006 Pb AQCD ( U.S. EPA, 2006 ) summarize d data on exposure

- response functions for freshwater and 

### 3
marine invertebrates (Table AX7

- 2.4.1) and freshwater and marine fish (Table AX7

- 2.4.2)

. The recent 

### 4

exposure

- response studies in this section expand on the findings from the 2006 Pb AQCD with 

### 5
information on newly

- tested organisms ( including microalgae, invertebrate, amphibian and fish species )

. 

### 6

A series of 72

- hour Pb toxicity tests were conducted with five marine microalgae species ( T. chuii

, 

### 7

R. salina , Chaetoceros sp., I. galbana and N. gaditana ) to determine the relative Pb sensitivities as 

### 8
measured by growth inhibition. The respective 72

- hour EC 50 values derived were 2,640, 900, 105, 1,340, 

### 9
and 740 μg Pb /L ( Debelius et al., 2009 ) . The authors noted that species cellular size, sorption capacity, or 

### 10
taxonomy did not explain differences i n sensitivity to Pb, leaving the mechanism of response still open to 

### 11
question . Additionally, t he aquatic freshwater microalgae Scenedesmus obliquus was significantly more 

### 12
susceptible to Pb exposure than Chlorella vulgaris algae, although the se authors stat ed that both appeared 

### 13
to be very tolerant of the heavy metal

. Laboratory 48

- hour standard toxicity tests were performed with 

### 14

both of these species and respective EC 50 values of 4,000 and 24,500 μg Pb /L were derived ( Atici et al., 

### 15

2008 ) . E xperiments with the blue

- green algae Spirulin a platensis produced a LC 50 value of 75.3 μg 

### 16

Pb /mL (95% CI : 58.5

, 97.0) ( Arunakumara et al., 2008 )

. 

### 17

In the 2006 Pb AQCD, adverse effects of Pb

- exposure in amphipods ( H. azteca ) and water fleas ( D. 

### 18

magna ) were reported at concentrations as low as 0.45 μg Pb /L. Effective concentrations for aquatic 

### 19
invertebrates were found to range from 0.45 to 8

, 000 μg Pb /L. Since the publication of the 2006 Pb 

### 20

AQCD, recent studies have identified the freshwater snail L. stagnalis as a species that is extremely 

### 21
sensitive to Pb exposure ( Grosell & Brix, 2009 ; Grosell et al., 2006b ) . Growth of juvenile L. stagna lis 

### 22

was inhibited at an EC 20 of < 4 μg Pb /L

. In contrast, freshwater juvenile ramshorn snail s M. cornuarietis 

### 23

were less sensitive to Pb with the same LOEC for hatching rate and LC 50

, calculated to be about 10

, 000 

### 24

μg Pb /L ( Sawasdee & Köhler, 2010 )

. 

### 25

Additional studies on Pb effects in aquatic invertebrates published since the 2006 Pb AQCD have 

### 26
indicated differences in susceptibility of different lifestages of aquatic organisms to Pb. In a series of 

### 27

seawater and sediment exposures using adult and juvenile amphipods Melita plumulosa , juveniles were 

### 28

more sensitive to Pb than adults ( King et al., 2006 )

. In the sea water

- only exposures, the 96

- h our LC 50 for 

### 29
adults was 3,000 μg Pb /L and 1,520 μg Pb /L for juveniles. Ten

- day exposures of adults in seawater 

### 30

resulted in an LC 50 of 1,270 μg Pb /L, an NOEC of 190 μg Pb /L and a LOEC of 390 μg Pb /L. In 

### 31
comparison, the LC 50 , NOEC, and LOEC value for the adults exposed in sediment was 3,560 μg Pb /L. 

### 32

Juvenile sediment tests results were LC 50 1,980, NOEC 580 and LOEC 1,020 μg Pb /L

. 

### 33May 2011 7

- 95 DRAFT

– DO NOT C ITE OR QUOTE In the freshwater mussel, Lampsilis siliquoidea (fatmucket) a Pb concentration response was 

### 1
observed in which newly transforme d (5

- day

- old) juveniles were the most sensitive lifestage in a 96

- hour 

### 2
toxicity test when compared to acute and chronic results with other lifestages ( N. Wang et al., 2010 ) . The 

### 3

96

- h our EC 50 values for the 5

- day

- old L. siliquoidea in two separate toxicity tests were 142 and 298 μg 

### 4

Pb /L (mean EC 50 220 μg Pb /L ) in contrast to older juveniles (2 months old) with an EC 50 >426 μg/L. The 

### 5

24

- h our median effect concentration for glochidia (larvae) of L. siliquoidea in 48

- hour acute toxicity tests 

### 6
was >299 μg/L. A 28 day exposure chronic value of 10 μg Pb /L was obtained from 2

- month

- old L. 

### 7
siliquoidea juveniles , and was the lowest genus mean chronic value ever reported for Pb ( N. Wang et al., 

### 8

2010 ) . A 96

- hour test on newly transformed juveniles was also conducted on Lampsilis rafinesqueana 

### 9
(Neosho mucket), a mussel that is a candidate for the enda ngered species list. The EC 50 for this species 

### 10
was 188 μg Pb/L. In contrast, a 24

- h our LC 50 of 4,500 μg Pb /L for adult black mussel ( M. 

### 11
galloprovincialis ) suggests that, in general, juvenile bivalves are more sensitive to Pb exposure than 

### 12

adults ( Vlahogianni & Valavanidis, 2007 )

. 

### 13
The acute toxicity of Pb to first

- instar C. riparius larvae was tested in soft water

, with hardness of 8 

### 14
mg/L as CaCO 3 . ( Bechard et al., 2008 ) . The 24

- h our LC 50 of 610 μg Pb /L f or first instar C. riparius larvae 

### 15
was much lower than previous values reported for later instars in hard er water. In a chronic test with 

### 16

Chironomus tentans

, ( 8 day

- old larvae exposed to Pb until emergence [ approximately 27 days ]) , the 

### 17
NOEC was 109, and the LOEC was 497 μg P b/L ( Grosell et al., 2006b )

. The EC 20 for reduced growth and 

### 18
emergence of the midge Chironomus d ilutus was 28 μg Pb /L

, observed in a 55

- day exposure

, while the 

### 19
same species had a 96

- h our LC 50 of 3,323 μg Pb /L ( M ebane et al., 2008 )

. The 24

- hour LC 50 for larvae of 

### 20
C. quinquefasciatus mosquito e s was 180 μg Pb /L ( Kitvatanachai et al., 2005 ) . A 48

- h our LC 50 of 5

, 200 

### 21

μg Pb /L was observed in water

- only exposures of the blackworm Lumbriculus variegatus ( Penttinen et 

### 22

al., 2008 )

. 

### 23
Cladocerans are commonly tested aquatic organisms

, with data from three species: D. magna

, D

. 

### 24
pulex and Cerodaphnia dubia , representing approximately 70% of available metal toxicological literature 

### 25
on this group ( L. C. Wong et al., 2009 ) . Since the publication of the 2006 Pb AQCD, additional studies 

### 26
have generated acute toxicity values for other cladocerans

. Median lethal concentrations for Moina 

### 27

micrura (LC 50 690 μg Pb /L), Diaphanosoma birgei (LC 50 3

, 160 μg Pb /L), and Alona rectangular (LC 50 

### 28

7

, 000 μg Pb /L) indicate differences in susceptibility to Pb in these freshwater species from Mexico 

### 29

( Garcia

- Garcia et al., 2006 )

. An acute study of Pb with D. pulex identified a 48

- h our LC 50 of 4

, 000 μg/L 

### 30
for this species ( Theegala et al., 2007 )

. 

### 31
Exposure

- response assays on other freshwater species have been conducted since the 2006 Pb 

### 32

AQCD

. In the mayfly Baeti s tricaudatus , the 96

- hour LC 50 was 664 μg Pb /L ( Mebane et al., 2008 ) . An 

### 33

EC 20 value of 66 μg Pb /L was derived f or B. tricaudatus by quantifying the reduction in the number of 

### 34

molts over a 10

- day exposure to Pb ( Mebane et al., 2008 )

. For rotifer Brachionus patulus neonates

, the 

### 35

24

- hour LC 50 was 6

, 150 μg Pb /L ( Garcia

- Garcia et al., 2007 )

. In a 48

- hour toxicity test with the rotifer 

### 36May 2011 7

- 96 DRAFT

– DO NOT C ITE OR QUOTE Brachionus calyc ifloru s, an NOEC (194 μg Pb /L), a LOEC (284 μg Pb /L), and an EC 20 of 125 μg Pb /L 

### 1
was established for this species ( Grosell et al., 2006b )

. 

### 2
Since the publication of the 2006 Pb AQCD, Pb toxicity to larval stages of marine species has been 

### 3

assessed at sublethal and lethal concentrations

. The effective con centrations at which Pb resulted in 50% 

### 4
of abnormal embryogenesis of the Asian clam ( M. meretrix ) was 297 μg Pb /L. The 96

- hour LC 50 for 

### 5
larvae of the same species was 353 μg Pb /L {Wang, 2009, 533439}. In comparison, juvenile Catarina 

### 6
scallop ( A. ventricosu s ) had a LC 50 of 830 μg Pb /L in a 96

- hour exposure ( A. S. Sobrino

- Figueroa et al., 

### 7

2007 )

. Morphological deformities were observed in 50% of veliger larvae of blacklip abalone ( Haliotis 

### 8

ru bra ) at 4,100 μg Pb /L following a 48

- hour exposure to Pb, suggesting this species is not as sensitive to 

### 9

Pb as other marine invertebrate larvae ( Gorski & Nugegoda, 2006 )

. 

### 10
In the marine polychaete H. elegans

, EC 50 values of gametes, embryos, larvae (blastula to 

### 11
trochophore and larval settlement)

, and adults

, exhibited dose

- re s ponses to Pb that reflected the 

### 12
differential sensitivity of various lifestages of this organism ( Gopalakrishnan et al., 2008 )

. The EC 50 

### 13

value s for sperm and egg toxicity were 380 and 690 μg Pb /L respectively. Larval settle ment measured as 

### 14
the metal concentration causing 50% reduction in attachment was most sensitive to Pb with an EC 50 of 

### 15
100 μg Pb /L, while the EC 50 for abnormal development of embryos was 1,130 μg Pb /L. The LC 50 values 

### 16

for adult worms in 24

- hour and 96

- hour tests were 25,017 and 946 μg Pb /L, respectively. Manzo et al. 

### 17

( 2010 ) established a LOEC of 500 μg Pb /L and a maximum effect at 3

, 000 μg Pb /L in an embryotoxicity 

### 18
assay with sea urchin P. lividu s . The EC 50 for developmental defects in this species was 1,150 μg Pb /L 

### 19
with a NOEL of 250 μg Pb /L. 

### 20

There have been only a few new exposure

- response studies in amphibians since the 2006 Pb 

### 21
AQCD. Southern leopard frog tadpoles exposed to Pb in sediment (45 to 7

, 580 mg/kg dry weight) with 

### 22
corresponding sediment pore water concentrations from 123 to 24,427 μg Pb /L from embryonic stage to 

### 23
metamorphosis exhibited concentration

- dependent effects on survival ( Sparling et al., 2006 )

. The LC 50 

### 24

value for Pb in sediment was 3

, 738 mg/kg

, which corresponds to 12,539 μg Pb /L i n sediment pore water

. 

### 25
In the same study, concentration

- dependent effects on skeletal development were observed. The 40 day



### 26

EC 50 for deformed spinal columns in the tadpoles was 1

, 958 mg Pb /kg (corresponding to 6,734 μg Pb /L 

### 27

sediment pore water) and the 60 day

- EC 50 was 579 mg Pb /k g (corresponding to 1,968 μg Pb /L sediment 

### 28
pore water) ( Sparling et al., 2006 ) . A 96

- hour LC 50 of 96,100 μg Pb /L was determined for X. laevis 

### 29
embryos exposed to a range of Pb concentrations from 8,600 to 220,500 μg Pb /L using the Frog Embryo 

### 30
Teratogenesis Assay ( Gungordu et al., 2010 )

. 

### 31
In the studies reviewed for the 2006 Pb AQCD, freshwater fish demonstrat ed adverse effects at 

### 32
concentrations ranging from 10 to >5,400 μg Pb /L, generally depending on water quality parameters ( e.g., 

### 33
pH, hardness, salinity) ( U.S. EPA, 2006 )

. Pb tended to be more toxic in longer

- term exposures and 

### 34

correlated to Pb

- uptake in tissue s

. Table AX7

- 2.4.2 of the 2006 Pb AQCD summarizes effects of Pb to 

### 35

freshwater and marine fish

. At the time of the 2006 Pb AQCD , there was a lack of exposure

- response data 

### 36May 2011 7

- 97 DRAFT

– DO NOT C ITE OR QUOTE in marine fish

. No new exposure

- response studies in marine fish have been conducted s ince the previous 

### 1

Pb review. 

### 2

A series of studies published since the 2006 Pb AQCD have been conducted and have further 

### 3

elucidate d the influence of water chemistry parameters on Pb uptake and toxicity in fathead minnow 

### 4
resulting in additional dose

- response data for this species. Grosell et al. ( 2006b ) conducted a series of 30

- 

### 5
day exposures with larval fathead minnow in whic h varying concentrations of Ca 2+ (as CaSO 4 ) and DOC 

### 6
were tested. The effects of reduced pH (6.7) and increased pH (8.1) compared to a control pH of 7.4 on 

### 7
Pb toxicity were also assessed in this study. DOC, CaSO 4 and pH influenced Pb toxicity considerably 

### 8
over the range of water parameters tested. The 30

- day LC 50 for low hardness (19 mg CaSO 4 / L) in basic 

### 9
test water was 39 μg dissolved Pb/L and the highest LC 50 value ( obtained from the protection from 

### 10

increased con centrations of DOC and CaSO 4 ) was 1

, 903 μg dissolved Pb/L ( Grosell et al., 2006a )

. 

### 11
Mager et al. ( 2010 ) conducted 300

- day chronic toxicity tests at 35 and 120 μg Pb /L with fathead 

### 12
minnow under conditions of varied DOC and alkalinity to assess the effects of these water quality 

### 13
parameters on fish growth and Pb

- uptake. In additional tests with fath ead minnow, Mager et al. ( 2011 ) 

### 14

conducted both 96

- hour acute and 30

- day chronic tests to further characterize Ca 2+ , DOC, pH, and 

### 15
alkalinity values on Pb toxicity . Increa sed Ca 2+ , DOC and NaHCO 3 concentration afforded protection to 

### 16
minnows in acute studies. The role of pH in Pb toxicity is complex and likely involves Pb speciation and 

### 17
competitive interaction of H + with Pb 2+ ( Mager et al., 2011 )

. 

### 18
In the 2006 Pb AQCD, fish size was recognized as an important variable in determining the adverse 

### 19

effects of Pb

. Acute (96

- hour) and chronic (60

- day) early

- lifestage test exposures were conducted with 

### 20
rainbow trout to develop ACR ’ s for this species ( Mebane et al., 2008 )

. Two e arly

- lifestage chronic tests 

### 21
were conducted, the first with an exposure range of 12

- 384 μg Pb /L (69 days) at 20 mg CaCO 3 /L water 

### 22
hardness and the second with an exposure range of 8 to 124 μg Pb /L (62 days) and a water hardness of 29 

### 23

mg CaCO 3 /L. In the 69

- d ay test, the following chronic values were observed for survival: NOEC=24 

### 24
μg/L, maximum acceptable toxicant concentration=36 μg/L, EC 10 =26 μg/L, EC 20 =34 μg/L

, and LC 50 =55 

### 25
μg/L. Results from the 62

- day test

, with fish length as the endpoint

, were NOEC=8 μg/ L, MATC=12 

### 26
μg/L, EC 10 =7μg/L, EC 20 =102 μg/L and LC 50 =120 μg/L

. In acute tests run concurrently with the chronic 

### 27

tests, 96

- hour LC 50 values were 120 and 150 μg/L, respectively

. Data from this study resulted in ACR ’ s 

### 28
for trout lower than previously reported. The low ACR values were due to the acute tests which produced 

### 29

LC 50 values that were 10 to 25 times lower than earlier studies with trout ( Mebane et al., 2008 )

. The 

### 30
authors speculate d that the lower LC 50 values were due to the age of the fish used in the study (two to four 

### 31
week old fry) and that testing with larger and older fish may not be protective of more sensitive lifestages. 

### 32May 2011 7

- 98 DRAFT

– DO NOT C ITE OR QUOTE 

## 7.3.6. Community and Ecosystem Effects

As discussed in the 2006 Pb AQCD, exposure to Pb is likely to have adverse impacts in aquatic 

### 1
environments via effects at several levels of ecological organization (individuals, populations, 

### 2
communities, or ecosystems). These adverse effects resulting from toxicity of Pb would be evidenced by 

### 3
chang es in species composition and richness, in ecosystem function, and in energy flow

. The 2006 Pb 

### 4
AQCD concluded that, in general, there was insufficient information available for single materials in 

### 5
controlled studies to permit evaluation of specific impacts on higher levels of organization (beyond the 

### 6
individual organism). Furth er more, Pb rarely occurs as a sole contaminant in natural systems making the 

### 7

effects of Pb difficult to ascertain

. New information on effects of Pb at the population, community and 

### 8

ec osystem level is reviewed below

. 

### 9
In laboratory studies, Pb exposure has been demonstrated to alter predator

- prey interactions, as 

### 10
well as feeding and avoidance behaviors

. Additionally, field studies have linked Pb contamination to 

### 11
reduced primary producti vity and respiration, and to altered energy flow and nutrient cycling

. However, 

### 12
because of the complexity inherent in defining such effects, there are relatively few available population, 

### 13
community, or ecosystem level studies that conclusively relate Pb ex posure to aquatic ecosystem effects

. 

### 14
In addition, most of the available work is related to point

- source Pb contamination, with very few studies 

### 15
considering the effects of diffuse Pb pollution. 

### 16
Both plant species and type of habitat were determined to be factors affecting the rate of Pb 

### 17

accumulation from contaminated sediments

. While the rooted aquatic plant E. canadensis was observed 

### 18
to accumulate the highest concentrations of Pb, the authors concluded that submerged macrophytes 

### 19
(versus emergent plants) a s a group were the most likely to accumulate Pb and other heavy metals 

### 20

( Kurilenko & Osmolovskaya, 2006 )

. This would suggest that certain types of aquatic plants, such as 

### 21
rooted and submerged species, may be more susceptible to aerially

- deposited Pb contamination, resulting 

### 22
in shifts in plant community composition as a result of Pb poll ution. 

### 23
Alteration of macrophyte community composition was demonstrated in the presence of elevated 

### 24
surface water Pb concentrations at three lake sites impacted by mining effluents

. A total of 11 species of 

### 25
macrophytes were collected

. Study sites 2 and 3 ex hibited similar dissolved Pb concentrations (78 to 92 

### 26

μg Pb /L, depending on season) and contained six and eight unique macrophyte species, respectively ( V. 

### 27

K. Mishra et al., 2008 )

. The site with the highest Pb concentrations (103 to 118 μg Pb /L) had the lowest 

### 28

number of resident macrophyte species; these included E. crassipes

, L. minor

, Azolla pinnata and 

### 29
Spirodela polyrrhiza

. Based on analysis of plant tissue Pb concentrations, the authors theorized that 

### 30
certain species may be more able to develop Pb tolerant eco

- types that can survive at higher Pb 

### 31

concentrations ( V. K. Mishra et al., 2008 )

. 

### 32
Exposure to three levels of sediment Pb contamination (322, 1,225, and 1

, 465 μg Pb /g dry weight) 

### 33
had variable effects on different species within an aquatic nematode community ( Mahmoudi et al., 2007 )

. 

### 34May 2011 7

- 99 DRAFT

– DO NOT C ITE OR QUOTE Abundance, taxa richness, and species dominance indices were altered at all Pb exposures when 

### 1
compared with unexposed communities

. Further, while the species Oncholaimellus mediterraneus 

### 2
dominated control communities (14% of total abundance), communities exposed to low and medium Pb 

### 3
concentrations were dominated by Oncholaimus campylocercoides (36%) and Marylynnia stekhoveni 

### 4
(32%), and O. campylocercoides (42%) and Chromadorina metulata ( 14%), respectively

. Communities 

### 5
exposed to the highest Pb sediment concentrations were dominated by Spirinia gerlachi (41%) and 

### 6
Hypodontolaimus colesi (29%)

. Given this, the authors concluded that exposure to Pb significantly 

### 7
reduced nematode diversity and resulted in profound restructuring of the community structure. 

### 8
The faunal composition of seagrass beds in a Spanish coastal saltwater lagoon was found to be 

### 9
impacted by Pb in sediment, plants, and biofilm ( Marín

- Guirao et al., 2005 ) . Sediment Pb concentrations 

### 10
ranged f rom approximately 100 to 5,000 mg Pb / k g and corresponding biofilm concentrations were 500 to 

### 11

1

, 600 mg Pb/kg , with leaf concentrations up to 300 mg Pb/kg . Although multiple community indices 

### 12
(abundance, Shannon

- Wiener diversity, Simpson dominance index) did not vary from site to site, 

### 13
multivariate analysis and similarity analysis indicated significant differences in macroinvertebrate 

### 14

communities between sites with different sediment, biofilm, and leaf Pb concentrations

. Differences were 

### 15
largely attributable to three amphipod species (Microdeutopus sp., Siphonoecetes sabatieri , Gammarus 

### 16
sp.). This indicates that, although seagrass abundance and biomass were unaffected by Pb exposure, 

### 17
organisms inhabiting these plants still may be adversely impacted. 

### 18

In certain freshwater habitats, exposure to Pb has been shown to result in significant alterations of 

### 19

invertebrate communities

. Macroinvertebrate community structure in mine

- influenced streams was 

### 20
determined to be significantly correlated to Pb sediment pore water c oncentrations

. Multiple invertebrate 

### 21
community indices, including Ephemeroptera, Plecoptera, Trichoptera (EPT) taxa richness, Missouri 

### 22

biotic index, and Shannon

- Wiener diversity index, were integrated into a macroinvertebrate biotic 

### 23

condition score ( Poulton et al., 2010 ) . These scores were determined to be significantly lower at sample 

### 24
sites downstream from mining sites where Pb pore water and bulk sediment concentrations were elevated. 

### 25

Rhea et al. ( 2006 ) examined the effects of multiple heavy metals in the Boulder River, MT, U.S., 

### 26
watershed biofilm on resident macroinvertebrate assemblages and community structure, and determined 

### 27
that, among all the metals, biofilm Pb concentrations exerted the greatest influence on the 

### 28
macroinvertebrate community indices

. Pb biofilm concentrations were significantly correlated with 

### 29
reduced EPT taxa richness, reduced EPT abundance, and an increase in Diptera species abundance

. 

### 30
Interestingly, Pb concentrations in invertebrate tissues were correlated to an increase in Hydrop sychidae 

### 31
caddisfly abundance, but this may have resulted from the intrinsically high variability in tissue Pb 

### 32

concentrations

. The authors concluded that Pb

- containing biofilm represented a significant dietary 

### 33
exposure for impacted macroinvertebrate species , thus altering invertebrate community metrics ( Rhea et 

### 34

al., 2006 )

. 

### 35May 2011 7

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– DO NOT C ITE OR QUOTE Kominkova and Nabelkova ( 2005 ) examined ecological risks associated with metal contamination 

### 1
(including Pb) in small urban streams

. Although surface water Pb concentrations in monitored streams 

### 2

were dete rmined to be very low, concentrations of the metal in sediment were high enough to pose a risk 

### 3
to the benthic community (e.g., 34 to 101 mg Pb /k g )

. These risks were observed to be linked to benthic 

### 4
invertebrate functional feeding group, with collector

- gath erer species exhibiting larger body burdens of 

### 5
heavy metals than other groups ( Kominkova & Nabelkova, 2005 )

. In contrast, benthic predators and 

### 6

collector

- filterers accumulated significantly lower metals concentrations

. Conse quently, it is likely that 

### 7

sediment

- bound Pb contamination would differentially affect members of the benthic invertebrate 

### 8
community, potentially altering ecosystems dynamics. 

### 9
Invertebrate functional feeding group may also affect invertebrate Pb body burde ns in those 

### 10
systems where Pb bioconcentration occurs. The predaceous zooplanktonic rotifer

, A. brigt h wellii collected 

### 11

from a Pb

- impacted reservoir in Mexico

, contained 384 ng Pb /mg and exhibited a water

- to

- tissue BCF of 

### 12

49,344. The authors theorized that P b bio magification may have been observed in this case because the 

### 13

cladoceran M. micrura is both a known Pb accumulator and a favorite prey item of the rotifer ( Rubio



### 14

Franchini et al., 2008 )

. They showed that M. micrura had twice the Pb body burden of D. similis , another 

### 15
grazing cladoceran species present in the reservoir. These two spec ies exhibited average Pb tissue 

### 16
concentrations of 57 and 98 ng Pb /mg, respectively, with respective water column BCFs of 9,022 and 

### 17

8,046

. Conversely, an examination of the simultaneous uptake of dissolved Pb by the algae P. subcapitata 

### 18

and the cladoceran D . magna suggests that the dietary exposure route for the water column filter

- feeder is 

### 19

minor

. Although Pb accumulated in the algal food source, uptake directly from the water column was 

### 20
determined to be the primary route of exposure for D. magna ( Komjarova & Blust, 2009c )

. 

### 21
For many invertebrate species, sediment Pb concentrations may be the most important driver in 

### 22
determining Pb uptake

. For instance, while Hg and Cd body burdens in lentic invertebrates were affected 

### 23
by lake ecological processes (e.g., eutrophication), a similar effect was not observed for Pb concentrations 

### 24
in crayfish tissue, despite a high variability between sites

. Although this may be a result of differing 

### 25
bioaccumulation tendencies, the authors suggested that other factors, including the potential for sediment 

### 26
exposures, may be responsible for Pb uptake in lentic invertebrates ( Larsson et al., 2007 )

. 

### 27

A risk assessment conducted in southern Florida freshwater canals determined that the 90th 

### 28
percentile of bulk Pb sediment concentrations in systems was 105 mg Pb /kg, which was predicted to 

### 29

result in a sediment pore water concentration of 2.6 μg Pb /L. This estimated pore water concentration was 

### 30
contrasted with acute 10th percentile toxic concentrations derived from a series of species sensitivity 

### 31

distributions : 8.7 μg Pb/L for arthropods, 223 μg Pb/L for fish sp ecies, and 116 μg Pb/L for all species 

### 32

( Rand & Schuler, 2009 )

. Although the predicted sediment pore water Pb concentration was below the 

### 33
derived acute toxicity hazardous concentration for 10% of species (HC10) values, it was considered 

### 34
possible that chronic exposure to such concentrations could impact some arthropod populations

. A 

### 35
chronic species sensitivity distribution constructed with Pb NOEC values for all aquatic species produced 

### 36May 2011 7

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– DO NOT C ITE OR QUOTE a chronic 10th percentile Pb NOEC value of 1.6 μg/L, a further indication that some aquatic species 

### 1
(likely arthropod species) may be impacted by the Pb contamination in southern Florida canals ( Rand & 

### 2

Schuler, 2009 )

. 

### 3

Caetano et al. ( 2007 ) investigated the mobility of Pb in salt ma r shes using total content and stable 

### 4
isotope signature. They found that roots had similar isotopic signature to sediments in vegetated zones 

### 5
indicating that Pb uptake by plants reflects the input in sediments. At one site, there was a high 

### 6
anthropogenic Pb content while at the other natural mineralogical sourc es dominated. The roots of S. 

### 7
fructicosa and S. maritima significantly accumulated Pb, having maximum concentrations of 2

, 870 mg 

### 8

Pb /kg and 1

, 755 mg Pb /kg, respectively, indicating that below

- ground biomass played an important role 

### 9
in the biogeochemical cyc ling of Pb. 

### 10
In addition to the ecological effects discussed above, there is additional evidence that Pb exposure 

### 11
could alter bacterial infection (and potentially disease transmission) in certain fish species

. Following 96

- 

### 12
hour exposures to 4

, 000 μg Pb /L, b acterial density in Channa punctatus fish was observed to be 

### 13
significantly altered when compared to non

- exposed fish. Bacteria population densities in fish spleen, 

### 14
gills, liver, kidneys and muscle tissues were higher following Pb exposure, with bacterial a bundance in 

### 15
the gills too numerous to quantify ( Pathak & Gopal, 2009 ) . In addition, bacteria inhabiting Pb

- exposed 

### 16
fish were more likely to exhibit antibacterial resistance than colonies isolated from non

- exposed fish. 

### 17
Although the mechanism remains unknown, this study suggests that Pb exposure may increase the 

### 18
likelihood of infection in fish, potentially af fecting fish abundance and recruitment. 

### 19
In summary, despite the fact that alterations of macrophyte communities may be highly visible 

### 20
effects of increased sediment Pb concentrations, several recently published papers propose that ecological 

### 21
impacts on inve rtebrate communities are also significant, and can occur at environmental Pb 

### 22
concentrations lower than those required to impact plant communities

. High sediment Pb concentrations 

### 23
were linked to shifts in amphipod communities inhabiting plant structures, an d potentially to alterations in 

### 24
ecosystem nutrient processing through selective pressures on certain invertebrate functional feeding 

### 25
groups (e.g., greater bioaccumulation and toxic effects in collector

- gatherers versus predators or filter



### 26
feeders)

. Increas ed sediment pore water Pb concentrations were demonstrated to likely be of greater 

### 27
importance to invertebrate communities, as well

. Interestingly, recent research also suggests that Pb 

### 28
exposure can alter bacterial infestations in fish, increasing both micr obial density and resilience, and 

### 29
potentially increasing the likelihood of serious disease outbreak. 

### 30



## 7.3.7. Critical Loads in Aquatic Systems

Since the publication of the 2006 Pb AQCD there is no new significant information on critical 

### 31
loads of Pb in aquatic sys tems

. Refer to Section 7.3.6 of the 2006 Pb AQCD for a discussion of critical 

### 32
loads of Pb in aquatic systems. 

### 33May 2011 7

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– DO NOT C ITE OR QUOTE 

## 7.3.8. Characterization of Sensitivity and Vulnerability

Data from the literature indicate that exposure to Pb may affect survival, reproduction, growth

, 

### 1
metabolism, and development in a wide range of aquatic species

. Often, species differences in 

### 2
metabolism, sequestration, and elimination rates control relative sensitivity and vulnerability of exposed 

### 3
organisms

. Diet and lifestage at the time of exposure also contribute significantly to the determination of 

### 4
sensitive and vulnerable populations and communities

. Further, environmental conditions in addition to 

### 5
those discussed as affecting bioavailability may also alter Pb toxicity

. The 2006 Pb AQCD reviewed the 

### 6
effects of genetics, age, and body size on Pb toxicity

. While genetics appears to be a significant 

### 7
determinant of Pb sensitivity, effects of age and body size are complicated by environmental factors that 

### 8
alter metabolic rates of aquatic organisms

. A review of the more recent literature corroborated these 

### 9
findings, and identified seasonally

- affected physiological changes and life

- stage as other important 

### 10
determinants of differential sensitivity to Pb. 

### 11

7.3.8.1. Seasonally

- Affected Physiological Changes

A study b y Duman et al. ( 2006 ) identified species and seasonal effects of Pb uptake in aquatic 

### 12
plants

. P. australis accumulated higher root Pb concentrations than S. lacustris . Additionally, the P. 

### 13

australis Pb accumulation factor was signi ficantly higher during the winter versus other seasons, while 

### 14

the Pb accumulation factor for S. lacustris was greatest in spring and autumn. The Pb accumulation factor 

### 15
for a third species, P. lucens , was greatest in autumn ( Duman et al., 2006 )

. Most significantly, these 

### 16
changes in bioaccumulation were not linked with biomass increases, indicating that species

- dependent 

### 17
seasonal physiological changes may control Pb uptake in aquatic macrophytes ( Duman et al., 2007 )

. 

### 18
Significant interspeci es differences in Pb uptake were observed for plants representing the same genus 

### 19
(Sargassum), indicating that uptake of Pb by aquatic plants also may be governed by highly species



### 20
dependent factors ( Jothinayagi & Anbazhagan, 2009 )

. 

### 21

Couture et al. ( 2010 ) investigated seasonal and decadal variations in Pb sources to mussels ( M. 

### 22

edulis ) from the French Atlantic shoreline. Pb concentrations in the mussels were 5

- 66 times higher than 

### 23
the natural background v alue for the north Atlantic. The 206 Pb/ 207 Pb signature indicated that the 

### 24
bioaccumulated Pb was anthropogenic in origin. The signature was not, however, the same as that emitted 

### 25
in western Europe as a result of leaded gasoline combustion, although that was a major emission source to 

### 26
the atmosphere during a large part of the study period (1985

- 2005). Instead, it was most similar to that of 

### 27
Pb released into the environment from wastewater treatment plants, municipal waste incinerators and 

### 28

industries such as m etal refineries and smelters. Thus continental runoff rather than atmospheric 

### 29
deposition was identified as the main source of Pb to the French coastal area. The strong seasonal 

### 30

variations in 206 Pb/ 208 Pb were used to conclude that resuspension of Pb trigger ed by high river runoff 

### 31
events was a key factor affecting bioaccumulation of Pb in M. edulis

. In another biota monitoring study, 

### 32May 2011 7

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– DO NOT C ITE OR QUOTE Pearce and Mann ( 2006 ) investi gated variations in concentrations of trace metals in the U.K. including Pb 

### 1
in the shells of pod razor shell ( Ensis siliqua ). Pb concentration varied from 3.06

- 36.2 mg Pb /kg and 

### 2
showed a regional relationship to known sources, e.g., former metal mining are as such as Cardigan Bay, 

### 3
Anglesey, and industrial activity in Liverpool Bay. Seasonal variations were also found for Pb in both 

### 4
Cardigan Bay and Liverpool Bay, relating to increased winter fluxes of Pb (and other metals) into the 

### 5

marine environment. Heier et al. ( 2009 ) established the speciation of Pb in water draining from a shooting 

### 6
range in Norway and looked at the time dependent accumulation in brown trout. They found that high 

### 7
molecular weight (>10 kDa l tons ) cationic Pb species correlated with high flow episodes and 

### 8
accumulation of Pb on gills and in the liver. Thus, high flow episodes can remobilize metals from a 

### 9
catchment and induce stress to aquatic organisms

. 

### 10

7.3.8.2. Increased Nutrient Uptake

Singh et al. ( 2010 ) proposed that metal

- resistant plants have the capacity to not only up

- regulate 

### 11
antioxidant synthesis, but also have the ability to increase nutrient consumption and uptake to support 

### 12
metal sequestration and detoxification via production of antioxidants ( Singh et al., 2010 ) . Therefore, it is 

### 13
likely that such plant species would be significantly less susceptible to Pb exposure than those species 

### 14

w ithout those abilities. 

### 15

7.3.8.3. Temperature and pH

Water temperature also appears to affect the toxicity of Pb to aquatic organisms, with higher 

### 16
temperatures leading to greater responses

. Pb toxicity to crayfish increased 7 to 10% when the water 

### 17
temperature was in creased by 4°C, and by 14% when the temperature increased by 7°C

. The authors 

### 18
determined that the increased toxicity was a result of the negative impact of Pb on crayfish respiration, 

### 19
which was exacerbated by the lower dissolved oxygen concentrations at higher water temperatures ( M. A. 

### 20
Q. Khan et al., 2006 )

. The sequestration ability of L. minor macrophytes was similarly impacted by 

### 21
increased surface water temperatu re; plants absorbed a maximum Pb concentration of 8.6 mg /g at 30°C, 

### 22
while uptake at 15°C was only 0.3 mg/g ( Uysal & Taner, 2009 ) . Decreased pH was also demonstrated to 

### 23
increase the uptake of environmental Pb in aquatic plants ( Uysal & Taner, 2009 ; C. Wang, X. Yan, et al., 

### 24

2010 )

. Additionally, Birceanu et al. ( 2008 ) determined that fish (specifically rainbow tr out) were more 

### 25
susceptible to Pb toxicity in acidic, soft waters characteristic of sensitive regions in Canada and 

### 26

Scandinavia

. Hence, fish species endemic to such systems may be more at risk from Pb contamination 

### 27
than fish species in other habitats. 

### 28May 2011 7

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– DO NOT C ITE OR QUOTE 

### 7.3.8.4. Life S tage

A comparison of C.riparius Pb LC 50 values derived from toxicity tests with different instars 

### 1
indicates a significant effect of lifestage on Pb sensitivity for aquatic invertebrates

. Bechard et al. ( 2008 ) 

### 2

calculated a first instar C. riparius 24

- hour LC 50 value of 613 μg Pb /L, and contrasted this value with the 

### 3

24

- hour and 48

- hour LC 50 s derived using later instar larvae

— 350,000 and 200,000 μg Pb/L, respectively

. 

### 4
This disparity would suggest that seasonal co

- occurrence of aquatic Pb contamination and sensitive early 

### 5
instars could have significant population

- level impacts ( Bechard et al., 2008 )

. Similarly , Wang et al. 

### 6
( 2010 ) demonstrated that the newly transformed juvenile mussels, L. siliquoidea and L. rafinesqueana , at 

### 7
5 days old were more sensitive to Pb exposure than were glochidia or tw o to six month

- old juveniles, 

### 8
suggesting that Pb exposure at particularly sensitive lifestages could have a significant influence on 

### 9
population viability ( N. Wang et al., 2010 )

. 

### 10
Pb exposures also differentially affected life

- stages of the marine polychaete H. elegans

. Pb water 

### 11
concentrations of 100 μg Pb /L and greater significantly affected fertilization and embryonic development, 

### 12
but the greatest effects were exhibited by 24

- hour

- old larvae ( Gopalakrishnan et al., 2007 )

. The authors 

### 13
suggested that timing of Pb exposure may have different impacts on marine polychaete populations, if life 

### 14
cycles are off

- set ( Gopalakrishnan et al., 2007 )

. Further, given that the adult lifestage is sedentary, 

### 15

reduction of the mobile early lifestage as a result of Pb exposures may disproportionally affect sessile 

### 16
polychaetes

. For instance, larval settlement was significantly reduced at Pb exposures of 50 μg Pb /L and 

### 17

greater ( Gopalakrishnan et al., 2008 )

. 

### 18

7.3.8.5. Species S ensitivity

Both inter

- and intra

- specific difference in Pb uptake and bioaccumulation may occur in 

### 19

macroinvertebrates of the same functional

- feeding group

. Cid et al. ( 2010 ) reported significant differences 

### 20

i n Pb bioaccumulation between field collected Ephoron virgo mayflies and Hydropysche sp, caddisflies, 

### 21
with only the mayfly exhibiting increased Pb tissue concentrations when collected from Pb

- contaminated 

### 22
sites; the caddisfly Pb tissue concentrations were s imilar between reference and Pb

- contaminated areas

. 

### 23
The authors also examined the lifestage specific accumulation of Pb for E. virgo mayflies, and although 

### 24
there was no statistical difference in Pb tissue concentrations between different lifestages, Pb 

### 25

bio accumulation did change as mayflies aged ( Cid et al., 2010 )

. Data from 20 years of monitoring of 

### 26

contaminant levels in filter

- feeding mussels Mytilus sp. and oysters Crassotrea virginica in coastal areas 

### 27
of the U.S. through the National Oceanic and Atmospheric Administration (NOAA) Mussel Watc h 

### 28
program indicate that Pb is on average three times higher in mussels than in oysters ( Kimbrough et al., 

### 29

2008 )

. 

### 30
Species

- specific Ca requirements have also been shown to affect the vulnerability of aquatic 

### 31
organisms to Pb. The snail, L. stagnalis , exhibits an unusually high Ca de mand due to CaCO 3 formation 

### 32May 2011 7

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– DO NOT C ITE OR QUOTE required for shell production and growth, and exposure to Pb prevents the uptake of needed Ca, leading to 

### 1
toxicity. Consequently, aquatic species that require high assimilation rates of environmental Ca for 

### 2

homeostasis are likel y to be more sensitive to Pb contamination ( Grosell & Brix, 2009 )

. Grosell and 

### 3
colleagues also noted that reduced snail growth following chronic Pb exposure was likely a result of 

### 4
reduced Ca uptake ( Grosell et al., 2006b )

. 

### 5
There is some indication that molting may comprise an additional sequestration and excretion 

### 6
pathway for aquatic animals exposed to Pb ( Bergey & Weis, 2007 ; Mohapatra et al., 2009 ; Soto

- Jiménez 

### 7

et al. ; Tollett et al., 2009 )

. Crab species U. pugnax ( Bergey & Weis, 2007 ) and Scylla serrata ( Mohapatra 

### 8

et al., 2009 ) , white shrimp L. vannamei ( Soto

- Jiménez et al. ) as well as Libellulidae dragonfly nymphs 

### 9

( Tollett et al., 2009 ) have been shown to preferentially sequester Pb in exoskeleton tissue, where it is later 

### 10
shed along with the hardened exterior tissue

. Consequently, aquatic arthropod species and those specie s 

### 11
that shed their exoskeleton more frequently may be able to tolerate higher environmental Pb 

### 12

concentrations than non

- arthropods or slow

- growing molting species, as this pathway allows them to 

### 13
effectively lower Pb body burdens. 

### 14
Some tolerant species of fis h (e.g., mummichog) have the ability to sequester accumulated Pb in 

### 15

metal

- rich granules or heat

- stable proteins ( Goto & Wallace, 2010 )

. Fish with such abilities are more 

### 16
likely to thrive in Pb

- contaminated environments than other species

. In contrast, the effect of Pb exposure 

### 17
on fish bacterial loads demonstrated by Pathak and Gopal ( 2009 ) suggest that infected fish populations 

### 18
may be more at risk to the toxic effects of Pb than healthier species

. Aqueous Pb was demonstrated to 

### 19
both increase bacteria density in several fish organs and to improve the likelihood of antibacterial 

### 20

resistance ( Pathak & Gopal, 2009 )

. 

### 21

Tole rance to prolonged Pb exposure may develop in aquatic invertebrates and fish

. Multi



### 22
generational exposure to low levels of Pb appears to confer some degree of metal tolerance in 

### 23

invertebrates such as Chironomus plumosus larvae; consequently, previous popul ation Pb exposures may 

### 24
decrease species ’ susceptibility to Pb contamination ( Vedamanikam & Shazilli, 2008 )

. However, the 

### 25
authors noted that metal tolerant larvae were significantly smaller than larvae reared under clean 

### 26

conditions, and that transference of Pb

- tolerant C. plumosus larvae to clean systems resulted in a 

### 27
subsequent loss of tolerance

. Evidence of acclimation to elevated Pb in fathead minnow was suggested in 

### 28
the variations in ionoregulatory parameters that were measured on day 10 and 30 in fish ex posed to 115 

### 29
μg Pb / L for 30 days. At the end of the experiment, whole body Ca 2+ was elevated while Na + and K + 

### 30
recovered from elevated levels at 30 days ( Grosell et al., 2006a )

. 

### 31
A series of species sensitivity distributions constructed by Brix et al. ( 2005 ) indicated that 

### 32
sensitivity to Pb was greatest in crustacean species, followed by coldwater fish, and warmwater fish and 

### 33
aquatic insects, which exhibited a similar sensitivity. Further, analysis of both acute and chroni c 

### 34

mesocosm data sets indicated that Pb

- contaminated systems exhibited diminished species diversity and 

### 35
taxa richness following both types of exposure ( Brix et al., 2005 ) . Wong et al. ( 2009 ) constructed Pb 

### 36May 2011 7

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– DO NOT C ITE OR QUOTE species sensitivity distributions for both cladoceran and copepod species

. A comparison of the two curves 

### 1
indicated that cladoceran species, as a group, were more sensitive to the toxic effects of Pb than were 

### 2
copepods, with respective hazardous concentration values for 5% of the species (HC5) values of 35 and 

### 3
77 μg Pb/L. This difference in sensitivities would indicate that cladoceran species are more likely to be 

### 4
impacted at lower environmental Pb concentrations than copepods, potentially altering community 

### 5
structures or ecosystem functions ( L. C. Wong et al., 2009 )

. 

### 6

7.3.8.6. Ecosystem Vulnerability

Relative vulnerability of different aquatic ecosystems to effects of Pb can be inferred from the 

### 7
information discussed above on species sensitivity and the influence of water quality variables on th e 

### 8
bioavailability and toxicity of Pb. It is, however, difficult to categorically state that certain plant, 

### 9

invertebrate or vertebrate communities are more vulnerable to Pb than others

, since toxicity is dependent 

### 10
on many variables and data from field studi es are complicated by co

- occurrence of other metals and 

### 11
alterations of pH, such as in mining areas. Aquatic ecosystems with low pH and low DOM are likely to be 

### 12
the most sensitive to the effects of atmospherically

- deposited Pb. Examples of such systems are acidic, 

### 13
soft waters such as sensitive regions in Canada and Scandinavia ( Birceanu et al., 2008 ) . In the U.S., 

### 14
aquatic systems that may be more susceptible to effects of Pb include habitats that are acidified due to 

### 15
atmospheric deposition of pollutants, runoff from mining activities or lakes and streams with naturally 

### 16
occurring organic acids. Hence, fish and invertebrate species endemic to such systems may be more at 

### 17
risk from Pb contamination than corresponding species in other habitats. 

### 18

19



## 7.3.9. Ecosystem Services

There are limited publications at this time that address Pb i mpacts to ecosystem services associated 

### 20
with aquatic systems and most of the available literature is for estuaries, salt marsh and freshwater 

### 21

wetlands rather than lakes and streams

. The evidence reviewed in the ISA illustrates that Pb can affect the 

### 22
ecolog ical effects in each of the four main categories of ecosystem services (Section 7.1.2) as defined by 

### 23

Hassan et al. ( 2005 ) . These effects are sorted into e cosystem services categories and summarized here: 

### 24

 Supporting: food for higher trophic levels, biodiversity 

### 25

 Provisioning: clean drinking water, contamination of food by heavy metals, decline in health 

### 26
of fish and other aquatic species 

### 27

 Regulating: water qua lity 

### 28

 Cultural: ecosystem and cultural heritage values related to ecosystem integrity and 

### 29
biodiversity, wildlife and bird watching, fishing 

### 30May 2011 7

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– DO NOT C ITE OR QUOTE A few recent studies consider the impact of Pb and other heavy metals on ecosystem services 

### 1
provided by estuaries ( Smith et al., 2009 ) and salt marsh {Bromberg Gedan, 2009, 672706}. These 

### 2
systems are natural sinks for metals and other contaminants. They provide habitat and bre eding areas for 

### 3
both terrestrial and marine wildlife and are locations for bird watching

. In a Monte

- Carlo modeling 

### 4
approach designed to assess the degree of risk of Pb and Hg to wading birds in estuarine habitats in the 

### 5
U.K., the authors found a high prob ability that Pb may pose an ecologically relevant risk to dunlin, 

### 6
Calidris alpina ( Smith et al., 2009 ) . However, the authors noted that a major source of uncerta inty in this 

### 7
study was the NOAEL values for Pb. Pb can be toxic to salt marsh plant species and decaying plant 

### 8
detritus may result in resuspension of Pb into the aquatic food chain {Bromberg Gedan, 2009, 672706}

. 

### 9
Ecological services provided by freshwater wetlands are similar to those provided by estuaries and 

### 10
are sinks for atmospheric Pb as well as Pb from terrestrial runoff ( Landre et al., 2010 ; Watmough & 

### 11

Dillon, 2007 )

. Several studies have addressed the response of natural wetlands to Pb ( Gambrell, 1994 ; 

### 12

Odum, 2000 )

. Recent reviews of pollution con trol by constructed wetlands ( Mander & Mitsch, 2009 )

, 

### 13
removal of metals by constructed wetlands ( Marchand et al., 2010 ) and phytoremediation of metals by 

### 14
wetland plants ( Rai, 2008 ) indicate that these systems can remove Pb from the aquatic environment. The 

### 15
use of plants as a tool for immobilization of Pb and other metals from the environment is not limited to 

### 16
wetland species. Recent advances in the phytoremediation of metals are reviewed in Dickinson et al. 

### 17

( 2009 )

. 

### 18
The impact of Pb on ecological services provided by specific components of aquatic systems has 

### 19

been considered in a limited number of studies

. Aquatic fauna can take up and bioaccumulate metals

. If 

### 20
the bioaccumulating species is a food source, the uptake of metals may make it toxic or more dangerous 

### 21
for people or other wildlife to consume

. For example, oysters and mussels bioaccumulate Pb from 

### 22
anthropogenic sources, including atmospheric deposition, and are a food source that is widely consumed 

### 23
by humans and wildlife ( Couture et al., 2010 )

. Their capacity to bioaccumulate Pb makes them good 

### 24
bioindicators of environmental contamination and they have been used as monitors of coastal pollutants 

### 25
by the NOAA Mussel Watch program since 1986. The conclusions of a recent assessment report are that 

### 26
the highest concentrations of Pb are found in mussels and oysters near urban and industrial centers and 

### 27
the only region that exceeded the Food and Drug Administrat ion action level for Pb in clams, oysters and 

### 28
mussels of 1.7 mg Pb /kg wet weight was Lake Michigan

, where maximum concentrations were 1.9 mg 

### 29

Pb /kg wet weight in the years 2004

- 2005 ( Kimbrough et al., 2008 )

. Although bioaccumulation may 

### 30
render aquatic fauna toxic to consumers, bio accumulation is a way to sequester the metals and remove 

### 31

them from waters and soils

. Sequestration for this purpose is an ecosystem service that has been 

### 32
quantified

. For example, the total ecological services value of a constructed intertidal oyster (Crass ostrea 

### 33
sp.) reef in improving water quality and sequestering metals including Pb was calculated in the Yangtze 

### 34
River estuary to be about $500,000 per year ( Quan et al., 2009 )

. Other aquatic organisms have been 

### 35May 2011 7

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– DO NOT C ITE OR QUOTE considered for their role in remediation of Pb in the environment

. Theegala et al. ( 2007 ) discuss the high 

### 1
uptake rate of Pb by D. pulex as the basis for a possible Daphnia

- based remediation for aquatic systems. 

### 2



## 7.3.10. Summary of Aquatic Effects

This summary of the effects of Pb on aquatic ecosystems covers information from the publication 

### 3
of the 2006 Pb AQCD to present

. Refer to Section 7.4: Causality determinations for Pb in Terrestrial and 

### 4
Aquatic Systems for a synthesis of all evidence dating back to the 1977 AQCD considered to determine 

### 5
causality. 

### 6

7.3.10.1. Biogeochemistry and Chemical Effects

Once the atmospherically

- derived Pb enters surface waters its fate and bioavailability are 

### 7
influenced by Ca concentration, pH, alkalinity, and total suspended solids, and DOC, including humic 

### 8

acids. Once in sediments , Pb bioavailability may be influenced by the presence of sulfides and Fe and Mn 

### 9
oxides, physical disturbance, the presence of other metals, biofilm and organisms. In many, but not all 

### 10
aquatic organisms, Pb dissolved in the water can be the primary exposur e route to gills or other biotic 

### 11
ligands. A more detailed understanding of the biogeochemistry of Pb in aquatic systems (both the water 

### 12
column and sediments) is critical to accurately predicting toxic effects of Pb to aquatic organisms. As 

### 13
recognized in th e 2006 Pb AQCD and further supported in this review, chronic exposures to Pb may also 

### 14
include dietary uptake, and there is an increasing body of evidence showing that differences in uptake and 

### 15
elimination of Pb vary with species

. Currently available models for predicting bio a vailability focus on 

### 16
acute toxicity and do not consider all possible routes of uptake

. They are therefore of limited applicability, 

### 17
especially when considering species

- dependent differences in uptake and bioaccumulation of Pb. 

### 18

7.3.10.2. Bioavail ability

There is evidence over several decades of research previously reviewed in Pb AQCDs and in recent 

### 19
studies reviewed in this ISA that Pb bioaccumulates in plants, invertebrates and vertebrates in aquatic 

### 20
systems, just as it does in terrestrial system s. According to the 2006 Pb AQCD, and further supported in 

### 21
this review, Pb adsorption, complexation, chelation, etc., are processes that alter bioavailability to aquatic 

### 22
biota. Given the low solubility of Pb in water, bioaccumulation of Pb by aquatic organ isms may 

### 23
preferentially occur via exposure routes other than direct absorption from the water column, including 

### 24
ingestion of contaminated food and water, uptake from sediment pore waters, or incidental ingestion of 

### 25

sediment

. 

### 26
As reviewed by Wang and Rainbo w ( 2008 ) and supported by additional studies reviewed in this 

### 27
ISA, there are considerable differences between species in the amount of Pb taken up from the 

### 28
environment and in the levels of Pb retained in the organism. The bioaccumulation and toxicity of Pb to 

### 29May 2011 7

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– DO NOT C ITE OR QUOTE aqua tic organisms are closely linked to the environmental fate of the metal under variable environmental 

### 1
conditions (Section 3.3) as they are highly dependent upon the proportion of free metal ions in the water 

### 2

column. 

### 3
Recent studies on bioavailability of Pb i n aquatic plants and algae support the findings of previous 

### 4

Pb AQCDs that all plants tend to sequester larger amounts of Pb in their roots than in their shoots and 

### 5
provide additional evidence for species differences in compartmentalization of sequestered P b and 

### 6
responses to Pb in water and sediments. Given that atmospherically

- derived Pb is likely to become 

### 7
sequestered in sediments, uptake by aquatic plants is a significant route of Pb removal from sediments, 

### 8
and a potential route for Pb mobilization into t he aquatic food web

. Although there are some similarities 

### 9
to Pb accumulation observed in terrestrial plants (e.g., preferential sequestration of the metal in root 

### 10
tissue), Pb appears to be more bioavailable in sediment than it is in soil

. Trees that inhabi t semi

- aquatic 

### 11

environments have also been shown to absorb Pb from contaminated sediments

. 

### 12
In the case of invertebrates, Pb can be bioaccumulated from multiple sources, including the water 

### 13
column, sediment, and dietary exposures, and factors such as amoun t of bioavailable Pb, lifestage, age, 

### 14

and metabolism can alter the accumulation rate. Additional studies have considered the relative 

### 15
importance of water versus dietary uptake of Pb in aquatic invertebrates. Use of stable isotopes ha s 

### 16

enabled simultaneous measurement of uptake and elimination in several aquatic species to assess the 

### 17
relative importance of water versus dietary uptake

. In uptake studies of various invertebrates, Pb was 

### 18
mainly found in the gills and digestive gland/hepatopancreas. 

### 19

There is mor e information now on the cellular and subcellular distribution of Pb in invertebrates 

### 20
than there was at the time of writing the 2006 Pb AQCD. Specifically, localization of Pb at the 

### 21
ultrastructural level has been assessed in the marine mussel ( M. edulis ), scallop and cuttlefish and was 

### 22
found to be bound principally to organelles ( Einsporn et al., 2009 ; Einsporn & Koehler, 2008 ) . Since the 

### 23
2006 Pb AQCD, BCF ’ s have been measured for several species in field studies and these BCF ’ s tend to be 

### 24
higher than calculated BCF ’ s from laboratory exposures (Table 7

- 4). 

### 25
Tissue accumulation of Pb via gill and dietary uptake has been further characterized in aquatic 

### 26
vertebrates and stable isotope techniques have been applied to further elucidate bioaccumulation of Pb in 

### 27
this ISA. The conclusions of the 2006 Pb AQCD that the gill is a major site of Pb uptake in fish and that 

### 28
there are species differences in the of Pb accumulation and distribution of Pb within the organism are 

### 29
supported in this review. In general, the accumulation of Pb in fish tissues is observed to be 

### 30
gill>kidney>liver. The anterior intestine ha s been newly identified as a site of uptake of Pb through 

### 31
dietary exposure studies ( Alves et al., 2006 ) . Additional detoxification strategies for Pb have been 

### 32
elucidated since the 2006 Pb AQCD. Mummichogs at more polluted sites stored a higher amount of Pb in 

### 33
metal rich granules as compared to other detoxifying ce llular components such as heat

- stable proteins, 

### 34

heat

- denaturable proteins and organelles ( Goto & Wallace, 2010 )

. 

### 35May 2011 7

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– DO NOT C ITE OR QUOTE There are few new studies on Pb uptake by amphibians and mammals. At the time of the 

### 1
publication of the 2006 Pb AQCD, trophic transfer of Pb through aquatic food chains was considered to 

### 2
be negligible

. Measured concentrations of Pb in the tissues of aquatic organisms we re generally higher in 

### 3
algae and benthic organisms than in higher trophic

- level consumers indicating that Pb was 

### 4
bioconcentrated but not biomagnified ( Eisler, 2000 ; U.S. EPA, 2006 )

. Some studies published since the 

### 5
2006 Pb AQCD support the potential for Pb to be transferred in aquatic food webs, while other studies 

### 6
indicate that Pb concentration decreases with increasing trophic level (biodilution). 

### 7

7.3.10.3. Biological Effects

Evidence in this review further supports the findings of the previous Pb AQCDs that waterborne Pb 

### 8
is highly toxic to aquatic organ isms, with toxicity varying with species and lifestage, duration of 

### 9
exposure, form of Pb, and water quality characteristics. 

### 10
Effects of Pb on algae reported in the 2006 Pb AQCD included decreased growth, deformation and 

### 11
disintegration of algae cells, and b locking of the pathways that lead to pigment synthesis, thus affecting 

### 12
photosynthesis

. Observations in additional algal species since the 2006 Pb AQCD support these findings. 

### 13
Effects on plants supported by additional evidence in this review and evidence fr om previous reviews 

### 14
include oxidative damage, decreased photosynthesis and reduced growth. The mechanism of Pb toxicity 

### 15
in plants is likely mediated by damage to photosystem II through alteration of chlorophyll structure

. 

### 16

Elevated levels of antioxidant enz ymes are commonly observed in aquatic plant, algae, and moss species 

### 17
exposed to Pb. 

### 18
As observed in terrestrial invertebrates, upregulation of antioxidant enzymes is a common 

### 19
biomarker of Pb exposure in aquatic invertebrates

. Since the 2006 Pb AQCD, there is additional evidence 

### 20
for Pb effects on antioxidant enzymes, lipid peroxidation, stress response and osmoregulation

. Studies of 

### 21
reproductive and developmental effects of Pb in aquatic invertebrates in this review provide further 

### 22
support for findings in th e 2006 Pb AQCD

. These new studies include reproductive endpoints for rotifers 

### 23
and freshwater snails as well as multigenerational effects of Pb in mosquito larvae

. Growth effects are 

### 24
observed at lower concentrations in some aquatic invertebrates since the 2 006 Pb AQCD, including 

### 25
juveniles of the freshwater snail L. stagnalis where growth is affected at < 4 μ g Pb /L ( Grosell et al., 

### 26

2006b )

. Behavioral effects of Pb in aquatic invertebrates reviewed in this ISA include decreased valve 

### 27
closing speed in scallops and slower feeding rate in blackworms. 

### 28
Additional mechanisms of Pb toxicity have been elucidated in the gill and the renal system of fish 

### 29
since the 2006 Pb AQCD. Further supporting evidence of reproductive, behavioral, growth effects and 

### 30
effects on blood parameters have become available since the 2006 Pb AQCD. The mitogen

- activated 

### 31
protein kinases, ER K1/2 and p38 MAPK were identified for the first time as possible molecular targets for 

### 32
Pb neurotoxicity in a tel e ost ( Leal et al., 2006 ) . Pb toxicity at the fish gill primarily involves disruption of 

### 33May 2011 7

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– DO NOT C ITE OR QUOTE Ca homeostasis as previously characterized in the 2006 Pb AQCD ( Rogers & Wood, 2004 ; Rogers & 

### 1

Wood, 2003 )

. In addition to this mechanism, Pb was found to induce ionoregulatory toxicity at the gill of 

### 2
rainbow trout through a binding of Pb with Na

- K, ATPase and rapid inhibition of carbonic anhydrase 

### 3
activity thus enabling noncompetitive inhibition of Na + and Cl - influx. 

### 4
In the 2006 Pb AQCD amphibians were considered to be relatively tolerant to Pb

. Observed 

### 5
responses to Pb exposure included decreased enzyme activity ( e.g., ALAD reduction) and changes in 

### 6

behavior summarized in Table AX7

- 2.4.3 of the 2006 Pb AQCD ( U.S. EPA, 2006 )

. Since the 2006 Pb 

### 7
AQCD, studies conducted at concentrations approaching environmental levels of Pb have indicated 

### 8
sublethal effects on tadpole endpoints including growth, deformity, and swi mming ability. 

### 9

7.3.10.4. Exposure and Response

Concentration

- response data on plants, invertebrates and vertebrates is consistent with findings in 

### 10
previous reviews of species differences in sensitivity to Pb in aquatic systems

. Growth in plants continues 

### 11

to be an en dpoint adversely affected by Pb exposure

. The lowest EC 50 for growth observed in marine 

### 12
microalgae and freshwater microalgae was in the range of 100 μg/L. 

### 13
In the 2006 Pb AQCD, concentrations at which effects were observed in aquatic invertebrates 

### 14
ranged fr om 5 to 8

, 000 μg Pb /L

. Several studies in this review have provided evidence of effects at lower 

### 15

concentrations

. Among the most sensitive species, growth of juvenile freshwater snail s L. stagnalis was 

### 16

inhibited at an EC 20 of <4 μg Pb /L. ( Grosell & Brix, 2009 ; Grosell et al., 2006b )

. A chronic value of 10 

### 17

μg Pb /L obtained in 28

- day exposures of 2

- month

- old L. siliquoidea juveniles was the lowest genus mean 

### 18
chronic value ever reported for Pb ( N. Wang et al., 2010 )

. 

### 19
In the 2006 Pb AQCD, adverse effects were found in freshwater fish at concentrations ranging from 

### 20

10 to >5

, 400 μg Pb/L, generally depending on water quality variables ( e.g., pH, hardness, salinity)

. 

### 21
Additional testing of Pb toxicity under conditions of varied alkalinity, DOC, and pH ha s been conducted 

### 22
since the last review. However, adverse effects in fish observed in recent studies fall within the range of 

### 23
concentrations observed in the previous AQC D

. 

### 24

7.3.10.5. Community and Ecosystem Effects

Since the publication of the 2006 Pb AQCD, additional evidence for community and ecosystem 

### 25
level effects of Pb have been observed primarily in microcosm studies or field studies with other metals 

### 26
present. One ecological effect reported in previous Pb AQCDs is a shift in community comp osition in Pb



### 27
impacted habitats towards more Pb

- tolerant species

. New studies in this ISA provide evidence in 

### 28
additional habitats for community composition shifts associated with Pb

. Alteration of aquatic plant 

### 29
community composition was demonstrated in the presence of elevated surface water Pb concentrations at 

### 30
three lake sites impacted by mining effluents

. Lakes with the highest levels of Pb had the lowest number 

### 31May 2011 7

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– DO NOT C ITE OR QUOTE of aquatic plant species when compared to sites with lower Pb concentrations. In an aquatic ma crophyte 

### 1
community, both plant species and type of habitat were determined to be factors affecting the rate of Pb 

### 2
accumulation from contaminated sediments. While the rooted macrophyte E. canadensis was observed to 

### 3
accumulate the highest concentrations of P b, the authors concluded that submerged macrophytes (versus 

### 4
emergent plants) as a group were the most likely to accumulate Pb and other heavy metals ( Kurilenko & 

### 5
Osmolovskaya, 2006 )

. This would suggest that certain types of aquatic plants, such as rooted and 

### 6
submerged species, may be most susceptible to atmospherically

- deposited Pb, re sulting in shifts in plant 

### 7
community composition as a result of Pb pollution. 

### 8
Despite the fact that alterations of macrophyte communities may be highly visible effects of 

### 9
increased sediment Pb concentrations, several recently published papers propose that ecological impacts 

### 10
on invertebrate communities are also significant, and can occur at environmental Pb concentrations lower 

### 11
than those required to impact plant communities. High sediment Pb concentrations were linked to shifts in 

### 12
amphipod communities inhab iting plant structures, and potentially to alterations in ecosystem nutrient 

### 13
processing through selective pressures on certain invertebrate functional feeding groups. 

### 14
Sensitive species may become locally extinct from habitats where Pb toxicity is greater. Birceanu et 

### 15

al

. ( 2008 ) determined that fish, specifically rainbow trout, were more susce ptible to Pb toxicity in acidic, 

### 16
soft waters characteristic of sensitive regions in Canada and Scandinavia. Hence, fish species endemic to 

### 17
such systems may be more at risk from Pb contamination than fish species in other habitats. A series of 

### 18
species sensi tivity distributions constructed by Brix et al. ( 20 05 ) indicated that sensitivity to Pb was 

### 19
greatest in crustacean species, followed by coldwater fish, and warmwater fish and aquatic insects, which 

### 20
exhibited a similar sensitivity. 

### 21

7.3.10.6. Critical Loads, Sensitivity and Vulnerability

Since the 2006 Pb AQCD there is no new significant information on critical loads of Pb in aquatic 

### 22

systems

. Recent studies have identified seasonally

- affected physiological changes and life

- stage as 

### 23
important determinants of differential sensitivity to Pb in aquatic organisms. Th ese factors are in addition 

### 24
to species differences in metabolism, sequestration, and elimination rates, diet, lifestage, genetics, age, 

### 25
and body size that were considered in the 2006 Pb AQCD

. Although evidence is available to support Pb 

### 26
impacts to supporti ng, provisioning, regulating and cultural ecosystem services, there is insufficient data 

### 27
available to adequately quantify these adverse effects. 

### 28May 2011 7

- 113 DRAFT

– DO NOT C ITE OR QUOTE 

# 7.4. Causality Determinations for Lead in Terrestrial and Aquatic Systems

This section presents key conclusions rega rding causality determinations for welfare effects of Pb 

### 1
(Table 7

- 6). Evidence considered in establishing causality was drawn from the 1977 ( U.S. EPA, 1977 )

, 

### 2

1986 ( U.S. EPA, 1986 ) and 2006 Pb AQCD ( U.S. EPA, 2006 ) for Pb where appropriate as well as the 

### 3

current ISA. EPA ’ s framework for causality described in Chapter 1 was applied and the causal 

### 4
determinations are highlighted. In this ISA, effects determined to be causal at the species level contribute 

### 5
to the body of evidence for causal effects at the community and ecosystem scale. Some of the effects of 

### 6
Pb observed in terrestrial and aquatic organisms are also considered in the chapters of the ISA that 

### 7
evaluate evidence for human health effects associated with Pb exposure

. 

### 8

Table 7

- 6. Summary of Pb causal determinations for plants, invertebrates and vertebrates

Effect Causality Determination

Bioaccumulation as it Affects Ecosystem Services

- All organisms Causal
Mortality

- Plants Inadequate
Mortality

- Invertebrates and Vertebrates Causal

Growth

- Plants Causal

Growth

- Invertebrates Causal

Growth

- Vertebrates Suggestive
Physiological Stress

- All organisms Causal
Hematological Effects

- Invertebrates and Vertebrates Causal
Development and Reproduction

- Inv ertebrates and V ertebrates Causal
Development and Reproduction

- Plants Inadequate

Neurobehavior

- Invertebrates and Vertebrates Causal
Community and Ecosystem Level Effects Causal



## 7.4.1. Bioaccumulation of Lead in Terrestrial and Aquatic Biota as it Affects Ecosystem Services

Pb deposited on the surface of, or taken up by organisms has the potential to alter the services 

### 9
provided by terrestrial and aquatic biota to humans. Ecosystem services are the benefits people obtain 

### 10
from ecosystems . They include supporting, provisioning, regulating and cultural services that are vital for 

### 11
the functioning of the biosphere and provide the basis for the delivery of tangible benefits to human 

### 12
society. There is compelling evidence over several decades of research that Pb bioaccumulates in plant s, 

### 13
invertebrates and vertebrates in terrestrial and aquatic systems. Generally, there are considerable 

### 14
differences between species in the amount of Pb taken up from the environment and in the amounts of Pb 

### 15May 2011 7

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– DO NOT C ITE OR QUOTE retained in the organism. In order for Pb to reach a biological receptor, the metal must first cross the 

### 1
membranes of organisms to the target organ or site of storage. This process varies between plants, 

### 2

invertebrates and vertebrates, and furthermor e, uptake and sequestration are at times similar in unrelated 

### 3
species , while substantially different between related one s. Uptake of Pb from environmental media is 

### 4
dependent upon the bioaccessibility of Pb (reviewed in Chapter 3) which is influenced by man y factors 

### 5
including, but not limited to, temperature, pH, presence of humic acid and dissolved organic matter

, 

### 6
presence of other metals

, and speciation of Pb. 

### 7
Terrestrial plants accumulate Pb via direct stomatal uptake into foliage and incorporation of 

### 8

at mospherically

- deposited Pb from soil into root tissue, followed by variable translocation to other 

### 9

tissues

. Near smelters and other point

- sources of Pb, leaf uptake may in some cases be greater than root 

### 10
uptake, otherwise root uptake is predominant. Transl ocation of soil Pb to shoots and leaves is limited in 

### 11
most species as plants sequester a large portion of Pb in root tissue. There are considerable species



### 12
dependent differences in rate of uptake and translocation of Pb to other parts of the plant. Uptake and 

### 13
sequestration of Pb primarily in roots of terrestrial plants is also observed in wetland species and algae. 

### 14
Rooted aquatic plants take up Pb primary from sediments while floating aquatic plants take up Pb from 

### 15

water

. 

### 16
Uptake of atmospherically deposite d Pb from soil is the primary exposure route in terrestrial plants 

### 17

and invertebrates

. Bioaccessibility of Pb to soil

- dwelling organisms is influenced by soil factors including 

### 18
soil type, amount of OM , pH, and CEC and there are considerable differences in u ptake among species

. 

### 19
Species exhibit different accumulation efficiencies and compartmentalize sequestered Pb differently. 

### 20
There is limited evidence of contributions to total Pb body burden via dietary exposures in primary and 

### 21
secondary consumers

. 

### 22
Aquatic organisms can uptake and bioaccumulate Pb from the water column, sediments or via 

### 23
dietary exposure

. However, as in terrestrial organisms, uptake and subsequent bioaccumulation of Pb in 

### 24
aquatic plants, invertebrates and vertebrates varies greatly between sp ecies and across taxa. Invertebrates 

### 25
may also sequester Pb in the exoskeleton, which is subsequently shed. Pb in aquatic invertebrates is 

### 26
primarily sequestered in the gill and hepatopancreas. Uptake of Pb in fish is well characterized and occurs 

### 27
primarily via direct uptake of dissolved Pb from the water column through gill surfaces and by ingestion 

### 28

of Pb

- contaminated diets. Pb in these organisms is primarily sequestered in the gill. In dietary exposures 

### 29
in fish, Pb also targets the anterior intestine indica ting the importance of water

- only versus dietary uptake 

### 30

exposures. 

### 31

Pb is bioaccumulated in plants, invertebrates and vertebrates inhabiting terrestrial and aquatic 

### 32
systems that receive Pb from atmospheric deposition. This represents a potential route for P b mobilization 

### 33

into the food web or into food products

. For example, Pb bioaccumulation in leaves and roots of an edible 

### 34
plant may represent an adverse impact to the provisioning of food, an essential ecosystem service. Recent 

### 35
research has suggested that d ietary Pb (i.e., Pb adsorbed to sediment, particulate matter, and food) may 

### 36May 2011 7

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– DO NOT C ITE OR QUOTE contribute to exposure and toxicity in primary and secondary order consumers (including humans). 

### 1

A lthough there is no consistent evidence of trophic magnification there is substant ial evidence of trophic 

### 2

transfer

. I t is through consumption of Pb

- exposed prey or Pb

- contaminated food that atmospherically 

### 3
deposited Pb reaches species that may have very little direct exposure to it. Overall, based on the 

### 4
consistency of findings across t axa, the evidence is sufficient to conclude that there is a causal 

### 5
relationship between Pb exposures and bioaccumulation of Pb that affects ecosystem services 

### 6

associated with terrestrial and aquatic biota. 

### 7



## 7.4.2. Mortality

The relationship between Pb exposure and mortality has been well demonstrated in terrestrial and 

### 8
aquatic species as presented in Sections 7.2.5 and 7.3.5 of this ISA and in the previous Pb AQCDs. 

### 9
Toxicological studies have established LC 50 values for some species of plants, invertebrates and 

### 10

ver tebrates

. From the LC 50 data on Pb in this review and previous Pb AQCDs a wide range of sensitivity 

### 11

to Pb is evident across taxa. However, the LC 50 is usually much higher than current environmental levels 

### 12
of Pb, even though physiological dysfunction that adversely impacts the fitness of an organism often 

### 13

occur s well below concentrations that result in mortality

. 

### 14
Pb is generally not phytotoxic to pla nts at concentrations found in the environment away from 

### 15
point

- sources , probably due to the fact that plants often sequester large amounts of Pb in roots, and that 

### 16
translocation to other parts of the plant is limited

. 

### 17
Invertebrates are generally more sens itive to Pb exposure than other taxa, with survival adversely 

### 18
impacted in a few species at concentrations occur r ing near point

- sources or at concentrations that 

### 19
approach ambient levels. These impacted species may include candidate or endangered species. Th e 

### 20

freshwater mussel Lampsilis rafinesqueana (Neosho mucket), is a candidate for the endangered species 

### 21

list. The EC 50 for foot movement (a measure of viability) for newly transformed juveniles of this species 

### 22
was 188 μg Pb/L

. ( N. Wang et al., 2010 )

. However, tolerance to Pb varies substantially among 

### 23
invertebrate species. As reviewed in the 2006 Pb AQCD, the LC 50 values for soil nematodes vary from 

### 24

10

- 1,550 mg Pb /kg dry w eight dependent upon soil OM content and soil pH ( U.S. EPA, 2006 ) . In 

### 25
earthworms, 14 and 28 day LC 50 values typically fall in the range of 2,400

- 5,800 mg Pb /kg depending 

### 26
upon the species tested. Toxicity of Pb to aquatic invertebrates is highly dependent on water quality 

### 27
parameters such as pH, DOC and Ca 2+ . For example, 48 hour LC 50 values for C. dubia range from 280 to 

### 28
>2,700 μg Pb /L when tested at varying pH levels ( U.S. EPA, 2006 ) . Other invertebrates such as odonates 

### 29
may be tolerant of Pb concentrations that greatly exceed environmental levels. Some invertebrates are 

### 30
able to detoxify Pb such as through sequestration of Pb in the exoskeleton which is subsequently molted. 

### 31
Early experiments from Pb mining areas indicate d local extinction of fish from streams

. Mortality 

### 32
in fish is dependent on the sensitivity of the species tested and on water quality parameters. Higher 

### 33May 2011 7

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– DO NOT C ITE OR QUOTE toxicity tends to occur in acidic waters where more free

- Pb ion is available for uptake. The interactio n 

### 1
between water quality parameters and Pb toxicity may result in a range of concentrations that cause 

### 2
equivalent toxicity. For example, 96

- hr LC 50 values in fathead minnow range from 810

- >5,400 μg Pb /L in 

### 3
varying pH and hardness ( U.S. EPA, 2006 )

. Increased mortality is also a function of age of the fish. Thirty 

### 4
day LC 50 values for larval fathead minnows ranged from 39 to 1,903 μg Pb /L in varying concentrations of 

### 5

DOC, CaSO 4 and pH ( Grosell et al., 2006b ) . In a recent study of rainbow trout fry at 2 to 4 weeks post



### 6
swim up, the 96

- hour LC 50 was 120 μg Pb /L at a hardness of 29 mg/L as CaCO 3 , a value much lower than 

### 7
in testing with older fish ( Mebane et al., 2008 )

. 

### 8
In terrestrial avian and mammalian species, toxic ity is observed in laboratory studies over a wide 

### 9
range of doses (<1 to >1

, 000 mg Pb/kg body weight

- day) as reviewed for the development of Eco

- SSL ’ s 

### 10
{U.S. EPA, 2005 #19390}. Mortality associated with Pb exposure in vertebrates is supported by the 

### 11

consiste ntly positive associations between Pb exposure and mortality observed in human epidemiologic 

### 12
studies (Section 5.3.1). 

### 13

The evidence is inadequate to conclude that there is a causal relationship between Pb 

### 14

exposure and mortality in plants. 

### 15

The evidence is s ufficient to conclude that there is a causal relationship between Pb exposures 

### 16

and mortality in sensitive terrestrial and aquatic animal taxa

. 

### 17



## 7.4.3. Growth Effects

Evidence for Pb effects on growth is strongest in plants with limited information on invertebrates 

### 18
and vertebrates. There is evidence over several decades of research that Pb inhibits photosynthesis and 

### 19
respiration in plants all of which reduce the growth of the plant ( U.S. EPA, 1977

, 1986

, 2006 ) . Ma ny 

### 20
laboratory toxicity studies report effects on plants ; however, there are few field studies

. Specifically, Pb 

### 21
has been shown to affect photosystem II with the hypothesized mechanism being that Pb may replace 

### 22
either Mg or Ca in chlorophyll, altering the p igment structure and decreasing the efficiency of visible 

### 23
light absorption in exposed plants

. Decreases in chlorophyll a and b content have been observed in 

### 24
various algal and plant species. The lowest 72

- hour EC 50 for growth inhibition reported for algae w as 105 

### 25

μg Pb /L in Chaetoceros sp. Most primary producers experience EC 50 values for growth in the range of 

### 26

1,000 to 100,000 μg Pb /L ( U.S. EPA, 2006 )

. 

### 27
In previous AQCDs, growth effects of Pb have been reported in fish (growth inhibition), birds 

### 28
(changes in juvenile weight gain), and frogs (delayed metamorphosis, smaller larvae). Growth effects 

### 29
observed in invertebrates and vertebrates underscore the impo rtance of lifestage to overall Pb 

### 30
susceptibility

. In general, juvenile organisms are more sensitive than adults

. Several studies since the 2006 

### 31
Pb AQCD have demonstrated adverse effects of Pb on growth at lower concentrations than in previous 

### 32

literature. A mong the animal taxa tested, aquatic invertebrates were the most sensitive to the effect of Pb, 

### 33May 2011 7

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– DO NOT C ITE OR QUOTE with adverse effects being reported as low as 4 μg Pb /L. Growth of juvenile freshwater snail s L. stagnalis 

### 1

was inhibited at EC 20 <4 μg Pb /L ( Grosell & Brix, 2009 ; Grosell et al., 2 006b ) . In the freshwater mussel, 

### 2
fatmucket ( L. siliquoidea ) juveniles were the most sensitive lifestage ( N. Wang et al., 2010 )

. A chroni c 

### 3
value of 10 μg Pb /L in a 28 day exposure of 2

- month

- old fatmucket juveniles was the lowest genus mean 

### 4
chronic value ever reported for Pb. Evidence is sufficient to conclude that there is a causal relationship 

### 5
between Pb exposures and growth effects in pl ants and invertebrates. Evidence is suggestive of a 

### 6
causal relationship between Pb exposures and growth effects in vertebrates

. 

### 7



## 7.4.4. Physiological Stress

In this review and in previous Pb AQCDs there is consistent and coherent evidence that Pb induces 

### 8
oxidative stress in plants, invertebrates, and vertebrates. This is consistent with evidence in humans and 

### 9
experimental animal studies for oxidative stress development, due in many instances t o the antagonism of 

### 10
normal metal ion functions (Section 5.2.4). This oxidative stress is characterized by increased reactive 

### 11
oxygen species and membrane and lipid peroxidation that can promote tissue damage, cytotoxicity, and 

### 12
dysfunction. Increased reactiv e oxygen species are often followed by a compensatory and protective 

### 13
upregulation in antioxidant enzymes, such that this observation is indicative of oxidative stress 

### 14
conditions. Additionally, continuous reactive oxygen species production may overwhelm thi s defensive 

### 15
process leading to further oxidative stress and injury. 

### 16
Building on this strong body of evidence presented previously, recent studies provide consistent 

### 17
and coherent evidence of upregulation of antioxidant enzymes and increased lipid peroxidati on associated 

### 18
with Pb exposure in many species of plants, invertebrates and vertebrates

. In plants, increases of 

### 19
antioxidant enzymes with Pb exposure occur in algae, aquatic mosses, floating and rooted aquatic 

### 20
macrophytes, and terrestrial species. There is considerable evidence for antioxidant activity in 

### 21
invertebrates, including gastropods, mussels, and crustaceans, in response to Pb exposure

. Markers of 

### 22
oxidative damage are also observed in fish and amphibians

. Across all biota, there are differences in t he 

### 23
induction of antioxidant enzymes that appear to be species

- dependent. 

### 24
Additional stress responses to Pb exposure observed in terrestrial and aquatic invertebrates 

### 25
including elevated heat shock proteins, osmotic stress, lowered metabolism and decreased g lycogen levels 

### 26
have been reported since the 2006 Pb AQCD

. Heat shock protein induction by Pb exposure has been 

### 27

observed in zebra mussels and mites

. Elevated expression of heat shock protein orthologs were reported 

### 28
for the first time in the hypothalamic and mesencephalic brain regions of Pb

- treated fish ( Giusi et al., 

### 29

2008 ) . Crayfish exhibit a Pb

- induced hypometabolism under conditions of environmental hypoxia in the 

### 30
presence of the metal ( Morris et al., 2005 ) . Tissue volume regulation is adversely affected in freshwater 

### 31

crabs ( Amado et al., 2006 ) . Glycog en levels in the freshwater snail B. glabrata were significantly 

### 32May 2011 7

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– DO NOT C ITE OR QUOTE decreased at near environmentally relevant concentrations of Pb (50 μg/L and higher) ( Ansaldo et al., 

### 1

2006 )

. 

### 2
Upregulation of antioxidant enzymes and increased lipid peroxidation are considered to be reliable 

### 3

biomarkers of stress, and suggest that Pb exposure induces a s tress response in those orga nisms which 

### 4

may increase susceptibility to other stressors and reduce individual fitness. Evidence is sufficient to 

### 5

conclude that there is a causal relationship between Pb exposures and physiological stress in plants, 

### 6

invertebrates and vertebrates

. 

### 7



## 7.4.5. Hematological Effects

Hematological responses are commonly reported effects of Pb exposure in invertebrates and 

### 8
vertebrates in both aquatic and terrestrial systems. In environmental assessments of metal

- impacted 

### 9

habitats, ALAD is a reco gnized biomarker of Pb exposure ( U.S. EPA, 2006 ) . ALAD activity is negatively 

### 10
correlated with total Pb concentration in bivalves. Lower ALAD activity has been correlated with 

### 11
elevated blood Pb levels in fish and mammals as well. In the 1986 AQCD, decreases in red blood cell 

### 12

AL AD activity following Pb exposure were well documented in birds and mammals. Further evidence 

### 13
from the 2006 Pb AQCD and this review of Pb effects on ALAD enzymatic activity

, including effects in 

### 14
bacteria, amphibians and additional field and laboratory stud ies on fish

, suggest this enzyme is an 

### 15
indicator for Pb exposure across a wide range of taxa

. Limited evidence of Pb effects on other blood 

### 16
parameters including altered serum profiles and changes in white blood cell counts in fish and amphibians 

### 17
support th e finding of the hematological system as a target for Pb in natural systems

. This evidence is 

### 18
strongly coherent with observations from human epidemiologic and animal toxicology studies (Section 

### 19

5. 7 )

. There is consistent toxicological and epidemiologic evid ence that exposure to Pb induces adverse 

### 20
effects on hematological endpoints, including altered heme synthesis mediated through decreased ALAD 

### 21

and ferrochelatase activities, decreased red blood cell survival and function, and increased red blood cell 

### 22

oxidat ive stress

. Taken together, the overall weight of epidemiologic and toxicological evidence is 

### 23
sufficient to conclude that a causal relationship exists between exposure to Pb and hematological effects 

### 24
in humans and laboratory animals (Section 5. 7 )

. Based on observations in both terrestrial and aquatic 

### 25
organisms and additionally supported by toxicological and epidemiological findings in laboratory animals 

### 26

and humans evidence is sufficient to conclude that there is a causal relationship between Pb exposures 

### 27

an d hematological effects in invertebrates and vertebrates. 

### 28



## 7.4.6. Developmental and Reproductive Effects

Evaluation of the literature on Pb effects in aquatic and terrestrial species indicates that exposure to 

### 29
Pb is associated with reproductive effects. Various e ndpoints have been measured in aquatic and terrestrial 

### 30
organisms to assess the effect of Pb on development, fecundity and hormone homeostasis

. Evidence in 

### 31May 2011 7

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– DO NOT C ITE OR QUOTE this review and the previous Pb AQCDs from invertebrate and vertebrate studies indicate that Pb is 

### 1

ad versely affecting reproductive performance in multiple species. However, there are typically only 

### 2

limited studies available from different taxa

. 

### 3
In plants, few studies are available that specifically address reproductive effects of Pb exposure

. 

### 4
One study with grass pea showed Pb exposure increased pollen sterility. 

### 5
Several studies with invertebrates provide evidence for adverse impacts to embryonic development, 

### 6
specifically in snails , clams and rotifers. In addition to affecting the embryo, Pb can alter de velopmental 

### 7
timing, sperm morphology and hormone homeostasis. In fruit flies, Pb exposure increased time to 

### 8
pupation and decreased pre

- adult development. Sperm morphology was altered in earthworms exposed to 

### 9

Pb

- contaminated soils. Pb may also disrupt hormo nal homeostasis in invertebrates as studies with moths 

### 10
have suggested

. 

### 11
Reproductive effects have also been observed in multi

- generational studies. Larval settlement rate, 

### 12
and rate of population increase was adversely impacted in rotifers. Rotifers have de creased fertilization 

### 13
rate associated with Pb exposure that appeared to be due to decreased viability of sperm and eggs. 

### 14

Evidence of multi

- generational toxicity of Pb is also present in terrestrial invertebrates, specifically 

### 15
springtails, mosquito e s, carab id beetles and nematodes where decreased fecundity in progeny of Pb



### 16
exposed individuals was observed. 

### 17
In aquatic vertebrates there is evidence for reproductive and developmental effects of Pb

. Pb



### 18
exposure in frogs has been demonstrated to delay metamorpho sis, decrease larval size and produce subtle 

### 19
skeletal malformations. Previous Pb AQCDs have reported developmental effects in fish, specifically 

### 20
spinal deformities in larvae

. In the 2006 Pb AQCD, decreased spermatocyte development in rainbow trout 

### 21

was obse rved at 10 μg Pb /L and

, in fathead minnow testicular damage occurred at 500 μg Pb /L. In fish, 

### 22
there is new evidence of Pb in this ISA on effects on steroid profiles and additional reproductive 

### 23
parameters. Reproduction in fathead minnows was affected in bre eding exposures following 300 day 

### 24
chronic toxicity testing

. However, reproductive performance was unaffected in zebrafish exposed to Pb



### 25
contaminated prey. Additional reproductive parameters in fish observed to be impacted by Pb include 

### 26
decreased oocyte diameter and density in toadfish associated with elevated Pb levels in gonad. 

### 27
In terrestrial vertebrates, evidence from Chapter 7 of this ISA and in previous Pb AQCDs indicates 

### 28
an association between observed adverse reproductive effects and Pb exposure

. Reproductive effects 

### 29

observed in birds near areas of Pb

- contamination or where Pb tissue concentration has been correlated 

### 30
with effects include declines in clutch size, number of young hatched, number of young fledged, 

### 31
decreased fertility, and decreased eg gshell thickness

. Decreased testis weight was observed in lizards

. In 

### 32
mammals, few studies in the field have addressed Pb specifically, due to most available data in wild or 

### 33
grazing animals being from near smelters, where animals are co

- exposed to other me tals. 

### 34
In Chapter 5 evidence from mammals demonstrates a consistency of adverse effects of Pb on sperm 

### 35
and the onset of puberty in males and females with strong evidence from both toxicology and 

### 36May 2011 7

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– DO NOT C ITE OR QUOTE epidemiology studies. Other reproductive endpoints including spontaneous abortions, pre

- term birth, 

### 1
embryo development, placental development, low birth weight, subfecundity, hormonal changes, and 

### 2
teratology were also affected, but less consistently. New toxicological data support trans

- generational 

### 3

effects, a findi ng that is also an area of emerging interest in ecology. The evidence presented in Section 

### 4

5. 8 is sufficient to conclude that there is a causal relationship between Pb exposure and reproductive 

### 5

effects. 

### 6
Adverse effects of Pb on reproduction in invertebrate s and vertebrates indicate that Pb is likely 

### 7
adversely affecting fecundity of Pb

- exposed organisms in both aquatic and terrestrial habitats, and 

### 8

therefore the evidence is sufficient to conclude that there is a causal relationship between Pb exposure s 

### 9

and r eproductive effects in terrestrial and aquatic invertebrates and vertebrates. In plants, the 

### 10

evidence is inadequate to conclude a causal relationship between Pb exposures and plant 

### 11
reproductive effects

. 

### 12



## 7.4.7. Neurobehavioral Effects

Evidence from laboratory stud ies and limited data from field studies reviewed in Chapter 7 have 

### 13
shown adverse effects of Pb on neurological endpoints in both aquatic and terrestrial animal taxa

. These 

### 14
include changes in behaviors that may decrease the overall fitness of the organism. There is also evidence 

### 15
from both invertebrate and vertebrate studies that Pb adversely affects behaviors that may decrease the 

### 16
ability of an organism to escape predators or capture prey. 

### 17
Central nervous system effects in fish recognized in previous Pb AQCD s include effects on spinal 

### 18
neurons and brain receptors. New evidence from this review identifies the MAPKs ERK1/2 and p38 MAPK 

### 19
as possible molecular targets for Pb neurotoxicity in catfish ( Leal et al., 2006 ) . Additionally, there is new 

### 20
evidence for neurotoxic action of Pb in invertebrates with exposure to Pb observed to cause changes in 

### 21
the morphology of GABA motor neurons in nematodes ( C. elegans ) ( Du & Wang, 2009 )

. 

### 22

Decreased food consu mption of Pb

- contaminated diet has been demonstrated in some invertebrates 

### 23
(snails) and vertebrates (lizards, pigs). Pb may also decrease the ability of an organism to capture prey or 

### 24
escape predation. For example, Pb exposure has been demonstrated to adversely affect prey capture 

### 25
ability of certain fungal and fish species. In limited studies available on snails, tadpoles, scallops, 

### 26
hatchling turtles and fish there is evidence that Pb may affect the ability to escape or avoid predation. The 

### 27
motility of nematodes was adversely affected in Pb

- contaminated soils ( D. Y. Wang & Xing, 2008 ) . In a 

### 28
laboratory study, Pb

- exposed gull chicks exhibited abnormal behaviors such as decreased walking, erratic 

### 29
behavioral thermoregulation and food begging that could make them m ore vulnerable in the wild ( Burger 

### 30

& Gochfeld, 2005 ) . Lizards exposed to Pb through diet in the laboratory exhibited abnormal coloration 

### 31
and posturing behaviors. Other behavioral effects affected by Pb exposure include increased hype ractivity 

### 32
in fish and hypoxia

- like behavior in frogs. 

### 33May 2011 7

- 121 DRAFT

– DO NOT C ITE OR QUOTE These findings are coherent with findings from studies in laboratory animals as described in 

### 1
Section 5.3 that show that Pb induces changes in learning, memory, attention and motor skills. Pb induced 

### 2

ne w behaviors including hyperactivity and mood disorders. Within the sensory organs, the visual and 

### 3
auditory systems are affected by Pb exposure. Changes in structure and function of neurons and 

### 4
supporting cells in the brain are detailed including effects on the blood brain barrier. Mechanisms 

### 5
including the displacement of physiological cations, oxidative stress and changes in neurotransmitters and 

### 6
receptors are detailed. Based on evidence from several cohort and cross

- sectional studies in diverse 

### 7
populations , the overall weight of the available evidence provides clear and consistent evidence of 

### 8
association between blood Pb concentrations and decrements in neurodevelopmental outcomes in young 

### 9
children (Section 5.1). In addition to the consistency of findings i n children, the evidence is strengthened 

### 10
by the coherence of findings with toxicological studies and by coherence of association of blood Pb with 

### 11
a spectrum of related endpoints including IQ, verbal and reading skills, motor coordination, mood and 

### 12

attentio n problems, and behavioral problems. The evidence presented in the health chapter is sufficient to 

### 13
conclude that there is a causal relationship between Pb exposure and neurobehavioral effects (Section 

### 14
5.3). These data from laboratory toxicology studies, es pecially neurobehavioral findings and structural 

### 15
changes are highly coherent with data from ecological studies. Overall, the evidence from aquatic and 

### 16
terrestrial systems is sufficient to conclude that there is a causal relationship between Pb exposures an d 

### 17

neurobehavioral effects in invertebrates and vertebrates. 

### 18



## 7.4.8. Other Physiological Effects

In addition to the above mentioned physiological effects of Pb on organisms in terrestrial and 

### 19
aquatic systems for which there is sufficient evidence to infer causality , there are a few recent studies that 

### 20

can be linked to effects observed in humans for which there is insuffic i ent evidence across taxa

. Pb 

### 21
exposure has been demonstrated to result in changes to DNA structure and chromosomal alterations in 

### 22
plants, gastropod s, mussels and fish

. DNA damage, chromosomal damage and aberrations, and 

### 23
micronucleus formation are also observed in humans and laboratory animals exposed to Pb (Section 

### 24

5. 10 )

. Additional new evidence in this review indicates that Pb can interfere with ren al function in fish, 

### 25
specifically with ionoregulation of Na and Cl and reabsorption Ca 2+ , Mg 2+ glucose and water ( Patel et al., 

### 26

2006 ) . In humans and labora tory animals, Pb is a recognized nephrotoxicant and is considered to be 

### 27
causal of kidney damage (Section 5.5). 

### 28



## 7.4.9. Community and Ecosystem Level Effects

Uptake of Pb into aquatic and terrestrial organisms and subsequent effects on survival, 

### 29
reproduction, gro wth, behavior and other physiological variables at the species scale are likely to result in 

### 30
effects at the population, community and ecosystem scale. The effects may include alteration of predator

- 

### 31May 2011 7

- 122 DRAFT

– DO NOT C ITE OR QUOTE prey dynamics, species richness, species composition, and biodiversity. There are few field studies 

### 1
available that directly consider effects of Pb on these measures of ecosystem health. Ecosystem

- level 

### 2
studies are complicated by the confounding of Pb exposure with other factors such as trace metals and 

### 3
acidic dep osition

. In natural systems, Pb is often found co

- existing with other stressors, and observed 

### 4
effects may be due to cumulative toxicity. 

### 5

Most direct evidence of community and ecosystem level effects is from near point sources. For 

### 6
terrestrial systems there are several decades of research on impacts to natural ecosystems near smelters, 

### 7

mines, and other industrial sources of Pb where Pb levels are elevated . Those impacts include decreases in 

### 8
species diversity and changes in floral and faunal community composi tion. For aquatic systems, the 

### 9
literature focuses on evaluating ecological stress from Pb originating from urban and mining effluents 

### 10
rather than atmospheric deposition. In laboratory studies and simulated ecosystems, where it is possible to 

### 11

isolate the ef fect of Pb, this metal has been shown to alter competitive behavior of species, predator

- prey 

### 12
interactions and contaminant avoidance. These dynamics may change species abundance and community 

### 13
structure at higher levels of ecological organization. Effects o f Pb on mortality, growth, physiological 

### 14
stress, blood, neurobehavior and developmental and reproductive endpoints at the individual level are 

### 15
expected to have ecosystem

- level consequences, and thus provide consistency and plausibility for 

### 16
causality in eco system

- level effects. 

### 17
Avoidance response to Pb exposure varies widely in different species and this could affect 

### 18
community composition. For example, frogs and toads lack avoidance response while snails and fish 

### 19
avoid higher concentrations of Pb ( U.S. EPA, 2006 ) . New evidence since the 2006 Pb AQCD indicates 

### 20
that some species of worms avoid Pb

- contaminated soils ( Langdon et al., 2005 ) 

### 21
In terrestrial ecosystems, most studies show decreases in microorganism abundance, diversity, and 

### 22
function with increasing soil Pb concentration

. Specifically, shifts in nematode communities, bacterial 

### 23

s pecies, and fungal diversity have been observed

. Furthermore, presence of arbuscular mycorr h izal fungi 

### 24
may protect plants growing in Pb

- contaminated soils. Increased plant diversity was shown to ameliorate 

### 25

effects of Pb contamination on a microbial communi ty. 

### 26
In aquatic ecosystems there are numerous field studies on reductions of species abundance, 

### 27
richness or diversity particularly in benthic macroinvertebrate communities coexisting with other metals. 

### 28
For example, in the 2006 Pb AQCD, the Coeur d ’ Alene Ri ver watershed in Idaho, U.S. was used as a case 

### 29
study for Pb effects at the population and community level

. Significant negative correlations were 

### 30

observed between Pb in water column and total taxa richness and EPT taxa richness in the river. In a 

### 31

simulate d aquatic microcosm a reduction in abundance and richness of protozoan species was observed 

### 32
with increasing Pb concentration from 50 to 1,000 μg Pb /L ( Fernandez

- Leborans & Antonio

- Garcia, 

### 33

1988 ) 

### 34

Since the last Pb AQCD , there is further evidence for effects of Pb in sediment

- associated 

### 35
communities. Exposure to three levels of sediment Pb contamination (322, 1,225, and 1,465 μg Pb / g dry 

### 36May 2011 7

- 123 DRAFT

– DO NOT C ITE OR QUOTE weight) in a microcosm experiment significantly reduced nematode diversity and resulted in profound 

### 1
restructuring of the community structure ( Mahmoudi et al., 2007 )

. Sediment

- bound Pb contamina tion 

### 2
appears to differentially affect members of the benthic invertebrate community, potentially altering 

### 3
ecosystems dynamics in small urban streams ( Kominkova & Nabelkova, 2005 ) . Although surface water 

### 4

Pb concentrations in monitored streams were determined to be very low, concentrations of the metal in 

### 5
sediment were high enough to pose a risk to the benthic community (e.g., 34 to 101 mg Pb /kg). These 

### 6
risks were observed to be linked to benthic invertebrate functional feeding group, with collector

- gatherer 

### 7
species exhibiting larger body burdens of heavy metals than benthic predators and collector

- filterers. 

### 8
In a new study conducted since the 2006 Pb AQCD, changes to aquatic plant community 

### 9
composition have been observed in the presence of elevated surface water Pb concentrations at three lake 

### 10
sites impacted by mining effluents. The site with highest Pb concentration (103

- 118 μg Pb /L) had lowest 

### 11
number of aquatic plant species when compared to sites with lower Pb concentrations (78

- 92 μg Pb /L) ( V. 

### 12

K. Mis hra et al., 2008 )

. This shift toward more Pb

- tolerant species is also observed in terrestrial plant 

### 13

communities near smelter sites ( U.S. EPA, 1986

, 2006 ) . Certain types of plants such as rooted and 

### 14
submerged aquatic plants may be more susceptible to aerially

- deposited Pb resulting in shifts in Pb 

### 15
community composition. High Pb sediment concentrations are linked to shifts in amphipod communities 

### 16
inhabiting plant structures. 

### 17
In many cases it is difficult to characterize the nature and magnitude of effects and to quantify 

### 18
relationships between ambient concentrations of Pb and ecosystem response due to existence of multiple 

### 19
stressors in natural systems. Howeve r, the evidence for Pb effects at higher levels of ecological 

### 20
organization is sufficient to conclude that there is a causal relationship between Pb exposures and the 

### 21
alteration of species richness, species composition and biodiversity in terrestrial and aq uatic 

### 22

ecosystems. 

### 23May 2011 7

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– DO NOT C ITE OR QUOTE 

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- 169. Adeyemo, O. K. (2008a). Histological alterations observed in the gills and ovaries of Clarias gariepinus exposed to environmentally relevant lead concentrations. Journal of Envi ronmental Health, 70 (9), 48

- 51. http://www.ncbi.nlm.nih.gov/pubmed/18517154 Adeyemo, O. K. (2008b). Histological alterations observed in the liver and brain of Clarias gariepinus exposed to chronic
sublethal dose of lead. Bulletin of the European Association of Fish Pathologists, 28 (3), 105

- 114. Ahlf, W., Drost, W., & Heise, S. (2009). Incorporation of metal bioavailability into regulatory frameworks: Metal exposure in
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- Khashman, O. A. (2007). Atmospher ic heavy metal pollution in Aqaba City, Jordan, using Phoenix dactylifera L. leaves. Atmospheric Environment, 41 (39), 8891

- 8897. http://dx.doi.org/10.1016/j.atmosenv.2007.08.028 Akerblom, S., Baath, E., Bringmark, L., & Bringmark, E. (2007). Experimentally induced effects of heavy metal on microbial activity and community structure of forest mor lay ers. Biology and Fertility of Soils, 44 (1), 79

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- eastern Australia. Environmental Pollution, 142 (1), 116

- 122. http://dx.doi.org/10.1016/j.envpol.2005.09.009 Alves Costa, J. R., Mela, M., da Silva de Assis, H. C., Pelletier, E., Randi, M. A., & de Olivei ra Ribeiro, C. A. (2007). Enzymatic inhibition and morphological changes in Hoplias malabaricus from dietary exposure to lead(II) or methylmercury. Ecotoxicology and Environmental Safety, 67 (1), 82

- 88. http://dx.doi.org/10.1016/j.ecoenv.2006.03.013 Alves, L. C., Glover, C. N., & Wood, C. M. (2006). Dietary Pb accumulation in juvenile freshwater rainbo w trout (Oncorhynchus mykiss). Archives of Environmental Contamination and Toxicology, 51 (4), 615

- 625. http://dx.doi.org/10.1007/s00244

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- 7 Alves, L. C., & Wood, C. M. (2006). The chronic effects of dietary lead in freshwater juvenile rainbow trout (Oncorhynchus mykiss) fed elevated calcium diets. A quatic Toxicology, 78 (3), 217

- 232. http://dx.doi.org/10.1016/j.aquatox.2006.03.005 Amado, E. M., Freire, C. A., & Souza, M. M. (2006). Osmoregulation and tissue water regulation in the freshwater red crab Dilocarcinus pagei (Crustacea, Decapoda), and the effect of waterborne inorganic lead. Aquatic Toxicology, 79 (1), 1

- 8. http://dx.doi.org/10.1016/j.aquatox.2006.04.003 Ancion, P. Y., Lear, G., & Lewis, G. D. (2010). Three common metal contamina nts of urban runoff (Zn, Cu & Pb) accumulate in freshwater biofilm and modify embedded bacterial communities. Environmental Pollution, 158 (8), 2738

- 2745. http://dx.doi.org/10.1016/j.envpol.2010.0 4.013 Anderson, M. B., Preslan, J. E., Jolibois, L., Bollinger, J. E., & George, W. J. (1997). Bioaccumulation of lead nitrate in red swamp crayfish (Procambru s clarkii). Journal of Hazardous Materials, 54 , 15

- 29. http://dx.doi.org/10.1016/S0304

- 3894(96)01852

- 3 Ansaldo, M., Nahabedian, D. E., Di Fonzo, C., & Wider, E. A. (2009). Effect of cadmium, lead and arsenic on the oviposition, hatching and embryonic survival of Biomphalaria glabrata. Science of the Total Environment, 407 (6), 1923

- 1928. http://dx.doi.org/10.1016/j.scitotenv.2008.12.001May 2011 7

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