A —United States
Environmental Protection
^^LbI Jr* Agency
EPA/600/R-23/061
March 2023
www.epa.gov/isa
Integrated Science
Assessment for Lead
Appendix 11: Effects of Lead in Terrestrial
and Aquatic Ecosystems
External Review Draft
March 2023
Health and Environmental Effects Assessment Division
Center for Public Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
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DISCLAIMER
1 This document is an external review draft for peer review purposes only. This information is
2 distributed solely for the purpose of predissemination peer review under applicable information quality
3 guidelines. It has not been formally disseminated by the Environmental Protection Agency. It does not
4 represent and should not be construed to represent any agency determination or policy. Mention of trade
5 names or commercial products does not constitute endorsement or recommendation for use.
6
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DOCUMENT GUIDE
This Document Guide is intended to orient readers to the organization of the Lead (Pb) Integrated
Science Assessment (ISA) in its entirety and to the sub-section of the ISA at hand (indicated in bold). The
ISA consists of the Front Matter (list of authors, contributors, reviewers, and acronyms), Executive
Summary, Integrated Synthesis, and 12 appendices, which can all be found at
https://cfpub.epa.gov/ncea/isa/recordisplay.cfm?deid=357282.
Front Matter
Executive Summary
Integrative Synthesis
Appendix 1. Lead Source to Concentration
Appendix 2. Exposure, Toxicokinetics, and Biomarkers
Appendix 3. Nervous System Effects
Appendix 4. Cardiovascular Effects
Appendix 5. Renal Effects
Appendix 6. Immune System Effects
Appendix 7. Hematological Effects
Appendix 8. Reproductive and Developmental Effects
Appendix 9. Effects on Other Organ Systems and Mortality
Appendix 10. Cancer
Appendix 11. Effects of Lead in Terrestrial and Aquatic Ecosystems
Appendix 12. Process for Developing the Pb Integrated Science Assessment
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CONTENTS
LIST OF TABLES v
LIST OF FIGURES vi
ACRONYMS AND ABBREVIATIONS vii
APPENDIX 11 EFFECTS OF LEAD IN TERRESTRIAL AND AQUATIC ECOSYSTEMS 1
11.1 Introduction, Scope, Concepts, and Tools 2
11.1.1. Scoping and Criteria for Study Inclusion 2
11.1.2. Introduction to Ecosystem Connections and Pb Transfers 6
11.1.3. Concentrations of Pb in Non-Air Media 7
11.1.4. Concepts Related to Ecosystem Effects of Pb 16
11.1.5. Ecosystem Services 17
11.1.6. Bioavailability 18
11.1.7. Risk Screening Tools 21
11.2 Terrestrial Ecosystems 25
11.2.1. Summary of New Information on Effects of Pb in Terrestrial Ecosystems and
Causality Determination Update Since the 2013 Pb ISA 25
11.2.2. Factors Affecting Bioavailability, Uptake and Bioaccumulation and Toxicity in
Terrestrial Biota 29
11.2.3. Environmental Concentrations of Pb in Terrestrial Biota and Ecosystems in the
United States at Different Locations and Over Time 52
11.2.4. Effects of Pb in Terrestrial Systems 55
11.2.5. Exposure and Response of Terrestrial Species 77
11.2.6. Terrestrial-Community and Ecosystem Effects 95
11.3 Freshwater Ecosystems 98
11.3.1. Summary of New Information on Effects of Pb in Freshwater Ecosystems and
Causality Determination Update Since the 2013 Pb ISA 98
11.3.2. Factors Affecting Bioavailability, Uptake and Bioaccumulation and Toxicity in
Freshwater Biota 104
11.3.3. Environmental Concentrations of Pb in Freshwater Biota and Ecosystems in the
United States at Different Locations and Over Time 123
11.3.4. Effects of Pb in Freshwater Systems 124
11.3.5. Exposure and Response of Freshwater Species 140
11.3.6. Freshwater-Community and Ecosystem Effects 166
11.4 Saltwater Ecosystems 169
11.4.1. Summary of New Information on Effects of Pb in Saltwater Ecosystems and Causality
Determination Update Since the 2013 Pb ISA 169
11.4.2. Factors Affecting Bioavailability, Uptake and Bioaccumulation, and Toxicity in
Saltwater Biota 173
11.4.3. Environmental Concentrations of Pb in Saltwater Biota in the United States at
Different Locations and Over Time 189
11.4.4. Effects of Pb in Saltwater Systems 191
11.4.5. Exposure and Response of Saltwater Species 199
11.4.6. Saltwater Community and Ecosystem Effects 211
11.5 References 213
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LIST OF TABLES
Table 11-1
Table 11-2
Table 11-3
Table 11-4
Table 11-5
Pb concentration in non-air media and biota.
Table 11-6
Table 11-7
Summary of Pb causality determinations for terrestrial plants,
invertebrates, and vertebrates
Studies of factors that affect the interpretability of exposure-response
experiments in terrestrial biota, since the 2013 Pb ISA.
Summary of Pb causality determinations for freshwater plants,
invertebrates, and vertebrates
28
83
102
Studies in freshwater biota with analytically verified Pb concentrations
and that report an effect on growth, reproduction or survival comparable
to, or lower than, the lowest effect concentrations reported in previous Pb
AQCDs or the 2013 Pb ISA. 147
Updated causality determinations for Pb in saltwater organisms and
ecosystems. 172
Studies in saltwater biota with analytically verified Pb concentration that
report an effect on growth, reproduction, or survival comparable to, or
lower than, the lowest effect concentrations reported in previous Pb
AQCDs or the 2013 Pb ISA. 204
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LIST OF FIGURES
Figure 11-1 Locations of the 4,857 soil sampling sites included in the U.S. Geological
Survey North American Soil Geochemical Landscapes Project conducted
from 2007 to 2010. 13
Figure 11-2 Conceptual diagram for evaluating bioavailability processes and
bioaccessibility for metals in soil, sediment, or aquatic systems. 19
Figure 11-3 Change in toxicity expressed as relative responses (i.e., response relative
to the mean of the corresponding control soil) for three different
laboratory soil treatments: freshly spiked; spiked, leached and pH-
corrected; and spiked, leached and pH- corrected with 5 years of aging. 34
Figure 11-4 Maps of Pb sampled from A-horizon (A.) and C-horizon (B.) soils, the
ratio of Pb observed in A-horizon to C-horizon soils (C.) and a map of
U.S. population density (D.). 54
Figure 11-5 Main forms of Pb in seawater as a function of pH at 25°C and salinity of
35 ppt. 176
Figure 11-6 Acute genus sensitivity distribution for saltwater biota from Church et al.,
(2017). 202
Figure 11-7 Comparison of chronic sensitivity distributions in saltwater biota for
dissolved Pb following the U.S. EPA and European Union methods. 203
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ACRONYMS AND ABBREVIATIONS
ACE abundance-based coverage estimator
AChE acetylcholinesterase
Ag silver
ALAD aminolevulinic acid dehydratase
AMF arbuscular mycorrhizal fungi
AQCD Air Quality Criteria Documents
As arsenic
ASTM American Society for Testing and
Materials
AVS acid volatile sulfides
AWCD average cell wall color development
AWQC ambient water quality criteria
BAF bioaccumulation factor
BCF bioconcentration factor
BEST Biomonitoring of Environmental Status
and Trends
BLM biotic ligand model
BMF biomagnification factors
BRT boosted regression tree
BSAF biota-sediment accumulation factors
Ca calcium
CAT catalase
CCA canonical correspondence analysis
CCC criterion continuous concentration
Cd cadmium
CEC cation exchange capacity
CF conversion factor
CMC criteria maximum concentration
CORT corticosterone
CRADA Cooperative Research and
Development Agreement
CSMW California State Mussel Watch
Cu copper
d day, days
DOC dissolved organic carbon
dpf days postfertilization
dph days posthatch
DOM dissolved organic matter
DT diatom + tetramin
eCEC effective cation exchange capacity
Eco-SSL ecological soil screening levels
EDTA ethylenediaminetetraacetic acid
FCORT fecal corticosterone
FCV final chronic value
FDA
GABA
GPx
GSH
GST
HAB
hpf
IC
ISA
LECES
LH
LOEC
LOAEL
LRMN
MATC
MBC
MDA
ME
Mg
MIC
MLR
mo
MRG
MTC
MW
NAAQS
NASGLP
NAWQA
NEC
NME
NOAA
NOEC
NOM
OC
OM
OP
OTU
Pb
PEC
PECOS
fluorescein diacetate hydrolysis activity
gamma-aminobutyric acid
glutathione peroxidase
glutathione
glutathione-s-transferase
harmful algal blooms
hours postfertilization
inhibitory concentration
integrated science assessment
Level of Biological Organization,
Exposure, Comparison, Endpoint and
Study Design
luteinizing hormone
lowest observed effect concentration
lowest observed adverse effect level
Large River Monitoring Network
maximum acceptable toxicant
concentration
microbial biomass carbon
malondialdehyde
mining ecotype
magnesium
minimum inhibitory concentration
multiple linear regression
month, months
metal-rich granules
maximum tolerable concentration
molecular weight
national ambient air quality standards
North American Soil Geochemical
Landscapes Project
national water quality assessment
no-effect concentration
nonmining ecotype
National Oceanic and Atmospheric
Administration
no observed effect concentration
natural organic matter
organic carbon
organic matter
omnivores-predators
operational taxonomic unit
lead
probable effects concentrations
Population, Exposure, Comparison,
Outcome and Study
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PMF Picher mine field
PNEC predicted no-effect concentrations
REACH Registration, Evaluation, Authorisation
and Restriction of Chemicals
ROS reactive oxygen species
SEM simultaneously extracted metal
SOD superoxide dismutase
SSD species sensitivity distribution
T3 triiodothyronine
T4 thyroxine
TBMF trophic biomagnification factor
TEC threshold effect concentrations
TRF terminal restriction fragments
TTF trophic transfer factor
USGS United States Geological Survey
WACAP Western Airborne Contaminants
Assessment Project
WEOC water-extractable organic carbon
wk week; weeks
WQC water quality criteria
YCT yeast, cereal leaves and trout pellets
yr year, years
Zn Zinc
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APPENDIX 11 EFFECTS OF LEAD IN
TERRESTRIAL AND AQUATIC
ECOSYSTEMS
Summary of Causality Determinations for Welfare Effects of Lead
This appendix characterizes the scientific evidence that supports causality determinations for lead
(Pb) exposure and the effects of Pb in terrestrial and aquatic ecosystems and biota. In assessing the overall
evidence, the strengths and limitations of individual studies were evaluated. More details on the causal
framework used to reach these conclusions are included in the Preamble to the Integrated Science
Assessments (U.S. EPA. 2015). The evidence presented throughout this appendix supports the following
causality determinations (bolded text indicates a change since the 2013 Integrated Science Assessment for
Pb).
Level
Effect
Terrestrial3
Freshwater3
Saltwater3
Community-
and Ecosystem
Community and Ecosystem Effects
Likely Causal
Likely Causal
Suggestive
Reproductive and Developmental Effects - Plants
Inadequate
Inadequate
Inadequate
(0
+-»
C
Reproductive and Developmental Effects -
Invertebrates
Causal
Causal
Likely
Causal
O
Q.
"O
c
LD
<1)
C
o
Reproductive and Developmental Effects -
Vertebrates
Causal
Causal
Inadequate
0
>
Growth - Plants
Causal
Likely Causal
Inadequate
_l
1
C
Growth - Invertebrates
Likely Causal
Causal
Inadequate
O
+-»
03
1 8>
Hematological Effects - Vertebrates
Causal
Causal
Inadequate
c c
03 O
o) a.
Physiological Stress - Plants
Causal
Likely Causal
Inadequate
£ 8
Physiological Stress - Invertebrates
Likely Causal
Likely Causal
Suggestive
w
Physiological Stress - Vertebrates
Likely Causal
Likely Causal
Inadequate
aBased on the weight of evidence for causal determination in Table II of the ISA Preamble (U.S. EPA, 2015).
The Executive Summary, Integrated Synthesis, and all other appendices of this Pb ISA can be found
at https://cfpub.epa.gov/ncea/isa/recordisplay.cfm?deid=357282.
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11.1 Introduction, Scope, Concepts, and Tools
This appendix synthesizes and evaluates the most policy-relevant scientific information on Pb
welfare effects to help form the foundation for the review of the secondary (welfare1-based) National
Ambient Air Quality Standards (NAAQS) for lead (Pb). The focus of this appendix is on studies
published since the 2013 Integrated Science Assessment (ISA) for Pb (2013 Pb ISA) U.S. EPA (2013)
that examine Pb interactions with the biotic components of terrestrial and aquatic ecosystems, including
effects on vegetation and wildlife. Pb transport through abiotic compartments (air, soil, water, and
sediment) is covered in Appendix 1: Lead Source to Concentration:
https://cfpub.epa.gov/ncea/isa/recordisplay. cfm?deid=357282. Section 11.1 of this appendix includes key concepts
and tools useful for characterizing the effects of Pb on biota. Section 11.2 examines the bioavailability,
bioaccumulation, and effects of Pb in terrestrial ecosystems. The effects of Pb in terrestrial environments
are followed by information on the bioavailability, bioaccumulation and effects of Pb in freshwater
(Section 11.3) and saltwater (Section 11.4) ecosystems.
11.1.1. Scoping and Criteria for Study Inclusion
This appendix builds upon the assessment of effects of Pb on ecosystems reported in the 2013 Pb
ISA (U.S. EPA. 2013) and in prior Air Quality Criteria Documents (AQCDs) from 1977 (U.S. EPA.
1977). 1986 (U.S. EPA. 1986). and 2006 (U.S. EPA. 2006a). The framework used to define the scope of
the ecological effects portion of the current ISA is modeled after the Population, Exposure, Comparison,
Outcome, and Study Design (PECOS) used for human health effects (Appendix 12
https://cfpub.epa.gov/ncea/isa/recordisplay.cfm?deid=357282). For the health
appendices, the PECOS statement defines the objectives of the review and establishes study inclusion
criteria, thereby facilitating identification of the most relevant literature to inform the ISA for each health
discipline. Similarly, the Level of Biological Organization, Exposure, Comparison, Endpoint, and Study
Design (LECES) statement aids in identifying the relevant evidence in the literature for the ecological
effects of Pb (Table 12-4; Appendix 12). Studies that reported the effects of Pb on biota were evaluated,
included, and discussed in this appendix if they satisfied the following LECES criteria:
1 Under The Clean Air Act (CAA) section 302(h) (42 U.S.C. § 7602(h)), effects on welfare include, but are not
limited to, "effects on soils, water, crops, vegetation, manmade materials, animals, wildlife, weather, visibility, and
climate, damage to and deterioration of property, and hazards to transportation, as well as effects on economic
values and on personal comfort and well-being."
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11.1.1.1. Level of Biological Organization
Studies considered for this appendix included those that reported Pb effects on species,
subspecies or populations of vegetation, microbes, invertebrates, or vertebrates at any lifestage on
biological communities or on ecosystems in terrestrial, freshwater, or saltwater environments and
transition zones present in the United States or similar to those in the United States. In the 2013 Pb ISA,
ecological effects were generally organized in order of increasing biological complexity (i.e., from the
subcellular and cellular levels through the individual organism and up to ecosystem-level effects) (U.S.
EPA. 2013). This appendix follows the same organizing principle. For effects that occur at the
suborganism scale such as perturbation of biomarkers of physiological stress or changes in hematological
parameters, emphasis was placed on studies that concurrently reported effects experimentally linked to
higher levels of biological organization. Organism-level endpoints such as growth, survival, and
reproductive output have been definitively linked to effects at the population level and above. Examples
of organism-level endpoints with direct links to population-level effects include mortality, gross
abnormalities, survival, fecundity, and growth (Sutcr et al.. 2004). Because of the complexity of processes
than can affect an ecosystem and considering that Pb rarely occurs as the only contaminant in natural
systems, it is difficult to attribute effects observed at higher levels of biological organization solely to Pb.
11.1.1.2. Exposure
The deleterious effects of any given concentration of Pb can vary greatly under different
environmental and experimental conditions, as well as the duration and pathway of exposure. Relevant
concentrations for this assessment take into consideration the range of Pb concentrations in environmental
media from U.S. locations (Table 11-1) and the available evidence for concentrations at which effects are
observed in microbes, plants, invertebrates, and vertebrates. Effects observed at or near environmental
concentrations of Pb measured in soil, sediment, and water are emphasized. For the studies included in
the 2013 Pb ISA, evidence from exposures or doses generally ranged "from current levels to one or two
orders of magnitude above current levels" (U.S. EPA. 2013). Concentration cutoffs for literature inclusion
were not applied in earlier EPA reviews of this metal. To focus on studies that are the most policy-
relevant with regard to current environmental concentrations of Pb in the United States, concentration
cutoff values were applied when evaluating the literature published since the 2013 Pb ISA (Appendix 12:
https://cfpub.epa.gov/ncea/isa/recordisplay.cfm?deid=357282). For soil, the cutoff
value for screening of terrestrial studies of Pb exposure and effects was set at a concentration of
approximately 230 mg Pb/kg of soil, although higher concentrations were considered if the study added
new information on a mechanism of action, or if the higher concentration was part of a series that
contributed exposure-response information and included other concentrations below 230 mg Pb/kg. For
aqueous exposures, the cutoff value for study screening was approximately 10 jxg Pb/L, although higher
concentrations were considered if the study added new information on a mechanism of action or if the
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higher concentration was part of a series that contributed exposure-response information. For sediments,
the literature cutoff value for study screening was approximately 300 mg Pb/kg dry weight or lower.
Studies at very high concentrations of Pb were excluded unless they were part of a series in an
experimental exposure-response study and at least one concentration in the test series was in the ranges
stated above. Justification for selection of these Pb concentration cutoff values and additional information
on scoping for the literature for ecological effects of Pb is provided in Appendix 12. Initial literature
search and screening steps for this review identified many studies conducted at higher concentrations of
Pb that were ultimately excluded from the draft ISA
(https://hero.epa.gov/hero/index.cfm/proiect/page/proiect id/4081).
All reported values for biological effects in this appendix are from exposures in which
concentrations of Pb were analytically verified unless they are stated to be nominal concentrations. For
consistency, concentrations of Pb in soil and sediment are reported in mg Pb/kg dry weight (unless
otherwise specified) and aqueous concentrations of Pb are reported as ng Pb/L. For study concentrations
originally in other units such as (iM or ppb, the values are converted to mg Pb/kg or |ig Pb/L, and original
reported units are retained in parentheses. Only a subset of the studies reporting Pb effects on biota
analytically verified the concentration of Pb in media and the test organisms investigated.
11.1.1.3. Comparison
Comparisons in the studies considered for inclusion in this appendix were to an unexposed laboratory
control, a reference population, or a site with no detectable exposure or with lower Pb exposure. For
ecological effects assessment, both laboratory and field studies (including field experiments and
observational studies) can provide useful data (U.S. EPA. 2015). As the number of factors that the study
holds constant increases, other than Pb exposure, so does the certainty with which observed variation in
outcomes can be attributed to exposure, while the size of effects that the study is capable of attributing to
exposure becomes smaller. The ability to hold other variables constant is expected to diminish with
increasing biological scale from subcellular processes to whole ecosystems and from laboratory to field.
In general, effects of Pb on ecological endpoints are reported in the ISA if they are statistically
significant.
11.1.1.4. Endpoint
The biological endpoints considered in this appendix are relevant to the levels of biological
organization discussed above. The endpoints encompass species or population-level effects including but
not limited to effects on growth, reproduction or development, neurobehavioral effects, reduced survival,
or fitness, and photosynthesis. At higher levels of biological organization, endpoints include but are not
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limited to changes in community composition, altered ecosystem processes and functions, shifts in
genotypes or species, species extirpation, declines in the total number of species or biomass, and
decreased species richness.
11.1.1.5. Study Design
Relevant study designs for assessing Pb effects on ecological receptors include laboratory,
mesocosm, observational or experimental field or gradient studies wherein observed effects are measured
and analyzed quantitatively, or mechanistic modeling studies that estimate the effect of Pb on an
organism, biological population, community, or ecosystem (U.S. EPA. 2015). Controlled exposure
studies in laboratory or small-to-medium-scale field settings provide the most direct evidence for the
effects of Pb exposure, but their scope of inference may be limited (U.S. EPA. 2013). Exposure-response
data from acute bioassays typically report effects on mortality, growth, or reproduction. Chronic
bioassays are designed to incorporate effects over the lifespan or partial lifespan of the study subjects,
including effects on reproduction. In contrast, mesocosms and field studies include potentially
confounding factors (e.g., other metals) or factors known to interact with exposure (e.g., pH), thus
increasing the uncertainty in associating the effects observed with exposure to Pb (U.S. EPA. 2013).
11.1.1.6. Additional Scoping
Topics within scope also include effects of Pb biogeochemistry on bioavailability in terrestrial,
freshwater, and saltwater environments as well as subsequent vulnerability of particular organisms,
populations, communities or ecosystems and studies that address key uncertainties and limitations in the
evidence identified in the previous review. Topics outside of the scope of this appendix included mixture
studies that did not assess Pb effects independently and site-specific studies in non-U.S. locations that do
not contribute novel insights on Pb biogeochemistry or effects. As in the 2013 Pb ISA, generally, studies
on mine tailings, industrial effluent, sewage, bioremediation of highly contaminated sites and ingestion of
Pb shot, pellets or fishing gear are not within the scope of this ISA due to the high concentration of Pb
and lack of a connection to air-related sources or processes. This is consistent with the 2006 AQCD,
which typically did not include "effects from irrelevant exposure conditions relative to airborne emissions
of Pb (e.g., Pb shot, Pb paint, injection studies, studies conducted on mine tailings and studies conducted
with hydroponic solutions)" (Section AX 7.1.3 of (U.S. EPA. 2006a)).
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11.1.2. Introduction to Ecosystem Connections and Pb Transfers
Metals, including Pb, occur naturally in the geosphere, and anthropogenic enrichment of these
elements can lead to elevated concentrations in terrestrial and aquatic ecosystems. Pb is a persistent metal
that, once emitted, may cycle through multiple environmental media compartments (e.g., air, soil, water,
sediment) prior to exposure to plants and animals, as discussed in Appendix 1:
https://cfpub.epa.gov/ncea/isa/recordisplay.cfm?deid=357282 (Section 1.3). In
terrestrial ecosystems, non-air media can receive Pb from atmospheric deposition or other sources. Once
deposited, Pb can be resuspended into the air or transferred among other environmental media (Section
1.3). Exposure of freshwater and estuarine organisms to Pb, and associated effects, are tied to terrestrial
systems via watershed processes. Atmospherically derived Pb can enter aquatic systems through erosional
transport of soil particles in runoff from terrestrial systems (Section 1.3.3) or via direct wet or dry
deposition over a water surface (Section 1.3.1.2). Once in the aquatic environment, Pb partitions between
various compartments (water column, sediment, biota; Section 1.3.3). Saltwater ecosystems include
habitats that encompass a range of salinities from just above that of freshwater to that of seawater. These
ecosystems may receive Pb contributions from atmospheric deposition (Section 1.3.1.2), riverine
transport (Section 1.3.3) and runoff (Section 1.3.3) from terrestrial systems. The contribution of
atmospheric Pb differs by location. Ecosystems in more urban areas are also influenced by non-air
sources of Pb such as paint, automobiles, wastewater, and industrial activities.
Although Pb is present in the natural environment, it has no biological function in plants or
animals. Terrestrial, freshwater, and marine/estuarine organisms have developed adaptive physiological
responses for living with metals. These adaptations may include intracellular sequestration (e.g., synthesis
of metallothioneins or metal-rich granules [MRG]), induction of enzymes involved in oxidative stress
response, and modification of metal uptake or elimination rates (Gismondi et al.. 2017). Anthropogenic
enrichment can result in concentrations that exceed the capacity of organisms to regulate internal
concentrations, causing atoxic response and potentially death. Across taxa, effects of Pb exposure are
likely mediated through common biological mechanisms. In the case of Pb, ecological receptors and
humans are linked via shared pathways of exposure and commonalities in biological response to this
metal (Lassiter et al.. 2015). Connections between the atmosphere, the abiotic and biotic compartments of
terrestrial and aquatic ecosystems, and humans are acknowledged for Pb. However, for the purposes of
this ISA, these topics are divided into different appendices. Within this Ecological Effects appendix,
terrestrial, freshwater, and saltwater ecosystems are considered separately because of different
environmental and physiological factors that influence Pb toxicity, such as bioavailability of the metal,
form of Pb, other water and soil chemistry factors, and organism adaptations.
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11.1.3. Concentrations of Pb in Non-Air Media
Organisms may be exposed to Pb in soil, water, sediment, and other biota (via diet). Food,
drinking water, and contact with contaminated soils are likely major routes of exposure for terrestrial
wildlife. Ingestion and water intake are major routes of exposure for aquatic fauna. Inhalation is thought
to be a minor pathway in wildlife, with the possible exception of exposures in proximity to Pb
atmospheric point sources, such as smelters. Due to the presence of Pb in various environmental media,
exposure to this metal can occur via multiple pathways.
To provide sufficient information to support development of air quality criteria for Pb that are
protective of terrestrial and aquatic systems, it is important to gain a general understanding of current
distribution and the concentrations of Pb in the environment. Information on environmental
concentrations of Pb at U.S. locations is tabulated in Table 11-1. This table updates Table 6-2 in the 2013
Pb ISA U.S. EPA (2013) on Pb concentration in non-air media and biota. Sources of environmental
concentration data in Table 11-1 were limited to regional or national-scale studies. Studies that reported
concentrations in environmental media for one or a very small number of locations would be considered
anecdotal for the purpose of this review. Measured concentrations of Pb in soils, sediment and water are
not necessarily representative of the amount of Pb available to elicit a toxic effect. For Pb to interact with
a biological membrane and be taken up into an organism, it must be in a bioavailable form (Section
11.1.6), which is dependent upon the physical, chemical, and biological conditions under which an
organism is exposed at a particular geographic location. In addition, caution must be taken while
comparing Pb concentrations in different studies of environmental media because reported concentrations
of Pb may not be directly comparable across studies, in part due to differences in sampling, collection and
measurement methods. For example, soil Pb measurements may vary between studies that used partial
and complete acid digestion. Furthermore, complete acid digestion is likely to overestimate the amount of
bioavailable Pb in many cases. In aquatic systems, measurements of dissolved Pb may vary among
collection methods, notably due to different sample filtration sizes, while the composition of sediment
samples of Pb is often influenced by sieving size. These are given as illustrative examples of how Pb
observations may be affected by methods, but a comprehensive discussion of Pb sampling, collecting, and
measuring methods is beyond the scope of this ISA.
Some surveys of Pb in environmental media in Table 11-1 predate the 2013 Pb ISA (U.S. EPA.
2013) and 2006 Pb AQCD (U.S. EPA. 2006a'). Although they may have used less optimal methods than
more recent studies, these data are not excluded from the ISA in cases wherein they remain the best
available information.
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Table 11-1
Pb concentration in non-air media and biota.
Media
Pb Concentration
Years Data
Obtained
References
Soil
Conterminous U.S. 0-5 cm depth soil:
Median: 18.1 ± 185 mg Pb/kg; range: <0.5-
12,400 mg Pb/kg; IQR: 13.5-23.9 mg Pb/kg (dry weight)
Conterminous U.S. A horizon soil:
Median: 17.8 ± 46.6 mg Pb/kg; range: <0.5-
2,200 mg Pb/kg; IQR: 13.2-23.2 mg Pb/kg (dry weight)
Conterminous U.S. C horizon soil:
Median: 14.9 ± 18.5 mg Pb/kg; range: <0.5-681 mg Pb/kg;
IQR: 11.1-19.2 mg Pb/kg (dry weight)
2007-2010
Smith et al.
(2013a)
Northeastern U.S. forest floor soil mean: 151 ±29 mg Pb/kg
(dry weight)
1980
Richardson et
al. (2014b)
Northeastern U.S. forest floor soil mean: 68 ± 13 mg Pb/kg
(dry weight) (resurvey of 16 of 25 1980 sites)
2011
Richardson et
al. (2014b)
Soil sampled at 54 sites in Los Angeles, Orange, San
Bernardino, and Riverside counties in California
Range: 5-70 mg Pb/kg
Mean: 23.9 ± 13.8 mg Pb/kg
2019
Mackowiak et
al. (2021)
Soil (freshwater
wetlands and salt
marshes)
Conterminous U.S. uppermost soil horizon mean:
20.15 ± 1.73 (95% CI) mg Pb/kg (dry weight)
2011
Nahlik et al.
(2019)
Cores from 35 U.S. lakes
1970s Median: 115 mg Pb/kg (dry weight)
1990s Median: 73 mg Pb/kg (dry weight)
1996-2001
Mahler et al.
(2006)
National Water Quality Assessment of lotic systems
Median: 28 mg Pb/kg (dry weight)
1991-2003
U.S. EPA
(2006a)
Freshwater
Sediment
National Water Quality Assessment of lotic systems
grouped by river basin land use:
Baseline (in low-population areas): median: 20 mg Pb/kg;
range: 2-200 mg Pb/kg (dry weight)
Agricultural sites: median: 20 mg Pb/kg; range: 6-
310 mg Pb/kg (dry weight)
Cropland sites: median: 19 mg Pb/kg; range: 8-
310 mg Pb/kg (dry weight)
Pasture sites: median: 20 mg Pb/kg; range: 6-49 mg Pb/kg
(dry weight)
Forested sites: median: 28 mg Pb/kg; range: 2-
200 mg Pb/kg (dry weight)
Rangeland sites: median: 18 mg Pb/kg; range: 6-
330 mg Pb/kg (dry weight)
1991-2001
Horowitz and
Stephens
(2008)
131 coastal conterminous U.S. rivers: Overall mean:
59 mg Pb/kg; median: 26 mg Pb/kg (dry weight)
Atlantic rivers: mean: 110 mg Pb/kg; median: 36 mg Pb/kg
(dry weight)
2010-2011
Horowitz et al.
(2012)
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Media
Pb Concentration
Years Data
Obtained
References
Gulf rivers: mean: 32 mg Pb/kg; median: 24 mg Pb/kg (dry
weight)
Pacific rivers: mean: 19 mg Pb/kg; median: 13 mg Pb/kg
(dry weight)
Global Range: 0.6-1,050 mg Pb/kg
U.S. Range (from Puget Sound): 13.4-52.8 mg Pb/kg
Reported in
studies dated
1977-1990
Sadia (1992)
Saltwater Sediment
U.S. Geometric Mean: 43 mg Pb/kg
Global Geometric Mean: 43 mg Pb/kg
Global Geometric Mean ("hot spot" data from contaminated
1984-1987
Cantillo and
O'connor
(1992)
sites removed): 34 mg Pb/kg
Median: 0.50 |jg Pb/L
Max: 30 |jg Pb/L, 95th percentile 1.1 |jg Pb/L
1991-2003
U.S. EPA
(2006a)
8 Texas rivers
Sabine: Mean: 0.04 ± 0.025 |jg Pb/L
Range: 0.013-0.098 |jg Pb/L
Neches: Mean: 0.036 ± 0.028 |jg Pb/L
Range: 0.01-0.099 |jg Pb/L
Trinity: Mean: 0.061 ± 0.067 |jg Pb/L
Range: 0.009-0.218 |jg Pb/L
Brazos: Mean: 0.02 ± 0.011 |jg Pb/L
Range: 0.008-0.061 |jg Pb/L
1997-1998
Jiann et al.
(2013)
Fresh Surface
Water
Colorado: Mean: 0.02 ± 0.009 |jg Pb/L
Range: 0.007-0.04 |jg Pb/L
(Dissolved Pb)
Guadalupe: Mean: 0.049 ± 0.059 |jg Pb/L
Range: 0.005-0.202 |jg Pb/L
San Antonio: Mean: 0.356 ± 0.235 |jg Pb/L
Range: 0.177-0.919 pg Pb/L
Nueces/Frio: Mean: 0.025 ± 0.034 pg Pb/L
Range: 0.008-0.166 pg Pb/L
Range: 0.0003-0.075 pg Pb/L
(Set of National Parks in western U.S.)
2002-2007
Field and
Sherrell (2003)
NPS (2011)
Appalachian headwater streams
(4 sites located in second- or third-order streams within the
Blue Ridge level III ecoregion)
2015-2017
Olson et al.
(2019)
Mean: <0.28 pg Pb/L
8 Texas rivers
Sabine: Mean: 27.76 ± 5.5 mg Pb/L
Fresh Surface
Range: 21.81-38.17 mg Pb/L
Jiann et al.
(2013)
Water (Particulate
Neches: Mean: 32.4 ± 4.55 mg Pb/L
1997-1998
Pb)
Range: 26.48-39.23 mg Pb/L
Trinity: Mean: 28.24 ± 3.82 mg Pb/L
Range: 22.87-33.24 mg Pb/L
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Media
Pb Concentration
Years Data
Obtained
References
Brazos: Mean: 22.45 ± 7.39 mg Pb/L
Range: 12.18^0.06 mg Pb/L
Colorado: Mean: 25.39 ± 12.33 mg Pb/L
Range: 13.4-72.92 mg Pb/L
Guadalupe: Mean: 20.2 ±5.17 mg Pb/L
Range: 14.2-35.8 mg Pb/L
San Antonio: Mean: 28.8 ± 5.23 mg Pb/L
Range: 21.97-38.34 mg Pb/L
Nueces/Frio: Mean: 22.33 ± 4.67 mg Pb/L
Range: 14.05-32.27 mg Pb/L
Saltwater
Global Range: 0.01-27 |jg Pb/L
Open-Ocean Range: 0.01-4.8 |jg Pb/L
Reported in
studies dated Sadiq (1992)
1977-1990
Vegetation
Lichens: 0.3-5 mg Pb/kg (dry weight) (Set of National Parks
in western U.S.)
2002-2007 NPS (2011)
Leaves from woody shrubs and trees from 54 sites in Los
Angeles, Orange, San Bernardino and Riverside counties in
California
Adenostoma fasciculatum
Mean: 0.17 ± 0.08 (SE) mg Pb/kg
Artemisia californica
Mean: 0.16 ± 0.01 (SE) mg Pb/kg
Baccharis salicifolia
Mean: 0.22 ± 0.03 (SE) mg Pb/kg
Encelia farinosa
Mean: 0.20 ± 0.02 (SE) mg Pb/kg
Eriogonum spp.
Mean: 0.23 ± 0.03 (SE) mg Pb/kg
Heteromeles arbutifolia
Mean: 0.42 ± 0.17 (SE) mg Pb/kg
Malosma luarina
Mean: 0.38 ± 0.06 (SE) mg Pb/kg
Quercus agrifolia
Mean: 0.29 ± 0.04 (SE) mg Pb/kg
2019
Mackowiak et
al. (2021)
Vertebrates
Fish (sampled from 111 sites in 9 river basins of large U.S.
rivers):
Mean: 0.07 mg Pb/kg (wet weight) (whole fish); Median:
0.10 mg Pb/kg (wet weight) (whole fish); 85th percentile:
0.27 mg Pb/kg (wet weight) (whole fish); Max:
9.29 mg Pb/kg (wet weight) (whole fish)
1995-2004
Hinck et al.
(2009)
Fish (96 sites in large U.S. rivers):
Female bass (Micropterus spp.): median: 0.04 mg Pb/kg;
mean: 0.06 ± 0.02 mg Pb/kg (wet weight) (whole fish)
Male bass (Micropterus spp.): median: 0.03 mg Pb/kg;
mean: 0.05 ± 0.01 mg Pb/kg (wet weight) (whole fish)
1995-2004
Hinck et al.
(2008)
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Media
Pb Concentration
Years Data
Obtained
References
Female carp (Cyprinus carpio)'. median: 0.10 mg Pb/kg;
mean: 0.11 ± 0.01 mg Pb/kg (wet weight) (whole fish)
Male carp (Cyprinus carpio)'. median: 0.09 mg Pb/kg; mean:
0.12 ± 0.01 mg Pb/kg (wet weight) (whole fish)
Dolphinfish (Coryphaena hippurus) in southern Gulf of
California (wet weight) (muscle tissue):
Mean: 0.059 mg Pb/kg
2006-2015
Gil-Manrique et
al. (2022)
Fish (from a set of national parks in western U.S.):
0.0033 (fillet) to 0.97 (liver) mg Pb/kg (dry weight)
2002-2007 NPS (2011)
Anna's hummingbirds (Calypte anna) surveyed in coastal,
valley and Sierra Nevada foothills regions of northern
California
Mean: 0.23 ± 0.25 mg Pb/kg; range: 0.00-1.35 mg Pb/kg
(body feathers; live) (dry weight)
Mean: 3.00 ± 7.64 mg Pb/kg; range: 0.28-46.0 mg Pb/kg
(body feathers; carcasses) (dry weight)
Mean: 1.01 ± 3.10 mg Pb/kg; range: 0.01-16.9 mg Pb/kg
(liver) (dry weight)
Mean: 0.94 ± 2.07 mg Pb/kg; range: 0.03-12.43 mg Pb/kg
(kidney) (dry weight)
Mean: 8.17 ± 36.27 mg Pb/kg (combined feathers) (dry
weight)
2015
Mikoni et al.
(2017)
Neotropic Cormorants (Phalacrocorax brasilianus) surveyed
in Lake Livingston, Texas:
Female mean: 4.92 ±4.11 (SE) mg Pb/kg (breast feathers)
(dry weight)
Male mean: 1.68 ± 0.822 (SE) mg Pb/kg (breast feathers)
(dry weight)
In Richland Creek Wildlife Management Area, Texas:
Female mean: 0.191 ± 0.044 (SE) mg Pb/kg (breast
feathers) (dry weight)
Male mean: 0.115 ± 0.015 (SE) mg Pb/kg (breast feathers)
(dry weight)
2014
Mora et al.
(2021)
7 earthworm species in northeastern U.S.
Overall mean: 29 ± 6 (SE) mg Pb/kg (dry weight)
Amynthas agrestis mean: 21 ±11 (SE) mg Pb/kg (dry
weight)
Aporrectodea rosea mean: 43 ± 5 (SE) mg Pb/kg (dry
weight)
Invertebrates Aporrectodea tuberculata mean: 30 ± 7 (SE) mg Pb/kg (dry
weight)
Dendrobaena octaedra mean: 43 ± 20 (SE) mg Pb/kg (dry
weight)
Lumbricus rubellus mean: 24 ± 5 (SE) mg Pb/kg (dry
weight)
Lumbricus terrestris mean: 14 ± 4 (SE) mg Pb/kg (dry
weight)
2013
Richardson et
al. (2015)
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Media
Pb Concentration
Years Data
Obtained
References
Octolasion cyaneum mean: 20 ± 8 (SE) mg Pb/kg (dry
weight)
Oysters (Crassostrea virginica) and mussels (Mytilus edulis)
in east coast U.S.
Range: 0.11-2.2 mg Pb/kg Pb (dry weight)
2003-2006
Shiel et al.
(2012)
Oysters (Crassostrea gigas) in west coast Canada
Range: 0.05-0.22 mg Pb/kg Pb (dry weight)
2002-2004
Shiel et al.
(2012)
CI = confidence interval; IQR = Interquartile range; Pb = lead; SE = Standard error.
This table updates Pb non-air media and biota concentration data from Tables 1-1 and 6-2 in the 2013 Pb ISA (U.S. EPA. 2013).
Sources of concentration data are limited to regional or national-scale studies.
Several large-scale surveys of soil Pb concentrations were identified for inclusion in the ISA. The
United States Geological Survey (USGS) North American Soil Geochemical Landscapes Project
(NASGLP) (Smith et al.. 2013a) is a recent soil survey that supplants Shacklette and Boerngen (1984).
the national soil survey cited in the 2013 Pb ISA, because of the larger size and extent, use of modern
geostatistical sampling methods, increased sampling resolution and documented data quality validation
(Smith et al.. 2013a). Shacklette and Boerngen (1984) collected 1,319 samples of Pb at a depth of 20 cm
along U.S. roadways between 1961 and 1976. The NASGLP provides a more comprehensive survey of
soil Pb in the conterminous United States because the survey employed a spatially balanced, sampling-
location selection method and collected soil samples from multiple depths at each selected location.
Samples were taken from depths of 0-5 cm in A-horizon and C-horizon soils at 4,857 sites systematically
selected using a generalized random tessellation stratified design in 2007-2010 (Figure 11-1). Soil Pb
concentrations were determined by inductively coupled plasma atomic emission spectroscopy and
inductively coupled plasma mass spectrometry analyses. Measurements were validated using documented
quality assurance and quality control procedures. A review of seven national-scale geochemical datasets
compared the NASGLP survey design to that of Shacklette and Boerngen (1984) and discussed the
methodological issues with other prior national-scale geochemical surveys that NASGLP was designed to
address (Smith et al.. 2013b). Summary statistics of conterminous U.S. soil Pb concentrations from Smith
et al. (2013a) are provided in Table 11-1. Regional studies of soil Pb, including Richardson et al. (2014b).
which provides information on temporal trends of Pb concentrations in northeastern forest floor soils, and
Mackowiak et al. (2021). which surveyed soil and vegetation Pb concentrations in four counties in
southern California, are summarized in Section 11.2.3.
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13CP 12CP 11CP IOC 9ff 805 70°
~1 T 1 T 1 1 T"
20° L I I I I
Base map from U.S. Geological Survey data
Lambert Conformal Conic projection 0 300 600 KILOMETERS
I 1 1 1
0 300 600 MILES
Source: Smith et al. (2013a)
Figure 11-1 Locations of the 4,857 soil sampling sites included in the U.S.
Geological Survey North American Soil Geochemical Landscapes
Project conducted from 2007 to 2010.
1 The 2006 Pb AQCD and 2013 Pb ISA reported representative Pb concentrations in fresh surface
2 water (median 0.50 |ig Pb/L, range 0.04 to 30 |ig Pb/L) and freshwater sediments (median 28 mg Pb/kg
3 dry weight, range 0.5 to 12,000 mg Pb/kg dry weight) in lotic systems in the United States based on a
4 synthesis of National Water Quality Assessment (NAWQA) data (U.S. EPA. 2013. 2006b). Another
5 analysis of the NAWQA data set provides additional detail to the prior 2006 Pb AQCD analysis by
6 stratifying the summary of Pb concentrations in freshwater sediment by land use within river basins
7 (Horowitz and Stephens. 2008). The baseline freshwater sediment concentration, comprising
8 measurements taken in low-population areas only, is reported to have a median of 20 mg Pb/kg with a
9 range of 2 to 200 mg Pb/kg. Land-use categories for agricultural, cropland, pasture, forested and
10 rangeland sites are reported in Table 11-1. A more recent survey of Pb concentrations in freshwater
11 sediment found higher concentrations in Atlantic rivers (mean 110 mg Pb/kg) compared with Pacific and
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Gulf of Mexico rivers (means of 19 and 32 mg Pb/kg, respectively) (Horowitz et al.. 2012). This observed
spatial variation in freshwater sediment Pb concentrations is likely driven by higher historical population
density and industrial activity associated with Pb emissions in the eastern United States compared with
the central and western regions of the country. Mahler et al. (2006) dated sediment cores and reported a
decline in Pb concentrations in sediment deposited between the 1970s and the 1990s, which corresponds
to the phasing out of widespread use of leaded gasoline. One additional regional survey of dissolved and
particulate Pb in fresh surface water was identified for inclusion in this ISA. In a study of water quality in
eight Texas rivers, Jiann et al. (2013) identified elevated particulate and dissolved Pb near areas with
greater anthropogenic influence and noted that Pb concentrations were decreased downstream of dams
and reservoirs, where slow-moving water causes suspended Pb to settle into sediment. Summary statistics
of the rivers included in Jiann et al. (2013) are included in Table 11-1. Additional information on
temporal trends observed in aquatic ecosystems is summarized in Sections 11.3.3 and 11.4.3.
No new surveys in coastal areas of the United States measuring dissolved Pb in saltwater or Pb in
saltwater sediment were identified for inclusion in this Pb ISA, although concentrations measured from
1984 to 1987 are included in Table 11-1 to provide additional information on Pb concentrations in
saltwater sediment (Cantillo and O'connor. 1992). The 2013 Pb ISA (U.S. EPA. 2013) reported saltwater
dissolved and sediment Pb concentrations from studies dated 1977 to 1990 summarized in Sadiq (1992).
which reports a global range of 0.6 to 1,050 mg Pb/kg in saltwater sediment, although the authors noted
that the maximum value reported was observed in an Australian inland saltwater lake. Observations from
only one U.S. saltwater sediment study were reported in Sadiq (1992). in which Pb concentrations
ranging from 13.4 to 52.8 mg Pb/kg from Puget Sound were recorded. Sadiq (1992) remains the only
study identified for inclusion in the ISA in which global dissolved saltwater Pb concentrations are
reported. Excluding observations from inland seas, open-ocean concentrations of dissolved Pb ranged
from 0.01 to 4.8 |ig Pb/L. Pb measurement methods have developed substantially in the last few decades,
and measurements of dissolved Pb from older studies may be less accurate than those measured using
modern methods. Table 11-1 summarizes the information available on concentrations of dissolved and
sediment Pb observed in U.S. saltwater aquatic ecosystems.
Information on Pb concentrations observed in regional surveys of U.S. biota at sites located far
from significant modern point sources of Pb have been collated in Table 11-1. The included surveys
provide a range of reference values which may provide context for Pb concentrations observed in similar
species and ecosystems. The Western Airborne Contaminants Assessment Project (WACAP) is the most
comprehensive database on contaminant transport and depositional effects in U.S. sensitive ecosystems
(U.S. EPA. 2013; NPS. 2011; Landers et al.. 2010). although it only covers locations in the western part
of the country. The project aimed to assess the locations where atmospheric pollutants were accumulating
due to deposition in remote ecosystems in the western United States and identify the most likely sources
of the identified pollutants. Pb (and other pollutants) was measured in sediment, snow, water, lichen, and
fish at eight western U.S. national parks. For species sampled across multiple national parks, Pb
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concentrations in biota in terrestrial and aquatic ecosystems surveyed in this project were reported in the
2013 Pb ISA and are included in Table 11-1.
Recent regional surveys of Pb in terrestrial ecosystems published in the peer reviewed literature
include Anna's hummingbirds (Calypte anna) surveyed in the coastal, valley and Sierra Nevada foothills
regions of northern California (Mikoni et al.. 2017) and cormorants (Phalacrocorax brasilianus) sampled
from two colonies in Lake Livingston and Richland Creek, Texas (Mora et al.. 2021). A summary of
feather Pb concentrations observed in each of these studies is included in Table 11-1. The study of Anna's
hummingbirds is unique in its investigation of bioaccumulation of metals in a nectar-feeding bird species.
The sources of Pb measured in hummingbird organs and feathers were not determined in this study, but
the authors listed absorption from food sources including plant and insect species, particularly those
living in urban environments, as the most likely routes of exposure (Mikoni et al.. 2017). Mora et al.
(2021) investigated the interaction between location and sex on Pb concentrations in cormorant feathers in
the Trinity River watershed in Texas and found no statistically significant effect for either variable.
A study of seven species of earthworms at nine sampling sites in the northeastern United States
was conducted alongside a concurrent soil survey that characterized the properties of the soil from which
the earthworm specimens were collected (Richardson et al.. 2015). This study provides an example of
how Pb from many sources in environmental media is distributed throughout a regional terrestrial
ecosystem, observed in both earthworms and the soil they inhabit. Earthworm Pb concentrations were
found to be poorly correlated with the Pb concentrations in the soil horizons they were sampled from,
which is explained in part by the selectiveness of earthworms' feeding and the unknown fraction of
bioavailable Pb in the measured soil Pb. Concentrations measured in earthworm species sampled in
Richardson et al. (2015) are summarized in Table 11-1.
Surveys of mussels (Mytilus sp.) and oysters (Crassostrea spp.) have been used to monitor Pb
concentrations in coastal ecosystems. The U.S. national Mussel Watch project (discussed in aquatic
temporal trends Section 11.4.3) has served as a biomonitoring network for Pb in coastal U.S. ecosystems
(Kimbrough et al.. 2008). An analysis of 2003-2006 Mussel Watch data including oysters (Crassostrea
gigas, Crassostrea virginica) and mussels (Mytilus edulis) identified a higher range of Pb concentrations
on the east coast of the United States relative to the west coast of Canada (Shiel et al.. 2012) (Table 11-1).
In this study, isotopic analysis and the covariance of cadmium (Cd) and zinc (Zn) were used to identify
the sources of Pb. Higher concentrations of Pb in the oysters and mussels on the east coast are attributed
to coal combustion and industries such as smelting and steelmaking.
The Large River Monitoring Network of the Biomonitoring of Environmental Status and Trends
(BEST-LRMN) surveyed fish from nine U.S. river basins from 1995-2004. This survey is the most recent
national-scale survey of Pb concentrations observed in biota in freshwater aquatic ecosystems, with
results summarized in two studies. Hinck et al. (2008) measured species-dependent Pb concentrations in
whole-fish common carp (Cyprinus carpio) and black bass (Micropterus spp.), and Hinck et al. (2009)
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presented average Pb concentrations measured across species including black bass, white bass (Morone
spp.), catfish (Ictaluridae), northern pike {lisax lucius), northern pikeminnow (Ptychocheilus
oregonensis), burbot (Lota lota), trout (Salmonidae), pikeperch (Sander spp.), and goldeneye (Hiodon
alosoides) (Table 11-1; summary statistics of Pb observations are presented with each included species
combined). The BEST-LRMN survey is the most comprehensive study of bioaccumulation of Pb in fish
from U.S. ecosystems.
11.1.4. Concepts Related to Ecosystem Effects of Pb
Organism exposure and response to Pb in the various environmental media must be considered in
the context of the ecosystem. An ecosystem is a functional unit consisting of living organisms, their
nonliving environment, and the interactions within and between them (Allwood et al.. 2014). The
boundaries of what could be called an ecosystem are somewhat arbitrary, depending on the focus of
interest or study. Thus, the extent of an ecosystem may range from very small spatial scales to, ultimately,
the entire biosphere (Allwood et al.. 2014). Ecosystems can be natural, cultivated, or urban (U.S. EPA.
1986) and may be defined on a functional or structural basis. "Function" refers to the suite of processes
and interactions among the ecosystem components that involve energy or matter. Examples include water
dynamics and the flux of trace gases such as rates of photosynthesis, decomposition, and nutrient cycling.
Biotic or abiotic structure may also define an ecosystem. Abiotic structure includes climatic and edaphic
components. Biotic structure includes species abundance, richness, distribution, evenness, and
composition measured at the population, species, community, ecosystem, or global scale. A species (for
eukaryotic organisms) is generally defined by a common morphology, genetic history, geographic range
of origin, and ability to interbreed and produce fertile offspring. A population consists of interbreeding
groups of individuals of the same species that occupy a defined geographic space. Interacting populations
of different species occupying a common spatial area form a community (Barnthouse et al.. 2008).
Community composition may also define an ecosystem type, such as a pine forest or a tall grass prairie.
Pollutants can affect the ecosystem structure at any of these levels of biological organization (Sutcr et al..
2005V
When an ecological receptor encounters Pb, this metal may affect uptake processes and/or
interact with biological membranes. In some instances, depending on the form of Pb and prevailing
environmental chemistry, Pb is taken up by biota which can then lead to a biological response. The
alteration of cellular ion status (including disruption of Ca2+ homeostasis, altered ion transport
mechanisms, and perturbed protein function through displacement of metal cofactors) appears to be the
major unifying mode of action underlying all subsequent modes of action in plants, animals, and humans
(U.S. EPA. 2013). Molecular mechanisms linked to oxidative stress may induce DNA damage and
generation of reactive oxygen species (ROS), leading to protein modification, lipid peroxidation, and
altered enzyme response. Initial perturbations such as cytological or biochemical changes associated with
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Pb exposure may cascade up to effects at higher levels of biological organization (i.e., from the
subcellular and cellular level through the individual organism and up to ecosystem-level processes). In
this ISA, biochemical (e.g., enzymes, stress markers) endpoints at the suborganism level of biological
organization are grouped under the broad endpoint of "physiological stress." Organism-level effects
include reproduction, growth, and survival. These endpoints also have the potential to alter population,
community, and ecosystem levels of biological organization (Suter et al.. 2004). Causality determinations
for ecological effects of Pb in the 2013 Pb ISA used biological scale as an organizing principle to
summarize effects on vegetation, invertebrates and vertebrates in terrestrial, freshwater and saltwater
environments. The same approach is applied in this appendix, focusing especially on the organism-level
endpoints of reproduction, growth, survival, and effects on ecosystems.
In natural environments, where many variables that may impact the effects of interest are left
uncontrolled, partitioning the variability of responses and attributing observed effects to Pb unequivocally
is difficult. The presence of confounding factors that is characteristic of field observational studies is also
compounded by high natural variability in organismal genetics and in abiotic seasonal, climatic, water
chemistry or soil-related factors (U.S. EPA. 2015). In natural environments, modifying factors affect Pb
bioavailability and toxicity, and considerable uncertainties are associated with generalizing effects
observed in controlled studies to effects at higher levels of biological organization. Differences in
environmental chemistry may enhance or inhibit uptake of metal from the environment, thus creating a
spatial patchwork of environments that are at greater risk than other environments. Similarly, organisms
vary in their degree of adaptation to, or tolerance of, the presence of metals. Generally, the correct
attribution of effects to Pb is expected to be most challenging in studies that examine its effects on entire
ecosystems, as they incorporate all of the ecological interactions among the various populations and all of
the chemical and biological processes that affect Pb bioavailability (Section 11.1.6). The fundamental
principles of how metals interact with organisms and ecosystems are described in detail in EPA's
Framework for Metals Risk Assessment (U.S. EPA. 2007).
11.1.5. Ecosystem Services
In general, both ecosystem structure and function play essential roles in providing goods and
services. "Ecosystem services" refers to the concept that ecosystems provide benefits to humans, directly
or indirectly (Costanza et al.. 2017). and that ecosystems produce socially valuable goods and services
deserving of protection, restoration, and enhancement (Bovd and Banzhaf. 2007). The concept of
ecosystem services recognizes that human well-being and survival are not independent of the rest of
nature, but rather that humans are an integral and interdependent part of the biosphere (Costanza et al..
2017). In some cases, ecosystem services analysis can result in attaching monetary values to ecosystem
outcomes. However, because ecosystem services are often public goods, their benefits can be difficult to
monetize. Although the ecosystem services literature has expanded since the 2013 Pb ISA, there are few
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publications that specifically link an ecological effect attributed to Pb to a change in an ecosystem
service. No new studies were identified that explicitly address Pb effects on ecosystem services associated
with terrestrial, freshwater, or saltwater systems.
11.1.6. Bioavailability
As discussed in prior AQCDs and sections 6.6.3 (terrestrial), 6.4.4 (freshwater) and 6.4.14
(saltwater) of the 2013 Pb ISA (U.S. EPA. 2013). bioavailability is a key concept for understanding Pb
effects on the biotic components of ecosystems. EPA defines bioavailability as "the extent to which
bioaccessible metals absorb onto, or into, and across biological membranes of organisms, expressed as a
fraction of the total amount of metal the organism is proximately exposed to (at the sorption surface)
during a given time and under defined conditions" (U.S. EPA. 2007). This section presents a general
overview of bioavailability and introduces modifying factors and models to estimate bioavailability.
Chemical and biological modifying factors affecting bioavailability and subsequent toxicity to biota are
considered in more detail in the following sections: Section 11.2.2 (terrestrial), Section 11.3.2
(freshwater) and Section 11.4.2 (saltwater).
Bioavailability increases with the amount of Pb available as free Pb ions (U.S. EPA. 2013).
Factors affecting bioavailability and subsequent effects of Pb on biota include chemical factors that can
be quantitatively linked to toxicity. In soils, these include but are not limited to pH, cation exchange
capacity (CEC) and organic carbon (OC) content. In aquatic systems, water chemistry conditions
including hardness, pH, alkalinity and colloidal or dissolved OC (DOC) as well as the presence of other
metals affect the availability of Pb at sites of action on biological membranes. In saltwater, higher levels
of ions additionally affect Pb bioavailability. In sediments, Pb bioavailability may be influenced by the
presence of other metals, sulfides, iron (Fe) and manganese (Mn) oxides, and physical disturbance. In
addition to chemical factors, biological factors (see Section 7.2.3(U.S. EPA. 2006b) and Section 6.4.9,
(U.S. EPA. 2013Y) affect bioavailability; however, they are more difficult to link quantitatively to
toxicity.
The bioavailability of a metal is also dependent upon the fraction of metal that is bioaccessible.
As stated in the Framework for Metals Risk Assessment (U.S. EPA. 2007). the bioaccessible fraction of a
metal is the portion (fraction or percentage) of environmentally available metal that interacts at the
organism's contact surface and is potentially available for absorption or adsorption by the organism. The
framework states that "the bioaccessibility, bioavailability, and bioaccumulation properties of inorganic
metals in soil, sediments, and aquatic systems are interrelated and abiotic (e.g., OC) and biotic
(e.g., uptake and metabolism) modifying factors determine the amount of an inorganic metal that interacts
at biological surfaces (e.g., at the gill, gut, or root tip epithelium) and that binds to and is absorbed across
these membranes. A major challenge is to consistently and accurately measure quantitative differences in
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bioavailability between multiple forms of inorganic metals in the environment." A conceptual diagram
presented in the Framework for Metals Risk Assessment (U.S. EPA. 2007) summarizes metals
bioavailability and bioaccumulation in aquatic, sediment, and soil media (Figure 11-2).
Bioaccessible Fraction (BF)a:
Percent soluble metal ion
concentration relative to total
metal concentration (measured in
solution near biomembrane)
Relative Bioavailability (RBA)b:
Percent adsorbed or absorbed
compared to reference material
(measure of membrane dynamics)
Absolute Bioavailability (ABA)c
Percent of metal mass absorbed
internally compared to external
exposure (measures systemic
uptake/accumulation)
Bioaccessibility
vailability
Environmental availability
Exp&gure ==
Bioaccumulation df metal
==========^ Effects
- Membrane .
uptake
¦~{Soluble species A
H.^nlnhlP ^npriP^ R I i fc ;^Uptake^-^
1
~I Soluble species C \ *
Benign
accumulation
Internal
Transport
and
Distribution
Toxicoloical
accumulation
Site of
Toxic
Action
aBF is most often measured using in vitro methods (e.g., artificial stomach), but should be validated by in vivo methods.
bRBA is most often estimated as the relative absorption factor, compared with a reference metal salt (usually calculated on the basis
of dose and often used for human risk, but can be based on concentrations).
°ABA is more difficult to measure and used less in human risk; it is often used in ecological risk when estimating bioaccumulation or
trophic transfer.
Source: ERG (2004) and U.S. EPA (2007)
Figure 11-2 Conceptual diagram for evaluating bioavailability processes and
bioaccessibility for metals in soil, sediment, or aquatic systems.
The development and continued refinement of models that predict toxicity by incorporating
factors affecting bioavailability in aquatic systems have advanced the field of risk assessment for metals
(Adams et al.. 2020). The physicochemical composition of the receiving water determines the
bioavailability and thus the toxicity of metals to aquatic organisms. Therefore, aquatic bioavailability
models must incorporate the effects of influential aspects of water chemistry on metal toxicity. The biotic
ligand model (BLM) is a mechanistically based model for predicting the toxicity of single metals under a
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large range of water chemistry conditions that considers complexations with inorganic ligands and
competition of active free metal ions with other cations, such as calcium (Ca) and magnesium (Mg), for
the site of action (i.e., biotic ligand) (Nivogi and Wood. 2004; Paquin et al.. 2002; Di Toro et al.. 2001). It
predicts both the bioaccessible and bioavailable fraction of Pb in the aquatic environment and can be used
to estimate the importance of environmental variables such as DOC in limiting uptake by aquatic
organisms (Alonso-Castro et al.. 2009). The U.S. EPA-recommended freshwater ambient water quality
criteria (AWQC) for copper (Cu) are based on the BLM. Deforest et al. (2017) proposed a BLM-based
freshwater aquatic life criteria for Pb (Section 11.3.5).
Another recent approach to describing and predicting bioavailability and subsequent toxicity of
metals in aquatic environments are empirically based multiple linear regression (MLR) models, which
take into consideration a wide range of endpoints and water chemistry parameters from large empirical
toxicity data sets (Brix et al.. 2020). Since the 2013 Pb ISA, some studies have focused on further
evaluating the suitability of bioavailability models for predicting the chronic toxicity of Pb to aquatic
biota (Deforest et al.. 2017; Nvs et al.. 2016b; Nvs et al.. 2014). while others have explored the
development and evaluation of bioavailability models to predict the acute and chronic toxicity of metals
mixtures, in which Pb is a component (Nvs et al.. 2017; Farley et al.. 2015; Santore and Ryan. 2015). A
detailed consideration of the advancements in metal bioavailability modeling approaches is beyond the
scope of this ISA. A recent EPA report titled Metals Cooperative Research and Development Agreement
(CRADA) Phase I Report: Development of an Overarching Bioavailability Modeling Approach to
Support US EPA's Aquatic Life Water Quality Criteria for Metals evaluates and compared BLM and
MLR approaches for the purpose of updating the AWQC for Pb and other metals and advocated for the
use of MLR models over the BLM in future AWQC for metals (U.S. EPA. 2022). A review of the current
status and regulatory applications of metal bioavailability models is provided in (Mebane et al.. 2020).
For historical perspective, refer to (Adams et al.. 2020) and see (Brix et al.. 2020) for empirical
bioavailability model development.
In terrestrial environments, predicting responses to Pb exposure under field conditions from
exposure-response experiments that use soluble salts of Pb to spike study soils has met longstanding
difficulties, chiefly because of the differences in the many interacting determinants of bioavailability and
the difficulty of identifying and quantifying those interactions. Ports et al. (2021) recently suggested that
two bioavailability corrections to the results of those experiments may be sufficient: one to adjust for
percolation and aging, and the other to correct differences in toxicity that arise from differing soil
properties. The authors demonstrated the derivation of predicted no-effect concentrations (PNEC)
according to the European Registration, Evaluation, Authorisation and Restriction of Chemicals
(REACH) Regulation Parliament and Council (2006) using the two corrections and data that conformed
to the REACH requirements.
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11.1.7. Risk Screening Tools
Risk assessors have developed tools for identifying the concentrations of Pb in environmental
media that are at or below the thresholds for effects on ecological receptors. The following sections
present ecological screening criteria available for evaluating Pb in atmospheric deposition, soil, water,
sediment, and biota.
11.1.7.1. Critical Loads for Atmospheric Deposition
The critical load concept is widely used as an organizing principle to relate atmospheric
deposition to ecological endpoints that indicate impairment (Pardo etal.. 2011; Bobbink et al.. 2010;
Porter and Johnson. 2007). The definition of a critical load is "a quantitative estimate of an exposure to
one or more pollutants below which significant harmful effects on specified sensitive elements of the
environment do not occur according to present knowledge" (Nilsson and Grennfelt. 1988). No recently
published critical loads for Pb from terrestrial ecosystems in the United States were identified for this
ISA. Several critical load studies from Europe reviewed in the 2013 Pb ISA (dc Vries and Groenenberg.
2009; Hall et al.. 2006; Morselli et al.. 2006) and a recent review study (Koptsik and Koptsik. 2022) noted
uncertainties inherent in a critical load approach to Pb risk assessment, such as soil type, critical
concentration of dissolved metal, adsorption coefficients of exposed soils, combined effects of different
metals in multimetal mixtures and the influences of a changing climate. Since the 2013 Pb ISA, critical
load studies for atmospheric deposition for aquatic systems have largely focused on eutrophication and
acidification associated with nitrogen (N) deposition, with no detailed assessments for Pb in freshwater or
coastal areas in Europe (RoTAP. 2012) or the United States. In the literature search for the current
assessment, no published critical loads for atmospheric deposition of Pb were identified for U.S. inland or
coastal waters.
11.1.7.2. Soil Screening Levels
Developed by EPA, ecological soil screening levels (Eco-SSLs) are maximum contaminant
concentrations in soils that are predicted to result in little or no quantifiable effect on terrestrial receptors.
The Pb Eco-SSL was completed in March 2005 and has not been updated since. Values for terrestrial
birds, mammals, plants, and soil invertebrates are 11, 56, 120 and 1,700 mg Pb/kg soil (dry weight),
respectively. These conservative values were developed so that contaminants that potentially present an
unacceptable hazard to terrestrial ecological receptors are reviewed during the risk evaluation process
while removing from consideration those that are highly unlikely to cause substantive effects. The studies
considered for the Eco-SSLs for Pb and detailed consideration of the criteria for developing the Eco-SSLs
are provided in the 2006 Pb AQCD (U.S. EPA. 2006b). Preference is given to studies using the most
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bioavailable form of Pb to derive values. Soil concentrations protective with respect to avian and
mammalian exposure through diet are calculated by first converting dietary concentration to dose (mg/kg
body weight per day) for a critical study, then using food (and soil) ingestion rates and conservatively
derived uptake factors to calculate a soil concentration that would result in unacceptable dietary doses.
This approach frequently results in Eco-SSL values below the average background soil concentration
(U.S. EPA. 2005a. 2003). as is the case with Pb for the birds Eco-SSL. Sample et al. (2019) used a re-
analysis of some of the early studies included in the 2005 derivation of the avian Eco-SSL to propose a
new value.
11.1.7.3. Ambient Water and Sediment Quality Criteria
AWQC represent surface water concentrations intended to be protective of aquatic communities,
including recreationally and commercially important species. The most recent AWQC for Pb were
developed in 1984 by the EPA Office of Water, which employed empirical regressions between observed
toxicity and water hardness to develop hardness-dependent equations for acute and chronic criteria for the
protection of aquatic biota (U.S. EPA. 1985a). These criteria are published pursuant to Section 304(a) of
the Clean Water Act and provide guidance to states and tribes to use in adopting water quality standards
for the protection of aquatic life and human health in surface water. The AWQC for Pb for aquatic life are
expressed as a criterion maximum concentration (CMC) for acute toxicity and criterion continuous
concentration (CCC) for chronic toxicity (U.S. EPA. 2009. 1985a). In freshwater, the CMC is 65 |ig Pb/L
and the CCC is 2.5 |ig Pb/L at a hardness of 100 mg/L.
The current EPA AWQC for Pb in freshwater, published in 1984, are hardness-based and the
chronic criteria were developed based on the acute-to-chronic ratio due to the lack of chronic toxicity tests
in freshwater biota at that time. Since the AWQC for Pb were first published, additional acute and chronic
toxicity data has become available and better characterization of factors that influence Pb bioavailability
including development of a BLM for Pb. In view of this information, several researchers have proposed
updated approaches for WQC derivation for this metal. Taking into account the range of surface water
chemistry across the United States and the inclusion of newer toxicity data, Deforest et al. (2017)
proposed a BLM-based acute Pb criteria range from 18.9 to 998 |ig Pb/L and chronic BLM-based Pb
criteria range from 0.37 to 41 |ig Pb/L for freshwater (Section 11.3.5). The lowest criteria were for water
with low DOC (1.2 mg/L), pH (6.7) and hardness (4.3 mg/L as calcium carbonate | CaCOs |). and the
highest criteria were for water with high DOC (9.8 mg/L), pH (8.2) and hardness (288 mg/L as CaCOs).
Compared to the current EPA AWQC for freshwater, the number of genera with acute toxicity data
increased from 10 to 32, and the number with chronic toxicity increased from 4 to 13, which enabled the
proposed chronic criteria to be based on bioassay data rather than an acute-to-chronic ratio. Furthermore,
DOC and pH are represented in BLM; these water quality factors have a significant influence on Pb
bioavailability and toxicity along with hardness and other water characteristics (Adams et al.. 2020).
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In comparison to the freshwater chronic criteria proposed by Deforest et al. (2017). Pb effect
thresholds to protect 95% of freshwater species calculated by Van Sprang et al. (2016) for seven selected
European freshwater scenarios were between 6.3 |ig Pb/L and 31.1 |ig Pb/L, based on chronic toxicity
datasets. There were several differences in development of the European thresholds for chronic Pb
toxicity compared with EPA guidelines, including the use of the 10% effect concentration (ECio) rather
than EC20 chronic toxicity data, selection of species mean values rather than genus mean values and
consideration of toxicity data for plants and algae in combination with bioavailability models to derive
effect thresholds. Furthermore, the range of water chemistries considered did not include the high
bioavailability conditions evaluated in (Deforest et al.. 2017).
For freshwater sediment, EPA guidance has not changed since the 2006 Pb AQCD, and a
summary of the guidance is provided here. EPA has recommended sediment quality benchmarks for Pb
that, although not truly regarded as criteria, are concluded to be protective of benthic organisms. Although
sediment quality criteria have not been formally adopted, EPA has published an equilibrium partitioning
procedure for developing sediment criteria for metals (U.S. EPA. 2005b). For freshwater sediment, the
two approaches first summarized in the 2006 Pb AQCD, based on either bulk sediment or equilibrium
partitioning, continue to be used and refined. The first approach is based on empirical correlations
between metal concentrations in bulk sediment and associated biological effects to derive threshold effect
concentrations (TEC) and probable effects concentrations (PEC) (Macdonald et al.. 2000). The TEC/PEC
approach incorporates numeric guidelines to compare against bulk sediment concentrations of Pb. The
equilibrium partitioning approach published by EPA for developing sediment criteria for metals (U.S.
EPA. 2005b) considers bioavailability by relating sediment toxicity to the porewater concentration of
metals. The amount of simultaneously extracted metal (SEM) is compared with the metals extracted via
acid volatile sulfides (AVS), since metals that bind to AVS (such as Pb) should not be toxic in sediments
where AVS occurs in greater quantities than SEM. The SEM approach was further refined in the
development of the sediment BLM (Di Toro et al.. 2005). An equilibrium partitioning sediment
benchmark for cationic metals, including Pb, was derived by Burgess et al. (2013). The mechanistic-based
sediment quality guideline was developed from the equilibrium partitioning theory, in which the
dissolved phase of Pb in sediment interstitial water serves as a surrogate for bioavailable Pb. In the
equation to derive the equilibrium partitioning sediment benchmark (Equation 1), AVS are subtracted
from SEMs to determine the amount of metal that could become bioavailable. The equation takes into
account interactions with both AVS and OC.
SEM. AVS = /foe FCV
" (X (Equation 1)
The final chronic value (FCV) (|ig/L) in the equation is calculated with the following formula
(Equation 2) using a conversion factor (CF) for Pb in freshwater. The FCV for Pb in saltwater is 8.1 |ig/L.
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p 1.273|Jm(hardncss)l -~4.705|C
1 1 (Equation 2)
The most recent aquatic life AWQC for Pb in saltwater were released in 1984 (U.S. EPA. 1985a)
by EPA's Office of Water. These criteria are published pursuant to Section 304(a) of the Clean Water Act
and provide guidance to states and tribes to use in adopting water quality standards for the protection of
aquatic life and human health in surface water. The AWQC for Pb are currently expressed as CMC for
acute toxicity and CCC for chronic toxicity (U.S. EPA. 2009). In saltwater, the CMC is 210 jxg Pb/L and
the CCC is 8.1 (ig Pb/L.
Since the most recent update of the EPA AWQC for saltwater, there are considerably more acute
and chronic toxicity data available for saltwater organisms, which reduce uncertainties related to Pb
toxicity and regulatory thresholds. For example, the 1985 CCC for saltwater was calculated based on
acute-to-chronic ratios from freshwater biota (Church et al.. 2017; U.S. EPA. 1985a). The EPA's
guidelines for derivation of AWQC indicate that when there are sufficient data, comparison of toxicity
data sets from different taxa using species sensitivity distributions (SSDs) can be performed to estimate
criteria values through a probabilistic approach and to set the level of protection (USEPA, 1985). The
minimum diversity required to develop SSDs has historically precluded this method for saltwater biota
due to lack of toxicity data. Using ECio acute toxicity data from sensitive early lifestages of 13 species
representing 7 taxa (phytoplankton, polychaetes, bivalves, crustaceans, echinoderms, chordates, fish)
inhabiting Atlantic European coastal ecosystems, Duran and Beiras (2013) derived an acute saltwater
quality criterion for Pb of 25.3 |ig Pb/L from SSD. This value, derived from the lower end of the 95%
confidence intervals of the 5th percentile of the SSD, is intended to protect 95% of species in 95% of
cases. Church et al. (2017) proposed an updated saltwater acute criterion of 100 |ig Pb/L and chronic
criterion of 10 |ig Pb/L based on genus mean toxicity values following U.S. EPA methodology (U.S.
EPA. 1985b) (Section 11.4.5).
Methods for establishing marine sediment guidelines and sediment quality values used globally
were recently reviewed by Birch (2018). Sediment quality values for U.S. waters were generally in the
range of the sediment quality threshold values reported by Macdonald et al. (1996). with a threshold
effects level of 30 mg Pb/kg and a probable effects threshold of 112 mg Pb/kg. A low effects threshold of
46.7 mg Pb/kg sediment and median effects threshold of 218 mg Pb/kg sediment were the sediment
quality guidelines developed for the National Oceanic and Atmospheric Administration (NOAA) National
Status and Trends Program (NOAA. 1999).
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11.2
Terrestrial Ecosystems
11.2.1. Summary of New Information on Effects of Pb in Terrestrial
Ecosystems and Causality Determination Update Since the 2013 Pb ISA
Since the 2013 Pb ISA (U.S. EPA. 2013). evidence has continued to accrue for many of the
effects of Pb on terrestrial ecosystems reported in the ISA and previous EPA assessments. This additional
support includes investigations of effects on species and communities that had not been studied, but none
of the additional evidence is sufficient to change any of the conclusions for terrestrial ecosystems that
were reached at the time. There are no changes to existing causality determinations for terrestrial
biota or ecosystems from the 2013 Pb ISA (Table 11-2).
Additional observational studies published after the 2013 Pb ISA (U.S. EPA. 2013). many of
which were anthropogenic environmental gradient studies, have linked Pb exposure and effects on
microbial community structure (e.g., abundance, diversity) and function (e.g., enzyme activities,
respiration rates). Many found mixed (negative, positive, or null) relationships between total or
bioavailable Pb soil concentration and the abundance of bacterial and fungal taxa. It remains difficult to
disentangle the effects of Pb exposure on microbial communities from the effects of other soil
contaminants using anthropogenic environmental gradient gradients, as other heavy metals and soil
physicochemical proprieties are significantly correlated with soil Pb concentration, and many of these
factors also influence microbial processes.
Studies published since the 2013 Pb ISA (U.S. EPA. 2013) continue to support previous findings
that plants tend to sequester larger amounts of Pb in roots as compared with shoots and that there are
species, ecotype, and cultivar-dependent differences in the uptake of Pb from soil and the atmosphere, and
in translocation of sequestered Pb. In the 2013 Pb ISA (U.S. EPA. 2013). the body of evidence was
sufficient to conclude there is a causal relationship between Pb exposure and plant physiological stress
and a causal relationship between Pb exposure and plant growth. Evidence was inadequate to determine
causal relationships between Pb exposure and both plant survival and plant reproduction. Recent studies
have continued to demonstrate various deleterious physiological effects of Pb exposure on plants,
particularly oxidative stress. Strong uncertainties remain regarding the concentrations at which these
effects would be observed in the environment. Recent studies have examined the protective effects of
mycorrhizae and of some plant nutrients when added in excess of the minimal requirements of the plants.
In terrestrial invertebrates, the evidence reviewed in the 2013 Pb ISA (U.S. EPA. 2013) was
sufficient to conclude that there is a causal relationship between Pb exposure and decreased survival and
between Pb exposure and reproductive and developmental effects, a likely causal relationship between Pb
exposure and decreased growth, neurobehavior effects and physiological stress, and the evidence is
inadequate to conclude that there is a causal relationship between Pb exposure and hematological effects.
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Evidence collected since then provides additional support for the effects of Pb exposure on organismal
and suborganismal responses including a decrease in survival, and decreased growth and fecundity.
Recently published studies on physiological responses to Pb include decreases in protein and lipid content
and increases in malondialdehyde (MDA) in earthworms. Acetylcholinesterase (AChE) activity decreased
in response to Pb in snails and honeybees while the effects on protein, glycogen, other enzymes, and
glutathione-s-transferase (GST) responses were variable depending on the site or species examined.
Several new studies quantified behavioral changes to Pb exposure in bees. Evidence also suggests that in
earthworms, Pb exposure can have lasting effects on growth even postexposure on earthworms and slow
the time to maturation. Pb exposure delayed onset of the breeding season and shortened duration in
isopods, as well as influenced mate selection in fruit flies. Evidence published after the 2013 Pb ISA
(U.S. EPA. 2013) includes new organisms as well as modifying factors of organism response such as
habitat, exposure history, and seasonality.
Effects of Pb commonly observed in terrestrial vertebrates include decreased survival, and
reproduction, as well as effects on development and behavior (U.S. EPA. 2006a'). The 2013 Pb ISA (U.S.
EPA. 2013) also provided evidence for Pb effects on hormones and other biochemical variables. In the
2013 Pb ISA (U.S. EPA. 2013) the body of evidence was sufficient to conclude that there is a causal
relationship between Pb exposure and reproductive and developmental effects, and between Pb exposure
and hematological effects, and a likely causal relationship between Pb exposure and decreased survival,
physiological stress, and neurobehavioral effects for terrestrial vertebrates. The evidence was inadequate
to conclude that the relationship between Pb exposure and growth is causal for terrestrial vertebrates.
Studies published since the 2013 Pb ISA provide additional evidence for effects on suborganism- and
organism-level endpoints, and specifically on hematological and physiological endpoints, but they do not
affect determinations of causality. New studies have expanded upon the relationship between Pb exposure
and ALAD activity by adding more species of birds, amphibians, and mammals to the evidence base.
More evidence of oxidative stress has been gathered, as well as evidence of effects on corticosterone
levels and immunity in birds. Literature since the 2013 Pb ISA continues to add to evidence relating to
reproductive effects at both the organism and suborganism levels including effects on lifetime breeding
success and some specific secondary sexual traits. New studies of behavioral effects included increased
aggression in mockingbirds.
Systematic studies of the validity of using results of Pb salt-addition experiments for estimating
effects of Pb exposure under field conditions have continued since the 2013 Pb ISA. As previously,
experiments showed that the form of Pb, pH, CEC, OC, Fe and Mn oxides, percolation, aging, and soil
composition are all strong modifiers of toxicity. Recent studies demonstrated additional interactions
among those variables and showed that their effects are at times mediated by additional variables such as
salinity. Those studies continue to support the conclusion that data from exposure-response experiments
in terrestrial environments conducted using spiking of soils with soluble salts of Pb, are unlikely to
generate accurate estimates of effects in contaminated natural environments. However, Ports et al. (2021)
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suggested that two corrections to the results of exposure-response experiments conducted with additions
of soluble salts of Pb to soil may be sufficient to derive predicted no-effect concentrations (PNEC)
according to the European REACH Regulation Parliament and Council (2006).
In the 2013 Pb ISA (U.S. EPA. 2013) the body of evidence was sufficient to conclude that there
is a likely causal relationship between Pb exposure and terrestrial-community and ecosystem effects.
Some new evidence of the effects of Pb at higher levels of biological organization is available, but it is
insufficient to change the determination of causality. Species interactions between tree species and their
pests, and between herbaceous plants and nectar robbers, worms and lepidopteran consumers were among
the new community and ecosystem endpoints for which effects of Pb were observed. Several studies
found negative relationships between Pb concentration along a pollution gradient and aspects of
invertebrate community structure, specifically in soil mites, potworms, insect communities on kale and
nematodes. Although evidence for effects on growth, reproduction, and survival at the individual
organism level and in simple trophic interactions makes the existence of effects at higher levels of
organization likely, direct evidence is relatively sparse and difficult to quantify. The presence of multiple
stressors, especially including other metals, continues to introduce uncertainties in attributing causality to
Pb at higher levels of organization.
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Table 11-2 Summary of Pb causality determinations for terrestrial plants,
invertebrates, and vertebrates
Level Effect Terrestrial
2013 Pb ISA3
2023 PbISA
Community and Ecosystem
Community and Ecosystem Effects
Likely Causal
Likely Causal
Reproductive and Developmental
Effects - Plants
Inadequate
Inadequate
Reproductive and Developmental
Effects - Invertebrates
Causal
Causal
Population-
level
Endpoints
Reproductive and Developmental
Effects - Vertebrates
Causal
Causal
Growth - Plants
Causal
Causal
Organism-level
Growth - Invertebrates
Likely Causal
Likely Causal
Responses
Growth - Vertebrates
Inadequate
Inadequate
Survival - Plants
Inadequate
Inadequate
Survival - Invertebrates
Causal
Causal
Survival - Vertebrates
Likely Causal
Likely Causal
Neurobehavioral Effects -
Invertebrates
Likely Causal
Likely Causal
Neurobehavioral Effects - Vertebrates
Likely Causal
Likely Causal
Hematological Effects - Invertebrates
Inadequate
Inadequate
Hematological Effects - Vertebrates
Causal
Causal
Suborganismal
Responses
Physiological Stress - Plants
Causal
Causal
Physiological Stress - Invertebrates
Likely Causal
Likely Causal
Physiological Stress - Vertebrates
Likely Causal
Likely Causal
aEcological effects observed at or near Pb concentrations measured in soil, sediment, and water in Table 6-2 of the 2013 Pb ISA
were emphasized and studies generally within one to two orders of magnitude above the reported range of these values were
considered in the body of evidence for terrestrial systems (Section 6.3.12) (U.S. EPA. 2013).
1 Previous AQCDs and the 2013 Pb ISA identified uncertainties with regard to the contribution of
2 Pb from current deposition to soil Pb concentration and subsequent toxicity to terrestrial biota, as opposed
3 to historic contributions. Historic Pb from gasoline and other sources as well as Pb from current air and
4 non-air sources is present in terrestrial systems and moves through the different environmental media
5 (e.g., soil, sediment, water, biota) confounding source apportionment. The contribution of atmospheric Pb
6 to specific sites is not clear (U.S. EPA. 2013). Furthermore, as stated in the 2013 Pb ISA, many factors,
7 including species and various soil physiochemical properties, interact strongly with Pb concentration to
8 modify effects. In terrestrial ecosystems, where soil is generally the main component of the exposure
9 route, Pb aging is a particularly important factor, and one that may be difficult to reproduce
10 experimentally. Without quantification of those interactions, characterizations of exposure-response
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relationships would likely not be transferable outside of experimental settings (U.S. EPA. 2013). Key
uncertainties with regards to Pb effects in terrestrial ecosystems in the last review included the
uncertainties expected from widening the scope of inference from controlled laboratory studies to
conditions in natural environments, where many modifying factors affect Pb bioavailability and toxicity.
This also applies when going from studies at low levels of biological organization to effects at higher
levels. In particular, available studies on community and ecosystem-level effects are usually from
contaminated areas where Pb concentrations are much higher than typically encountered in the
environment and where multiple contaminants are present.
Studies that characterize bioavailability, uptake, bioaccumulation, and effects of Pb in terrestrial
ecosystems or that decrease uncertainties identified in the prior Pb NAAQS review and were published
since the 2013 Pb ISA (literature cutoff for inclusion in the 2013 Pb ISA was September 2011) are
presented throughout the following sections. Brief summaries of conclusions from the 1977 Pb AQCD
(U.S. EPA. 1977). 1986 Pb AQCD (U.S. EPA. 1986). 2006 Pb AQCD (U.S. EPA. 2006a) and 2013 Pb
ISA (U.S. EPA. 2013) are included where appropriate. Recent research on the bioavailability and uptake
of Pb into terrestrial biota including plants, invertebrates and vertebrates is presented in Section 11.2.2.
Environmental concentrations in terrestrial biota and ecosystems in the United States at different locations
and over time are discussed in Section 11.2.3. The toxicity of Pb to terrestrial biota (Section 11.2.4) is
followed by data from exposure-response studies (Section 11.2.5). Responses at the community and
ecosystem levels of biological organization are reviewed in Section 11.2.6.
11.2.2. Factors Affecting Bioavailability, Uptake and Bioaccumulation and
Toxicity in Terrestrial Biota
Long-range atmospheric transport of Pb and natural rock weathering are the primary sources of
Pb in natural systems away from anthropogenic point sources. Non-urban terrestrial ecosystems
potentially affected by Pb deposition include natural forests, managed forests, grasslands, pastures, and
cropland. Once deposited, Pb can be resuspended into the air or transferred among other environmental
media. Pb atmospheric inputs into terrestrial ecosystems include direct deposition as well as resuspension
and transport of historically deposited Pb from nearby roads and contaminated soils (Appendix 1
https://cfpub.epa.gov/ncea/isa/recordisplay.cfm?deid=357282). In terrestrial systems,
Pb is distributed between biota, soil, and soil porewater. Mobility of Pb into biotic components of the
ecosystem is a function of the chemical speciation of Pb and subsequent bioavailability. Bioavailability of
Pb in soils (Section 11.1.6) depends on local soil physicochemical properties including pH, CEC, organic
matter (OM), inorganic compounds, salinity, clay content and aging. Uptake experiments with terrestrial
plants and invertebrates generally show increases in Pb uptake with increasing Pb concentration in the
medium but with strong effects from several interacting factors (U.S. EPA. 2013. 2006a). Below, factors
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that affect bioavailability of Pb in terrestrial systems are summarized along with information that
advances understanding of Pb uptake in terrestrial biota since the 2013 Pb ISA.
11.2.2.1. Factors Affecting Bioavailability of Pb in Terrestrial Biota
The 2013 Pb ISA described the bioavailable fraction of Pb in soil as being strongly dependent on
the fraction of Pb dissolved in soil porewater, which is primarily controlled by processes related to
partitioning of Pb between liquid and solid phases: (1) solubility equilibria; (2) adsorption-desorption
relationship of total Pb with inorganic compounds (e.g., oxides of aluminum (Al), Fe, silicon (Si), Mn;
clay minerals); (3) adsorption-desorption relationship reactions of dissolved Pb phases on soil OM; (4)
pH; (5) CEC; and (6) aging (U.S. EPA. 2013). The 2013 Pb ISA summarized studies that confirmed the
role each of these six factors plays in the sequestration and release of Pb in soil porewater (U.S. EPA.
2013). Total metal loading is described by the 2013 Pb ISA as the most influential factor controlling
adsorption and desorption, with higher concentrations of Pb corresponding to an overall decrease in the
fraction of Pb adsorbed to organic and inorganic surfaces (U.S. EPA. 2013). However, even as the
adsorbed fraction decreases with increasing metal loading, the rate of that decrease and the fraction of
adsorbed Pb will vary considerably between different soil types. This variability can be attributed to
differences in soil physicochemical properties, pH, CEC, OM, inorganic compounds, salinity, and aging.
These physicochemical properties are based on soil forming factors: climate, organisms, parent material,
relief, time, and anthropogenic input. Soils that differ in these factors will subsequently have different
physicochemical properties and considerable differences in the environmentally available fraction of Pb.
In addition, although predictions of bioavailability and toxicity based on environmentally available
fractions using extractable or porewater concentrations are still generally supported, evidence from recent
studies suggests that there may be limitations in predicting toxicity from environmentally available
concentrations represented as either porewater or calcium chloride (CaCl2)-extractable concentrations
(Lanno et al.. 2019; Buretal.. 2012; Pauget et al.. 2011).
11.2.2.1.1. pH and Cation Exchange Capacity
The 2013 Pb ISA cited a study conducted by Smolders et al. (2009) wherein models of metal
bioavailability calibrated from 500+ soil toxicity tests on plants, invertebrates and microbial communities
indicated pH and CEC were the most important factors governing both metal solubility and toxicity.
Recent literature confirms these findings and continues to highlight the important influence that pH and
CEC have on Pb bioavailability. To identify the main physicochemical factors controlling Pb
bioavailability in earthworms, Tang et al. (2018) conducted toxicity experiments on earthworms exposed
to 13 soils with low-level Pb contamination and varying physicochemical properties. Bioaccumulation
factors (BAFs) were calculated for each of the 13 soils and stepwise multiple linear regression and path
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analyses were used to assess the relationships between soil physicochemical properties and BAFs. Results
showed that the Pb BAFs of earthworms in soils with pH<5.5 were higher than those in other soils. OC,
pH and total Pb in soil were identified as the most important physicochemical parameters controlling Pb
bioavailability. The authors concluded that their results confirmed that low pH increases Pb mobility,
which promotes uptake and subsequent bioaccumulation (Tang et al.. 2018). Romero-Freire et al. (2015)
demonstrated the important influence of pH on bioavailability by measuring Pb toxicity to plants and
bacteria exposed to aqueous extracts from seven soils with different physicochemical properties. Both Pb
solubility and toxicity were significantly correlated with pH, CO3 and OC. Of the seven soils that were
assessed, sandy acidic soil with the lowest pH was associated with the highest extractable Pb
concentration and the lowest EC50 value for the plant bioassay. Wiiavawardena et al. (2015) investigated
the relationship between soil properties and relative bioavailability in swine exposed to 11 different soils
spiked with Pb. Freundlich partition coefficients (Kd) were calculated for each soil, and stepwise
regression analysis was used to evaluate the relationships between different soil properties and relative
bioavailability as well as Kd partition coefficients. Regression models showed that pH and clay content
were the most influential soil properties, accounting for 85% and 54% of variability in Kd and the relative
bioavailability of Pb, respectively. Lanno et al. (2019) examined the effects of physicochemical properties
on the toxicity of Pb to two different soil invertebrates, collembolans (Folsomia Candida) and earthworms
(Eisenia fetida), in seven different soils spiked with Pb salts at varying concentrations. EC50 values varied
considerably amongst the different soil types, ranging from 35-5,080 mg/kg for earthworms and 389 to
>7,190 mg/kg for collembolans. BAFs were also calculated for earthworms and varied with a > 10-fold
range across the different soil types. Effective CEC (eCEC) and soil properties related to eCEC including
total C, exchangeable Ca and Mg and clay content had a significant effect on both Pb toxicity and
bioaccumulation as well as the toxicity thresholds EC10 and EC50 in earthworms. However, there were no
correlations between soil properties and Collembola toxicity threshold concentrations. The authors
suggested that reduced toxicity in Collembola may be attributed to species-dependent differences in Pb
uptake across epidermal surfaces, specifically the sclerotized cuticles of collembolans may reduce the
uptake of Pb2+ across epidermal surfaces, limiting uptake to intestinal absorption from ingestion of soil
porewater. The study also assessed whether variability in toxicity values was better explained using
exposure estimates based on environmental available fractions (measured as Pb2+ in porewater or as total
dissolved Pb in porewater) rather than total Pb in soil. The results showed greater variation in EC50 values
based on environmentally available fractions compared with EC50 values based on total Pb soil
concentrations. These results combined with significant correlations between earthworm endpoints and
eCEC, but not pH, may suggest that eCEC reduces Pb uptake by cation exchange of Pb2+ in both clay and
OC coupled with competition for uptake between multiple cations at the surface of the earthworm
epithelium. The competition for cation uptake at the epithelial surface may also extend to H+, which may
help explain why toxicity thresholds were not correlated with pH. Additional explanations for greater
toxicity variability in porewater may also be due to unexplained chemical interactions between Pb2+ and
soil porewater as well as the physiological mechanisms of earthworm absorption and metabolism. Similar
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results were reported in a study that examined Pb and Cd bioavailability in soils located in the vicinity of
a smelter; uptake rate constants of Pb in earthworms were significantly greater at higher pH. Giska et al.
(2014) suggested that higher pH may be associated with a decrease in competition between heavy-metal
ions and H+ ions for binding sites on biotic ligands.
11.2.2.1.2. Organic Matter and Inorganic Compounds
The 2013 Pb ISA described the significant roles that both organic and inorganic soil constituents
play in immobilizing Pb and decreasing bioavailability. Surfaces of both OM and inorganic materials
(clays and sesquioxide minerals) contain negatively charged functional groups, which serve as sites of Pb
adsorption. In addition, Pb can form immobile precipitates with CO3, phosphate and sulfate that may also
be present in soil porewater. Shaheen and Tsadilas (2009) noted that soils with higher clay content, OM,
total CaCC>3 equivalent and total free sesquioxides also exhibited higher total Pb concentration, indicating
that less Pb had been taken up by resident plant species.
While recent studies confirm findings from the 2013 Pb ISA regarding the roles of OM and
inorganic surfaces in Pb immobilization, they also suggest that OM is capable of increasing or decreasing
Pb mobility. Shahid et al. (2012) reviewed the role of humic substances on Pb phytoavailability and
toxicity and concluded that the overall role of humic substances in Pb bioavailability is complex due to
the heterogenous nature of humic substances and varying soil physicochemical properties. Depending on
both of these factors, humic substances may exist as dissolved OM (DOM) capable of binding free Pb2+ in
soil porewater, as solid constituents with high adsorption affinity for Pb or as DOM capable of increasing
the extractable and bioavailable fractions of Pb. de Santiago-Martin et al. (2014) used bioassays with
romaine and iceberg lettuce grown in calcareous Mediterranean soils with low levels of OM that were
spiked with Pb, Cu, Cd and Zn to assess the contribution of soil physicochemical properties toward
bioavailability. CO3, OM and fine mineral fractions accounted for 85% of the variance in bioavailability,
and OM was the most important variable explaining Pb and Cd bioavailability patterns. However, OM
seemed to exert contrary effects on Pb and Cu bioavailability. At lower concentrations of the metals, OM
and bioavailability were negatively correlated, but a positive correlation was observed at higher
concentrations. The authors suggested that differences in the role OM had at different concentrations may
be attributed to competitive binding between Pb and Cu onto humic acids, resulting in a larger
bioavailable fraction at higher concentrations due to saturation of binding sites on humic acids (de
Santiago-Martin et al.. 2014). Similar results of the contradictory role that OM may have on
bioavailability were reported by Zeng etal. (2011). whereby OM was observed to have a positive
correlation with ethylenediaminetetraacetic acid (EDTA)-extractable chromium (Cr), Cu, Fe, Mn, Pb and
Zn, and both positive and negative correlations with concentrations in rice straw grown in the
contaminated soils. Paugetet al. (2011) evaluated the influence of pH, OM and clay content on chemical
availability and bioavailability of Pb to land snails (Cantareus aspersus) exposed to nine contaminated
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soils, each differing by a single characteristic (pH, OM, or clay content). The results demonstrated that an
increase in both pH and OM decreased Pb bioavailability to snails. However, clay did not have a
significant influence. It is worth noting that the clay mineral used for this assessment was kaolinite.
Kaolinite is 1:1 clay with no interlayer spaces and only external exchange sites at the edges of tetrahedral
and octahedral sheets. As a result, kaolinite has a low CEC compared to other clay minerals. Other clay
minerals (2:1) with both external and internal exchange sites in interlayer spaces may have had more
influence on bioavailability. The authors of the study acknowledged this limitation and other studies have
conveyed the important role that clay can have in decreasing metal mobility (dc Santiago-Martin et al..
2013). Pauget et al. (2016) investigated the contributions of soil and lettuce to bioavailability in garden
snails (Cantareus aspersus) and the influence of soil properties, pH, and OM on the contribution of each
source. Results indicated that soil contributed to 90% of Pb bioavailability in snails exposed to both soil
and lettuce, and increasing OM content further increased the contribution by an additional 6%. The
authors suggested that increasing OM may have also resulted in increased DOM, which may have
increased the soluble fraction of Pb through formation of DOM-Pb complexes in soil solution. An
additional explanation suggested for the increased bioavailability in soil with higher OM may be an
increase in ingestion rate caused by a decrease in nutrients following the addition of OM.
11.2.2.1.3. Salinity and Aging
In addition to the physicochemical properties described above, Pb mobility and bioavailability
can also be influenced by salinity. Application of CaCk MgCl or NaCl salts to field-collected soils
containing 31 to 2,764 mg Pb/kg increased the proportion of the mobile metal fraction. As the strength of
the salt application was increased from 0.006 to 0.3 M, the proportion of released Pb increased from less
than 0.5% to over 2% for CaCh and from less than 0.5% to over 1% for MgCl (Acosta et al.. 2011).
However, the majority of salinity-induced effects occurred in soils containing less than 500 mg Pb/kg,
and the proportion of released Pb decreased with increasing total soil Pb concentrations. Recent literature
continues to show that laboratory soils spiked with Pb2+ salts, which are commonly used in toxicity
studies, may overestimate toxicity in corresponding field-contaminated soils (Figure 11-3) due to lack of
aging as well as increases in salinity and acidification that occur after the soil has been spiked with Pb2+
salts (Smolders etal.. 2015). Smolders et al. (2015) compared Pb toxicity between three groups of soils:
(1) aged 5 years, leached and pH-corrected; (2) leached and pH-corrected and (3) freshly spiked soils with
no leaching or pH corrections. Leaching, pH correction and aging after spiking reduced toxicity to plant,
microbial and invertebrate receptors by a factor of 8 (median value) based on ECio values. ECio values
were often near background levels for freshly spiked soils, but after leaching, pH correction and 5 years
of aging, the majority of ECio values were above 1,000 mg/kg. The authors concluded that salinity stress,
rather than acidification or aging, is the main factor explaining increased Pb toxicity in freshly spiked and
unleached soils and suggested that researchers performing future toxicity tests consider spiking soils with
lead monoxide (PbO) fine powder rather than PbCh salt to exclude confounding salt effects. PbO fine
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1 powder would also be more representative of Pb that contaminates soil through atmospheric deposition.
2 Similar results demonstrating the importance of aging were reported by Zalaghi and Safari-Sincgani
3 (2014). In the study, soils were spiked with 0, 600, 1,200 and 1,800 mg/kb Pb as lead nitrate (Pb(NC>3)2),
4 and the environmentally available fraction of Pb and microbial toxicity were measured at select time
5 increments across a 90-day period. The concentrations of Pb in the environmentally available fraction and
6 microbial toxicity showed a considerable decrease over the 90-day period of the study. The authors
7 concluded that this decrease in bioavailability was due to the transfer of Pb into CO3 and residual
8 fractions that occurred as a result of aging. Similar results demonstrating a decrease in Pb bioavailability
9 following soil aging were reported by (Zhang and Van Gestel. 2019a).
20 30 40 60 200 200 300 500 1000 2000 4000 7000
Added Pb (mg Pb/kg soil)
Source: Smolders et al. (2015)
Figure 11-3 Change in toxicity expressed as relative responses (i.e., response
relative to the mean of the corresponding control soil) for three
different laboratory soil treatments: freshly spiked; spiked,
leached and pH-corrected; and spiked, leached and pH- corrected
with 5 years of aging.
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11.2.2.1.4. Biological Factors
The severity of Pb effects on terrestrial biota depends in part upon species differences in
metabolism, sequestration, and elimination rates. Because of the effects of soil aging and other
bioavailability factors discussed above, in combination with differing species assemblages and biological
accessibility, ecosystems may also differ in their sensitivity and vulnerability to Pb. The 2006 Pb AQCD
and 2013 Pb ISA reviewed these factors, including nutritional factors, soil aging and bioavailability.
Sensitivity to Pb exposure was found to vary widely among terrestrial species, even among closely related
organisms. It was noted that in many species of birds and mammals, dietary factors can exert significant
influence on the uptake and toxicity of Pb. Since the 2013 Pb ISA, new information on soil aging has
further expanded understanding of factors that modify soil bioavailability under natural conditions.
To disentangle the effects of salinity, acidification, and aging on the sensitivity of microbial communities,
plants, and invertebrates to Pb, Smolders et al. (2015) conducted an experiment in which toxicity to these
groups was tested in soils spiked with Pb2+ salts, leached and aged. Uncontaminated soils were collected
from grasslands and agricultural lands in Spain, the United Kingdom and Belgium and were exposed to 0,
250, 500, 1,000, 2,000, 4,000 or 8,000 mg Pb/kg using PbCh Some of the soil was set aside (treatment:
freshly spiked), while the rest was incubated for a week, leached using artificial rainwater and pH-
corrected to maintain soil pH within 0.2 pH units within each Pb concentration using CaO (treatment:
leached and pH-corrected). Five years prior to spiking soils with PbCk additional soils were exposed to
the same Pb gradient using Pb(NO;,): and stored in perforated pots which were left outdoors to age. After
5 years, pH was corrected using CaO (treatment: aged, leached and pH-corrected). Soil solution Pb
concentration, i.e., porewater Pb concentration, increased in a dose-dependent manner with spiked soils,
followed by leached soils and finally aged soils containing the least soil solution Pb (except in aged soils
from Spain). Toxicity was then tested in microbial communities, earthworms (E. fetida), Collembola (/¦'.
Candida), tomato (Lycopersicon esculentum) and barley (Hordeum vulgare). Toxicity was highest in
freshly spiked soils (mean ± S.E., EC50 for all organisms tested: 2,300 ± 145 mg/kb Pb), followed by
leached and pH-corrected soils (6,500 ± 750 mg Pb/kg) and then aged soils (>10,000 mg Pb/kg);
however, the effects of leaching with pH correction and aging with pH correction were inconsistent
among organisms and toxicity tests. Depending on the origin of the soil, leaching and pH correction
reduced toxicity based on EC10 values by a factor of 1.9-2.3 compared with freshly spiked soils, while
aging and pH correction reduced toxicity by a factor of 2.7-13. Microbial activity (potential nitrification
rate, substrate-induced nitrification, and respiration rate), invertebrate reproduction and plant growth were
negatively correlated with total soil Pb concentration, porewater Pb concentration, Pb2+ ion activity and
porewater ionic strength. With the exception of E. fetida reproduction, these factors were positively
correlated with soil pH. Given porewater ionic strength had the strongest influence on toxicity across all
tested organisms, the authors suggest that salt stress may modify the toxicity of Pb, as acidification and
aging were unable to explain variation in toxicity.
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11.2.2.1.5. Summary
In summary, studies published since the 2013 Pb ISA continue to substantiate the important role
that soil geochemistry plays in sequestration or release of Pb and its bioavailability to organisms.
Environmentally available concentrations, measured either in soil porewater or as extractable Pb, are
generally still a useful predictor of bioavailability, although predictions cannot be transferred between
experiments with soluble salts of Pb and field conditions. pH is still considered the most important factor
influencing the concentration of Pb in this fraction due to its important role in Pb solubility. However,
several studies have reported results that suggest limitations in using the environmentally available
fraction to predict bioavailability and toxicity. These studies suggest species-dependent uptake and
metabolism mechanisms as well as other soil physicochemical properties that may be involved in
chemical interactions between soil porewater and biological receptors should be taken into account.
Inorganic compounds, including clay minerals and sesquioxides, particularly Fe and Mn oxides are still
considered to play important roles in Pb sequestration, and CEC is still a reliable measure of a soil's
ability to sorb and exchange cations, which is an important function for Pb sequestration. The role of OM
in Pb sequestration and mobility remains complex. Depending on the nature of the OM and soil
physicochemical properties, Pb may bind to solid OM surfaces, decreasing Pb mobility. Alternatively,
OM may enhance Pb release into soil solution through the formation of Pb-DOM complexes or following
OM decomposition. Studies published since the 2013 Pb ISA also continue to highlight limitations in
using laboratory soils spiked with Pb salts to predict toxicity in field-contaminated soils. Many of these
studies have demonstrated that the use of Pb2+ salts in laboratory soils without adequate leaching, pH
correction and aging greatly affects Pb bioavailability and leads to overestimating the toxicity that would
be expected to occur in field-contaminated soils with similar concentrations of Pb.
11.2.2.2. Uptake and Bioaccumulation in Terrestrial Plants
Studies published since the 2013 Pb ISA continue to support previous findings that plants tend to
sequester larger amounts of Pb in roots as compared with shoots, and that there are species-, ecotype-, and
cultivar-dependent differences in uptake of Pb from soil and the atmosphere and translocation of the
sequestered Pb (U.S. EPA. 2013. 2006a. 1977). Further, many species of plants accumulate heavy metals
in environments with extreme soil concentrations and are therefore used for phytoremediation at such
sites. Although occasional phytoremediation studies may be informative with respect to the mechanisms
of Pb uptake and tolerance, most do not add further evidence with respect to the effects of atmospheric
Pb. The same applies regarding mosses and lichens as biomonitors of atmospheric Pb. Despite Pb not
being a plant nutrient, it is taken up from soils through the symplastic route, the same active ion transport
mechanism used by plants to take up water and nutrients and move them across root cell membranes
(U.S. EPA. 2006^. As with all nutrients, only the proportion of a metal present in soil porewater is
directly available for uptake by plants. In addition, soil-to-plant transfer factors in soils enriched with Pb
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have been found to better correlate with bioavailable Pb soil concentration, defined as diethylenetriamine
pentaacetate-extractable Pb, than with total Pb concentration (U.S. EPA. 2006b).
Previous reviews (U.S. EPA. 2013. 2006a. 1977) noted that terrestrial plants accumulate
atmospheric Pb primarily via two routes: direct stomatal uptake into foliage and incorporation of
atmospherically deposited Pb from soil into root tissue, followed by variable translocation to other
tissues. It was recognized that most Pb taken up from soil remains in the roots and that distribution to
other portions of the plant is variable among species. Most of the Pb absorbed from soil remains bound in
plant root tissues either because (1) Pb may be deposited within root cell wall material or (2) Pb may be
sequestered within root cell organelles, which may be a protective mechanism for the plant. Studies since
the 2013 Pb ISA have generally confirmed that Pb taken up from soil largely remains in the roots (Naikoo
et al.. 2019; Zhou et al.. 2019; Zhou et al.. 2015; Meiman et al.. 2012; Rossato et al.. 2012).
Previous findings have shown that Pb translocation to stem and leaf tissues does occur at
significant rates in some species, including some crops and herbaceous species (e.g., rattlebush,
buckwheat, Chinese cabbage, pak-choi, and water spinach). There is broad variability in uptake and
translocation among plant species, and interspecies variability has been shown to interact with other
factors such as soil type. These results indicate significant interspecies differences in Pb uptake, as well as
potential soil-dependent differences in Pb bioavailability (U.S. EPA. 2013).
Although exposures are often high, field studies carried out in the vicinity of Pb smelters and
other industrial point sources have determined the relative importance of direct foliar uptake and root
uptake of atmospheric Pb deposited in soils, with greater overall uptake corresponding to closer proximity
to the source (Angelova et al.. 2010; Hu and Ding. 2009; Cui et al.. 2007). Hu and Ding (2009) concluded
that metal accumulation in some leafy greens grown in the vicinity of a smelter were greater in shoot than
in root tissue, which suggested both high atmospheric Pb concentration and direct stomatal uptake into
the shoot tissue. Similarly, evidence since the last review shows substantial accumulation of Pb in needles
in areas with high contributions of atmospheric Pb (Kandziora-Ciupa et al.. 2016; Gandois and Probst.
2012). Studies also noted a significant difference between Pb concentrations in washed and unwashed
leaves, indicating that aerial deposition and surface dust is likely a significant source of plant-associated
Pb (Ugolini et al.. 2013; El-Rjoob et al.. 2008). Foliar Pb may include both incorporated Pb (i.e., from
atmospheric gases or particles) and surficial particulate Pb deposition. The plant may eventually absorb
the surficial component; however, the main importance of surficial Pb is its likely contribution to the
exposure of plant consumers or to leaf litter. The consideration of these Pb exposures to humans via
consumption of food crops is briefly discussed in Section 2.1.3 of Appendix 2.
Because of their long life spans, certain trees can provide essential information regarding the
sources of bioavailable Pb. A Scots pine (Pinus sylvestris) forest in northern Sweden was found to
incorporate atmospherically derived Pb pollution directly from ambient air, accumulating this Pb in the
bark, needles, and shoots (Klaminder et al.. 2005). More recent studies have also shown that accumulation
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in the bark of some species is a useful bioindicator of exposure to atmospheric Pb (Janta and Chantara.
2017; Palowski et al.. 2016). Metal content can also vary in relation to altitude as a result of long-range
transport. Korzeniowska et al. (2021) found that metal content in the moss (Pleurozium schreberi (Willd.)
Mitten) and in Norway spruce (Picea abies (L.) H. Karst) in the Tatra National Park in the Carpathian
Mountains of Poland was greater with increasing altitude.
Dendrochronology (tree-ring analysis) is an important tool for measuring the exposure of trees to
environmental Pb (Watmough. 1999). While effectiveness may vary by species investigated, tree-ring
studies reviewed in the previous AQCDs and ISAs showed that trees could be used as indicators of
increasing environmental Pb concentrations with time (U.S. EPA. 2013. 2006a. 1977). Trees accumulate
and sequester atmospheric Pb in close correlation with the rate of point-source emissions (Guvcttc et al..
1991). Studies published since the 2013 Pb ISA continue to demonstrate dendrochronology is a useful
tool for monitoring historical uptake of Pb into trees exposed to atmospheric or soil Pb (Scnsula et al..
2017; Dinis et al.. 2016; Beramendi-Orosco et al.. 2013; Doucet et al.. 2012) (Section 11.2.3).
In the 2013 Pb ISA (U.S. EPA. 2013). plant-associated arbuscular mycorrhizal fungi (AMF) were
found to protect the host plant from Pb uptake. Additional evidence indicates that the presence of AMF or
bacteria hosts can influence Pb accumulation in and alleviate Pb stress on plants. Inoculation of David's
mountain laurel (Sophora davidii, previously Sophora viciifolia) with the AMF Funneliformis mosseae
resulted in lower concentrations of Pb in belowground and aboveground biomass (Xu et al.. 2016a). S.
davidii seeds collected from around the Qiandongshan Pb and Zn mine in northwest China were grown in
pots receiving 0, 50, 500, or 1000 mg Pb/kg (aqueous Pb(NO,)2). Half of the pots with S. davidii plants
were inoculated with F. mosseae. After 4 months, mycorrhizal colonization, Pb accumulation, plant
height, diameter, aboveground and belowground biomass, and root characteristics were recorded (Section
11.2.4.2). Vesicular, arbuscular, hyphal, and total root colonization of S. davidii decreased with increasing
Pb treatment. Both mycorrhizal and nonmycorrhizal plants showed increasing Pb content in their roots
and aboveground tissue in a dose-dependent manner, but belowground and aboveground Pb
concentrations were lower for mycorrhizal plants. Pb concentration in aboveground tissue of mycorrhizal
plants was 54-66 % less Pb than that in nonmycorrhizal plants, while roots contained 15-85 % less,
depending on Pb exposure. The root-to-shoot Pb concentration of mycorrhizal plants increased with Pb
exposure while nonmycorrhizal plant root-to-shoot concentration decreased with increasing Pb exposure,
suggesting that Pb was sequestered in the root following inoculation with F. mosseae. Furthermore,
transmission electron micrographs and X-ray microanalysis of S. davidii roots under different Pb and
mycorrhizal treatments suggested Pb in the cytoplasm was sequestered in the cell walls and vacuoles of F.
mosseae, while Pb was transported into the root cells and intracellular space of nonmycorrhizal plants.
Pot marigolds (Calendula officinalis) inoculated with Glomus mossea and G. intradices
accumulated more Pb relative to nonmycorrhizal plants, yet experienced greater fitness-(Tabrizi et al..
2015). Calendula officinalis were grown in pots and received 0, 150, or 300 mg Pb/kg (aqueous
Pb(NC>3)2). Half of the plants were inoculated with a mixture of G. mossea and G. intradices. Root
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colonization, Pb accumulation, plant growth, reproduction flavonoid contents and nutrients were
analyzed. Root colonization decreased with increasing Pb exposure in a dose-dependent manner, as root
colonization in the control (0 mg Pb/kg) was 56% higher than in the high Pb treatment (300 mg Pb/kg).
Pb concentration in the roots and the shoots (mg Pb/plant) increased with increasing Pb exposure.
Inoculated Calendula officinalis had 10.3% more Pb in the roots compared with noninoculated plants,
while shoots of inoculated and noninoculated plants contained the same amount of Pb. The interaction
between Pb exposure and inoculation did not influence Pb uptake in aboveground or belowground
biomass.
In another example, the AMF Gigaspora margarita increased bioaccumulation of Pb but reduced
Pb-induced stress of silver banner grass (Miscanthus sacchariflorus) (Sarkar et al.. 2018).Miscanthus
sacchariflorus rhizomes and soil were collected from sites around the Ara River, Japan and placed in the
greenhouse. The collected soil contained 0.12 mg Pb/kg .Miscanthus sacchariflorus received 0, 100, or
1000 mg Pb/kg additional Pb (aqueous), and half of the plants were inoculated with G. margarita. After
4 months, root colonization, bioaccumulation of Pb and plant growth, survival, hormones, enzymes,
nutrients, and chlorophyll content were characterized. Root colonization ofM sacchariflorus by G.
margarita decreased with increasing Pb concentration for both inoculated and noninoculated plants. The
Pb content of the belowground biomass of inoculated M. sacchariflorus was higher than the Pb content of
noninoculated M. sacchariflorus. A similar pattern was observed for aboveground biomass, wherein
inoculated plants contained equal or higher concentrations of Pb than noninoculated plants.
Inoculation of black alder (Alnus glutonisa) by an actinobacteria. Frankia, affected Pb uptake in
roots and shoots (Belanger etal.. 2015). Alnus glutonisa seedlings were grown from seeds in the
laboratory and half were inoculated with Frankia alni (ACN 14a), isolated from Alnus viridis ssp. crispa
in Quebec, Canada. Half of the inoculated and noninoculated control plants were exposed to Pb(NO?h
(0.10 mM). Pb exposure did not affect the nodule development of inoculated plants and Pb root
concentration was 4.3 times lower in roots and 6.3 times higher in shoots compared with inoculated .4.
gultonisa not exposed to Pb.
In a recent study, (Gao et al.. 2021) reported that the type of mycorrhizal fungi (AMF versus
ectomycorrhizal fungi [EMF]) associated with seven tree species in an evergreen broad leaf forest in
China docs not affect uptake of Pb from roots to leaves. Foliar and root tissues were collected and
analyzed for Pb concentrations as well as phosphorus (P), potassium (K), Ca, Mg, Fe, Mn, Cu, Zn,
strontium (Sr), total C, and total N. Elemental concentrations in the tree were analyzed according to their
mycorrhizal type (AMF versus EMF), plant organ (leaves versus roots) and an interaction term. Pb
concentrations were significantly higher in the roots compared with the leaves. The elemental Pb
concentrations between the roots and the leaves were uncorrelated for AMF-associated trees, EMF-
associated trees, and all species, suggesting that mycorrhizal type does not influence Pb uptake in the
roots or the leaves.
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11.2.2.3. Uptake and Bioaccumulation in Terrestrial Invertebrates
At the time of publication of the 2006 Pb AQCD (U.S. EPA. 2006a). little information was
available regarding the uptake of atmospheric Pb pollution by terrestrial invertebrate species. Evidence in
the 2013 Pb ISA indicated that invertebrates, especially snails and earthworms, can accumulate Pb via
diet, exposure through soil, or from both exposure routes in the case of earthworms and snails. In the
2013 Pb ISA, snail Pb concentrations were reported to be lower than soil concentrations and uptake and
bioaccumulation were reported to be lower than the corresponding values for other metals (U.S. EPA.
2013). Exposure routes for soil organisms are through food consumption and soil exposure; soil variables,
such as pH and OM, influence uptake. Similarly, earthworm uptake is influenced by soil physicochemical
properties, genus, and the vertical position earthworms occupy within the soil profile (i.e., epigeic, epi-
endogeic, endogeic, anecic). Furthermore, earthworm activity in soil acts as a control on Pb
bioavailability and its uptake by earthworms, potentially other soil organisms, and plants. In addition to
providing supporting information on the uptake and availability of Pb to snails and earthworms, recent
literature has examined the bioavailability and accumulation of Pb with many other invertebrates
including lepidoptera, spiders and bees; in addition to soil factors (such as pH and OM), field
characteristics, organism sex and season may also influence uptake and accumulation. Since new
information has become available on organisms not discussed in previous assessments, these studies are
included despite being non-U.S. based.
11.2.2.3.1. Snails
In support of the 2013 Pb ISA conclusions regarding Pb uptake by snails, recent literature
continues to show snail tissue concentration is typically lower than soil concentration values. One recent
study found that when Pb was examined in soil, leaves, and snail tissues at increasing distance to metal
smelters, Pb in soils was, in general, highest closest to smelting plants and decreased with increasing
distance. Pb content in stinging nettle leaves (Urtica dioica) followed the same general pattern of
decreasing Pb concentration with distance as did European land snail (Cepaea nemoralis) digestive gland
tissue. The concentration in plant tissue was positively correlated with soil level, and snail tissue
concentration was positively correlated with plant tissue concentration. Patterns persisted over 4 months
of exposure. Nettles are the preferred food source of C. nemoralis and exposure to Pb appears to be
primarily through consumption. While bioaccumulation factors (BAF) were not calculated, Pb
concentration in snail tissue was considerably lower than soil concentrations but was typically 2.5-3.5
times higher than plant tissues after 16 weeks of exposure (Boshoff et al.. 2015) [see also (Nica et al..
2012)1. However, one recent study suggests some snail species may be greater accumulators than others.
Vrankovic et al. (2020) sampled Roman snails (Helixpomatia) foot muscles and hepatopancreas tissue
across a three-location urban gradient of soil Pb levels. Soil Pb varied from approximately 15 mg Pb/kg at
the reference (forest), approximately 30 mg Pb/kg at the medium pollution site and approximately
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110 mg Pb/kg at the high pollution site. Foot muscle and hepatopancreas tissue concentration increased
with increasing exposure levels. More Pb was stored in the hepatopancreas than the foot tissue, and
hepatopancreas levels were generally higher than soil contamination. BAF values were less than 1 for foot
muscle (0.47, 0.9, and 0.42) and greater than 1 for hepatopancreas tissue at the low and medium pollution
sites (1.61, 1.72, 0.76). The greater concentration found in the hepatopancreas indicates greater uptake via
food. Concentrations reported within snail tissues in this study were higher than those reported in studies
examining other snail species, suggesting uptake and accumulation are partly species-specific; see also
(Mleiki et al.. 2017).
New literature further supports that Pb uptake by snails is influenced by soil characteristics as
well as being dose- and duration-dependent. The concentration in the digestive gland of the green garden
snail (Cantareus apertus) increased with increasing exposure level after 1 week of exposure for low
(25 mg Pb/kg), medium (100 mg Pb/kg) and high (2500 mg Pb/kg, nominal values reported) exposure
levels (Mleiki et al.. 2016). However, tissue concentration was not significantly greater in the 2500
mg Pb/kg treatment compared with the 100 mg Pb/kg treatment. Similarly, after eight weeks of exposure,
digestive gland tissue concentration was higher under Pb exposure compared with the control, but the
highest concentrations were found under the 100 mg Pb/kg exposure. An observational field study
examining the uptake and elimination kinetics of Pb by the common garden snail (Cantareus aspersus)
found soil Pb concentration (positive), CEC (positive) and soil OC content (negative) have a multivariate
effect on Pb bioavailability. Similarly, soil silt (positive), sand (positive) and OC content (negative)
modulate Pb uptake by snails (Pauget et al.. 2013b). In another study, soil Pb concentration was correlated
with Pb concentration in juvenile C. aspersus but when OC content and Al and Fe oxides were included
in the model, R2 increased from 0.37 to 0.56. The most polluted plots (i.e., plots with the highest Pb
concentration) did not have the highest Pb transfer to snails. OC content is known to influence metal
mobility and bioavailability for soil organisms (Pauget et al.. 2013a).
11.2.2.3.2. Earthworms
In the 2013 Pb ISA, studies of bioaccumulation of Pb in earthworms reported that many soil
physicochemical properties, including pH, OM and CEC, affect metal bioavailability for these organisms;
recent studies confirm these observations. Following 4 weeks in soil spiked with a solution of Pb (NO;,):
(40, 250, 500, 1000, 2500 mg Pb/kg, nominal values reported), juvenile E. fetida body Pb concentration
increased with exposure concentration. BCFs ranged from 0.14 to 0.3, indicating either low
bioavailability of Pb in the soil or low ability to accumulate Pb within tissues. After 4 weeks of recovery
(no Pb exposure), earthworm body Pb was significantly lower than the value at the end of the exposure
period but was still higher than the control and positively correlated with exposure values (Zaltauskaitc et
al.. 2020). A study on native Eisenoides lonnbergi earthworms in Maryland found E. lonnbergi can
accumulate extraordinarily high levels of Pb, with a BAF of 83 recorded (Bever et al.. 2018).
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Accumulation was driven by soil Ca levels and indirectly by pH and clay content, not by soil Pb content
or availability. In acidic, low Ca soils, Pb uptake and accumulation is greater. Over soil Ca concentrations
ranging from 49 to 1695 mg Pb/kg, E. lonnbergi can maintain body Ca concentrations between 4000 and
8000 mg Pb/kg. Thus, even in Ca-poor soils, E. lonnbergi can uptake enough needed Ca to maintain
necessary body concentrations. The Ca BCF was 3.3 in high Ca soils and 117 in low Ca soils. The Pb
concentration factor was 1.02 in high Ca soils and 83 in low Ca soils, suggesting Pb is absorbed by the Ca
transport system, which is known to occur in vertebrates (Bever et al.. 2018).
In E. fetida earthworms exposed to a range of soil Pb values from 125-350 mg Pb/kg across a
range of pH, Pb concentration in the worms was higher in low pH (<5.5) soils than in neutral or alkaline
soils with similar Pb concentrations (Tang et al.. 2018). Following 4 weeks in soil spiked with a solution
of Pb (NC>3)2 (40, 250, 500, 1000, 2500 mg Pb/kg, nominal values reported), juvenile E. fetida body Pb
concentration increased with exposure concentration. BCF varied from 0.14 to 0.3 indicating either low
bioavailability of Pb in the soil or low ability to accumulate Pb within tissues (Zaltauskaitc et al.. 2020).
After 4 weeks of recovery (no Pb exposure), earthworm body Pb was significantly lower than at the end
of the exposure period but was still higher than the control and positively correlated with exposure values.
In another study examining earthworm Pb concentrations, BAFs in low pH soils were also higher than
those in other soils but all BAFs were less than one (Richardson et al.. 2015). Soil Pb, OC, and pH
together gave the best predictive model outcome on earthworm Pb concentration. Earthworm ecotype can
influence Pb tissue concentrations as well. Endogeic and epigeic species were found to have higher Pb
tissue concentration than epi-endogeic and anecic earthworms (Richardson et al.. 2015). A recent meta-
analysis by Richardson et al. (2020) examined the influence of soil concentration, soil characteristics,
earthworm genus and ecotype on trace metal uptake. They found soil concentration did not predict
earthworm tissue concentration but ecophysiological group, earthworm genus, metal source, exposure
duration, and soil OM were important predictors.
In studies cited in the 2013 Pb ISA, earthworm feeding and burrowing behavior altered the
bioavailability, mobility, and uptake of Pb by earthworms and other soil biota. Recent studies further
elucidate the effects of earthworms on soil Pb processes. One study examined the decomposition of
Amynthas agrestis and Lumbricus rubellus earthworms and the subsequent release of Pb in different
fractions within the soil column over 60 days (Richardson et al.. 2016b). Both species had similar Pb
tissue concentrations but due to the greater mass of A. agrestis added to experimental soils on a dry
weight basis, A. agrestis contributed a larger pool of Pb to the soil column. Leachate from both
earthworm treatments was significantly higher in Pb than leachate from control (no earthworm) soils.
Exchangeable Pb pools were greater under both earthworm treatments but only at days 7 and 21. By
day 60, there was only slightly more exchangeable Pb under the A. agrestis treatment compared with the
control. The stable Pb pool was greater under earthworm treatments across all sampling dates and the
majority of Pb under earthworm treatments was in the stable fraction. In a lab experiment using field-
collected polluted soils, Lumbricus terrestris earthworms were exposed to a high Pb-contaminated soil
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(4550 mg Pb/kg), a medium polluted soil (988 mg Pb/kg) and a low polluted soil (109 mg Pb/kg) for
28 days (Sizmur et al.. 2011a). By the end of the exposure period, earthworms had consumed less than
2% of the bulk soil. Soil pH and water-extractable OC were higher in earthworm casts compared to
control soils. Earthworm casts had greater extractable and residual Pb pools and lower reducible pools.
Porewater from earthworm-inhabited highly contaminated soils had higher Pb concentrations compared
with control soils. Under the medium contamination Pb soils, there was more Pb2+ and inorganic Pb, but
less organic Pb compared with control soils. In low pollution earthworm soils, there was less Pb2+ but
more organic and inorganic Pb compared with control soils. While earthworms only processed a small
portion of the soil during the 28-day exposure treatment, the greater solubility of Pb from casts shows
earthworms can alter Pb bioavailability and is tied to the changes in pH and OC of the casts.
The effect of two invasive, but widespread, species of earthworms in northeastern U.S. forests
(Amynthas agrestris and Lumbricus rubellus) on litter decomposition, metal exchange, and metal
bioaccumulation was examined in a laboratory experiment using forest floor material (collected from
New Hampshire) with and without earthworms (Richardson et al.. 2016a). Both species dwell at the soil
surface either in or just below the litter layer. Pb levels in forest floor and soil were approximately 26 and
16 mg Pb/kg, respectively. After 80 days, litter mass, percent carbon, and carbon mass were all lower in
the forest floor material when earthworms were present. Earthworm presence also resulted in lower
exchangeable Pb fraction concentrations but there was no difference between earthworm treatments and
control on the stable Pb fraction. Tissue concentration increased over time, with a BAF of 2.32 for A
agrestris after 80 days and 2.39 forZ. rubellus. The BAF for the exchangeable fraction only was 104.2
for A. agrestis and 88.3 for L. rubellus. Both worms increased litter decomposition and carbon loss and
lowered the exchangeable Pb fraction. However, the stable Pb pool did not respond to earthworm
presence. Both earthworm species did accumulate Pb at greater concentrations than the forest floor and,
as mentioned by the authors, at levels higher than the maximum tolerable level approved for poultry and
mice feed, therefore posing a contamination risk to birds and small mammals. In an observational
experiment in New England forests, Pb soil concentrations and pools were examined in the presence or
absence of nonnative earthworms (Aporrectodea rosea, Dendrobaena octaedra, Aporrectodea
tuberculata were most common) (Richardson et al.. 2017). Like the previous study, Pb in New England
soils sampled in this study represent background Pb levels in an area of the country with a history of
metal enrichment via pollution. Pb concentration was lower in the Oa horizons at high abundance sites
compared with low abundance sites; however, within the A and E horizons, Pb was higher at high
abundance sites. Organic horizon Pb pools were negatively correlated with earthworm biomass, but total
soil Pb pools showed no relationship with earthworm biomass.
In a study that examined earthworm effects on the bioavailability and mobility of metals in soil,
leachates at the end of a 112-day exposure period had greater Pb concentration in the presence of L.
terrestris earthworms (1.9 |ig Pb/L) compared with control soils (1.0 |ig Pb/L) (Sizmur et al.. 2011b). Pb
leachate from under L. terrestris consisted of 98.4% Pb2+ as free ions and 0.9% as fulvic-acid-complexed
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Pb compared with 95.7% and 4.0%, respectively, in control soil leachate. Soil pH was lower under all
earthworm species at the end of the experiment compared with the control. Perennial ryegrass (Lolium
perenne) was planted 28 days prior to soil sampling and harvested 21 days later. Ryegrass shoots had
greater Pb concentrations when grown on columns with L. terrestris compared with grass grown in
control soils. The dry mass of plant shoots did not differ between treatments. The results showed
earthworms can increase Pb mobility and availability to plants, increasing sequestration. Over a 6-week
experiment, there was no effect of Pb on lettuce growth but when grown in soils with earthworms, lettuce
biomass increased with increasing concentrations (significantly higher at 3730 mg Pb/kg concentration)
(Lcvcquc et al.. 2014). Earthworms also increased lettuce Pb concentration but only at exposure
concentrations of 2822 and 3730 mg Pb/kg.
11.2.2.3.3. Other Invertebrates
For the 2013 Pb ISA, studies of bioavailability and uptake comprised earthworms, snails and
arthropods including bees and beetles. Since the 2013 Pb ISA, new literature has examined additional
invertebrate groups including spiders, and butterflies. Pollen, honey, and bees from 16 honeybee (Apis
mellifera) apiaries were sampled twice a year for 2 years for Pb contamination across an urban-cultivated-
hedgerow-natural environmental gradient in France (Lambert et al.. 2012). Pb concentration in pollen was
influenced by sampling season but not by landscape characteristics. Thirty percent of honey samples were
below detection limits, and the rest had very low concentration values. Pb concentrations in honey from
apiaries surrounded by a hedgerow matrix were two times higher than those in other landscapes
measured, with honey from cultivated sites having the lowest concentrations (most were below the
detection limit). Pb in honey was higher in the 2009 season compared with the 2008 season. Pb
concentrations ranged from 0.001 to 1.896 mg Pb/kg in bees, from 0.004 to 0.798 mg Pb/kg in pollen and
from 0.004 to 0.378 mg Pb/kg in honey. Seasonality may influence bee Pb concentration, as levels were
higher in bees sampled during the June-October sampling period for one of the years studied. There was
no clear relationship of contamination between the three biological compartments (pollen, bees, honey).
In general, apiaries in urban and hedgerow locations had higher Pb contamination than apiaries in
cultivated or island landscapes. There was variation across the year, and contamination was typically
higher during the dry (summer) season. Honeybees are exposed to Pb contamination via direct contact
with Pb atmospheric deposition on flowers and through food contamination. Pb contamination patterns in
bees were similar to contamination levels in pollen, suggesting deposition contact contamination.
Seasonal differences may be explained by changes in floral availability.
Following 20 km-pollution gradients away from active Zn or metal smelters in Russia and
Poland, bumblebee (Bombus spp.) Pb levels (0.21-3.3 mg Pb/kg) and soil Pb levels (13.6-
814.2 mg Pb/kg) both decreased with increasing distances from the pollution source (Szcntgvorgvi et al..
2011). In another study, bee body, bee bread, propolis, and honey Pb content was examined across
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different geologic areas (Golubkina et al.. 2016). Sites included an unpolluted control located in the
Ribnitsa district in Moldavia (located away from industry or major highways), a selenium (Se)-deficient
area in the Henty province of Mongolia and the Voskresensk district of Moscow region, which is an area
of fertilizer production. Bee body Pb concentration was lowest at the unpolluted Moldavia location
(0.51 mg Pb/kg), higher in Mongolia (0.94 mg Pb/kg), 0.97 mg/kg away from fertilizer production area
(Novoselki, Russia) and over 4 times higher near fertilizer production (2.16 mg Pb/kg, Lopatino). There
was a positive correlation between Pb content in bees and bee bread for the Lopatino and Moldavia sites.
Pb content in the propolis was highest in Mongolia (16.07 mg Pb/kg) and much lower in the other
locations (2.08, 1.52, 3.18 mg Pb/kg, Moldavia, Novoselki, Lopatino, respectively) and was not as closely
correlated with bee body content. Honey Pb content was low across all sites (approximately 0.2 mg Pb/kg
or less).
Wolf spiders (Lycosidae) are common ground-dwelling arachnids and are known to accumulate
metals. An observational study in Korea found that while Pb in soil did not differ by season
(31.13 mg Pb/kg averaged across seasons), Pb was significantly greater in spiders from an autumn brood
(7.83 mg Pb/kg) compared with that in a spring brood (1.52 mg Pb/kg). While overall BCF was below 1,
the difference in brood accumulation suggests that while spiders accumulate Pb at low levels, seasonality
may affect accumulation (Conti et al.. 2018). Jung and Lee (2012) measured Ariadna spider Pb
accumulation in Namibia in relation to uranium (U), Cu, and gold mines. Overall, Ariadna spiders do
accumulate heavy metals in relation to their environment (in this case burrowing spiders and sand
contamination), but Pb levels were higher in sand compared to the levels in spider bodies, indicating Pb is
not readily bioaccumulated.
In the common cutworm (Spodoptera litura), Pb accumulation in body tissue generally increased
with increasing Pb exposure concentration across all development stages (Shu et al.. 2015). Larvae were
exposed to increasing Pb concentration via diet at 0, 12.5, 25, and 50 mg Pb/kg (nominal values reported)
and larvae were raised for five generations at each exposure concentration. Growth stage (larvae, pupae,
adult), Pb exposure concentration, and their interaction explained Pb accumulation, but generation did not
(F1 versus F5), nor were there any significant interactions with generation. Within development stages,
Pb accumulation was highest during the 6th instar stage, second highest in adults, and lowest in pupae (Pb
accumulation was only significantly higher at 50 mg Pb/kg treatment for pupae and adults). Within 6th
instar larvae, Pb exposure and tissue type mattered but sex did not. Overall, Pb accumulated primarily
within the midgut, and overall gut accumulation (mid, fore, and hindgut) was greater than that in the
hemolymph, head, or body fat. Accumulation also increased with exposure. In a trophic uptake study, Pb
accumulation in the roots, stems and leaves of mulberry (Morus alba) increased with increasing soil Pb
exposure (0, 200, 400, 800 mg Pb/kg, nominal values) (Zhou et al.. 2015). In turn, Pb in silkworm
(Bombyx mori) larvae and moths as well as in feces and silk excretions increased with increasing Pb
content in the mulberry leaves (in response to increasing Pb in soil). However, larvae (0.63, 4.08, 5.74,
and 11.16 mg Pb/kg) and moths (0.6, 2.95, 4.39, 6.23 mg Pb/kg) had lower body content than leaves
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(5.54, 41.79, 51.21, 60.26 mg Pb/kg) while Pb in feces was higher than that in leaves (9.85, 187.96,
230.44, 279.8 mg Pb/kg), indicating that while silkworms accumulate more Pb in response to increasing
exposure, Pb is not biomagnified, and the majority of Pb consumed is excreted instead.
A study that examined soil, plant, and grasshopper Pb concentrations at increasing distance to a
Zn smelter in China Zhang et al. (2012) found Pb content in all compartments decreased with increasing
distance. Soil Pb ranged from 49.9 to 973.5 mg Pb/kg. Plant Pb concentration ranged from approximately
5 to approximately 65 mg Pb/kg and varied by species (all species serve as a food source for
grasshoppers). Leaf Pb content was greatest in Japanese millet (Echinochloa crusgalli), followed by
Siberian elm (Ulmuspumila) and green foxtail (Setaria viridis). Grasshopper (Locusta migratoria
manilensis and Acrida chinensis) Pb content ranged from 1.07 to 46.95 mg Pb/kg (8.83 average). Soil and
plant contamination significantly decreased at 4000 m distance but Pb content in grasshoppers was
significantly higher within only 2000 m to the smelter.
Whole-body Pb content in isopods (Armadillidium granulatum) was positively correlated with Pb
food exposure (100, 500, 1000 mg Pb/L, nominal values), but concentrations were much lower than food
contamination levels, indicating isopods do not biomagnify Pb (Mazzci et al.. 2013). Simon et al. (2016)
examined soil, leaf litter, and beetle (Carabus violaceus and Pterostichus oblongopunctatus) Pb
concentrations along an urbanization gradient in Hungary. Pb concentration in soils was highest in the
urban locations but not different between rural and suburban locations. There was no difference in Pb
concentration within beetle species across sites but .P. oblongopunctatus (19.6 mg Pb/kg) had higher Pb
concentrations compared with C. violaceus (not detected). Within P. oblongopunctatus, Pb concentration
was higher in males compared with females (when pooled across sites). The BAF for .P.
oblongopunctatus was 1.26 in urban environments, 1.48 in suburban environments and 1.37 in rural
environments.
Vinegar fruit flies (Drosophila melanogaster) also display Pb accumulation differences based on
sex. Females had higher Pb accumulation compared with males (Peterson et al.. 2017). Both sexes
exposed to approximately 109 mg Pb/kg (250 (j,M Pb, nominal value) had higher Pb body concentration
(18.44 ng per female versus 7.32 for males) compared with controls (0.2 ng per male or female), but
females had greater concentration values. Furthermore, exposure of either male or female parent did not
lead to generational uptake effects. Pb loads in unexposed F1 generations with a Pb-exposed parent were
no different from those in F1 adults with control-treated parents. However, in another study by Peterson et
al. (2020). when D. melanogaster were reared in the same conditions but across an increasing gradient of
Pb exposure of approximately 109, 217, and 434 mg Pb/kg (250, 500, and 1000 |iM Pb nominal values),
they found no effect of sex on Pb accumulation nor a sex-Pb exposure interaction. Body Pb accumulation
did increase with increasing exposure concentrations, but the response was similar across both sexes.
Additional work is needed to determine the effect organism sex has on Pb uptake and accumulation in D.
melanogaster.
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Overall, literature since the 2013 Pb ISA adds additional supporting evidence of the importance
of soil variables on uptake and accumulation by soil invertebrates as well as new information on
additional arthropod groups and modifying factors such as season, and possibly, generation. Snails
typically accumulate Pb at lower concentrations than those found in soil or vegetation, but a higher
concentration of Pb in the hepatopancreas compared with that in the snail foot show uptake via
consumption leads to greater Pb accumulation than uptake through the soil-skin interface. Similarly,
grasshoppers and silkworms readily accumulate Pb but at levels lower than those in both food and soil
contamination. CEC and soil organic content interact with soil Pb concentration on driving uptake by the
common garden snail while pH and Ca content influence uptake and accumulation in earthworms.
Earthworm uptake also depends upon ecotype due to differences in feeding and burrowing behavior. As
discussed in previous assessments, there is an abundance of information examining the effects of
earthworms on Pb mobility and bioavailability due to these feeding and burrowing behaviors. Earthworm
casts, for example, were found to have higher pH and water-extractable OC. Literature since the 2013 Pb
ISA provides new information on the uptake and accumulation of Pb by spiders and butterflies, and
additional information on bees. Generally, Pb concentration is higher in bee bodies compared to honey
and pollen. Two spider genera examined show low accumulation levels in relation to soil contamination,
suggesting spiders do not readily bioaccumulate Pb. Lastly, there appear to be interactions of generation
and sex on Pb uptake by common cutworms and fruit flies, but the results are variable and the overall
effects remain unclear.
11.2.2.4. Uptake and Bioaccumulation in Terrestrial Vertebrates
The 2013 Pb ISA provided evidence of the accumulation of Pb in blood, bones, and a variety of
different tissues in birds and mammals. In studies of birds in the 2013 Pb ISA, the focus was mainly on
ingestion of man-made materials (e.g., Pb shot). In mammals, multiple species were found to accumulate
Pb from contaminated soils as well as from plants grown in contaminated soils. In birds, low dietary Ca2+
concentrations were linked to increased accumulation of Pb in liver, bone, kidney, muscle, and brain
tissues.
New information has become available on the uptake of Pb in terrestrial reptiles and amphibians
since the 2013 Pb ISA. A study of northern pine snakes (Pituophis melanoleucus melanoleucus) in the
pine barrens of New Jersey found that Pb was accumulated in a wide variety of tissues including liver,
kidney, muscle, skin, heart, as well as in blood, with the highest mean Pb concentration in muscle
(0.393 ±0.131 jxg/g wet weight) (Burger et al.. 2017). The pathway of exposure was not determined in
this study, but the authors suggested that consumption of prey items was the most likely pathway, as pine
snakes are a top predator in their food web. Pb was found to accumulate in the blood of giant toads
(Rhinella marina) captured at industrial, urban, and rural sites in Mexico (Ilizaliturri-Hernandez et al..
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2013). Blood Pb levels ranged from 10.8 to 70.6 (ig/dL and were found to increase with increasing soil Pb
levels.
Since the 2013 Pb ISA, new studies have been published that support findings of Pb accumulation
in different mammalian tissues. Tete et al. (2014) and Camizuli et al. (2018) both found evidence of Pb
accumulation in the kidneys and livers of wood mice (Apodemus sylvaticus). Kidney concentration ranged
from values under the limit of detection to 268.3 jxg/g dry weight, and liver concentrations ranged from
values under the limit of detection to 281.7 jxg/g dry weight. Another study on Pb accumulation in
mammalian tissues evaluated brain tissue from nine mesocarnivore species in Europe (Kalisinska et al..
2016). Eurasian otters (Lutra lutra), badgers (Meles meles), pine martens (Martes martes), beech martens
(Martes foina), European polecats (Mustela putoris), red foxes (Vulpes vulpes), feral and ranch American
minks (Neovison visori), raccoons (Procyon lotor), and raccoon dogs (Nyctereutes procyonoides) were all
sampled during this study. Brain tissue Pb was highest in raccoons (0.47 mg/kg dry weight) and lowest in
ranch American minks (0.072 mg/kg dry weight). The study's authors speculated that carrion with
hunting ammunition is likely to be an important source of Pb for omnivores and partial scavengers, while
organic Pb incorporated in the diet and Pb contained in the soil, earthworms, and dusted food may also be
possible sources of exposure.
Studies of bioaccumulation and uptake in birds tend to support information provided in the 2013
Pb ISA and provide additional evidence for Pb accumulation in a variety of different tissues. Soil remains
an important source of Pb exposure in many bird species. French et al. (2017) identified soil consumption
as one of the most common routes of Pb exposure in American woodcocks (Scolopax minor). Woodcocks
use their long bills to probe the soil for earthworms, with their dietary intake comprising as much as 10%
ingested soil, indicating that Pb-contaminated soil may be an important exposure pathway. Additionally,
the consumption of earthworms is another pathway of exposure, as earthworms can bioaccumulate metals
from the soil. Other species with similar feeding habits to woodcock such as American robins (Turdus
migratorius) may be exposed to Pb through these same pathways.
Birds of prey such as bald eagles (Haliaeetus leucocephalus) and California condors (Gymnogyps
californianus) have also been shown to accumulate Pb in blood and different tissues. A study of bald
eagle nestlings in the western Great Lakes region found blood Pb concentrations ranging from below the
limit of detection to 26.4 (ig/dL wet weight and feather Pb concentrations ranging from below the limit of
detection to 371 jxg/g wet weight (Bruggeman et al.. 2018). The authors speculated that Pb air pollution,
as well as Pb shot and Pb paint may all be sources of exposure. A study of California condors found that
between 1997 and 2010, the annual percentage of condors with blood Pb levels higher than 0.1 |ig/m L
(originally reported as 100 ng/mL) ranged from 50% to 88% (Finkelstein et al.. 2012). However, this
study found that the majority (79%) of condors had blood Pb isotope ratios that were not significantly
different from Pb-based ammunition. This indicates that Pb ammunition is likely the primary source of Pb
exposure in California condors. Behmke et al. (2015) examined bone Pb as a measure of chronic exposure
and Pb in liver as an indicator of more recent exposure in American black vultures (Coragyps atratus)
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and turkey vultures (Cathartes aura) collected in Virginia. Bone Pb was significantly higher than Pb in
liver in both species indicating that Pb in the birds was primarily associated with long-term exposure.
Possible sources of Pb in these long-lived birds based on comparison of Pb isotope ratios in femur bones
and Pb isotope ratios associated with Pb sources included ammunition, coal-fired power plants, leaded
gasoline, and zinc smelting operations.
In summary, literature since the 2013 Pb ISA (U.S. EPA. 2013) adds support to existing evidence
of Pb accumulation in blood, bones, and a variety of different tissues in terrestrial vertebrates. Pine snakes
accumulated Pb in liver, kidney, muscle, skin, and heart tissue, with the highest concentrations found in
the muscles. In toads, Pb was found to accumulate in blood and increased with increasing soil Pb levels.
New evidence continues to support findings of the accumulation of Pb in tissues from a wide range of
mammalian species. Pb ammunition continues to be a prevalent source of Pb contamination in both
mammals and birds. Consumption of prey species has also been found to be an important route of Pb
exposure especially in species that consume earthworms such as woodcocks and robins.
11.2.2.5. Uptake and Bioaccumulation Through Food Web
In the 2006 Pb AQCD (U.S. EPA. 2006a) and the 2013 Pb ISA (U.S. EPA. 2013). various studies
suggested that Pb might be transferred through terrestrial food webs, with lower Pb concentrations
occurring in each successive trophic level. Having data on bioavailable or bioaccessible concentrations of
Pb at every trophic level would lead to more accurate estimates of trophic transfer within food webs.
Since the 2013 Pb ISA (U.S. EPA. 2013). there have been more observational and experimental examples
of gradual attenuation of Pb concentrations with increasing trophic level; however, this depends on Pb
concentration, the presence of other heavy metals, ecosystem, and organism sensitivity to Pb exposure.
Although most of the following studies were conducted in non-U.S. locations or in proximity to point
sources, they further elucidate biotransfer processes for Pb.
Pb was transferred through a soil, nettle, snail food web in Antwerp, Belgium (Boshoff et al..
2015). In a microcosm field experiment, adult European land snails (Cepaea nemoralis) from an
uncontaminated site were exposed to sites varying in distance from the Umicore Precious Metal Refinery,
a nonferrous smelter in Antwerp, Belgium. The snails were sampled along with nettle (Urtica dioica), one
of their food sources. Cepaea nemoralis were placed in microcosms at each site and allowed to feed on
soil, litter, and vegetation for 16 weeks. A subset of snails was collected at weeks 0, 1,2, 4, 8, and 16 for
metal analysis (Pb, arsenic [As], Cd, Cu, Zn, nickel [Ni]) and morphological and physiological biomarker
response (Section 11.2.4.4). Nettle (U. dioica) samples were collected three times throughout the
experiment for trace metal analysis. Pb concentration in the soil was the only significant factor explaining
Pb concentration in U. diocia. Pb concentrations in the digestive glands of the C. nemoralis varied
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spatially and temporally, as there was a statistically significant interaction between site and time. Pb
concentration in the soil was higher than that in U. diocia, while the concentrations of Pb in the digestive
glands of C. nemoralis were similar to or higher than Pb concentrations in U. diocia.
Detoxification may be an important mechanism behind biodilution of Pb with trophic level in the
food web. Silkworms (Bombyx mori) were shown to excrete Pb when fed Pb-exposed mulberry (Morus
alba) (Zhou et al.. 2015). Soils collected from an agricultural field in China were exposed to nominal
concentrations of 0, 200, 400, or 800 mg Pb/kg via Pb (NO,):. Morus alba was planted in the Pb-spiked
soils for 3 months, and the leaves were collected and fed to fifth instar larvae of B. mori. The available
fraction of Pb in the soils, the total concentration in mulberry leaves, shoots and roots, and B. mori larvae,
silk, feces, and adult moth increased with increasing soil Pb addition in a dose-dependent manner. Roots
sequestered the most Pb, followed by stems, and leaves. The translocation factor was highest for the
transfer of Pb from the soil to the root in the 400 mg Pb/kg treatment, followed by 1.60 in the
800 mg Pb/kg treatment, and 1.13 in the 200 mg Pb/kg treatment. All other translocation factors between
the soil and plant (root-soil, stem-soil, leaves-stem, stem-root, leaf-root, leaves-stem) were below 1.0 or
near 1.0 for the control (0 mg Pb/kg). Across all treatments, the subcellular distribution of Pb in the leaves
was greatest in the cell wall, followed by the soluble fraction, and organelles. Pb treatment did not affect
silkworm survival or mean weight, but, increasing Pb treatment negatively affected the silkworm growth
rate. Specifically, the body weight of silkworms was significantly lower at the end of the experiment in
the 800 mg Pb/kg treatment compared to the control and the 200 mg Pb/kg treatment. Pb concentration in
the silkworm increased with increasing treatment. Pb concentration in the feces was the greatest, followed
by the concentration in the peel, the larvae, the silk moths and finally the silk. Metallothionein synthesis
increased in B. mori when fed with Pb-treated leaves. Metallothionein content in the midgut was more
sensitive to lower Pb exposure (200 mg Pb/kg) than metallothionein in the posterior of the silk gland and
in the fat body, both of which increased in the high Pb exposures (400 and 800 mg Pb/kg). These results
suggest that B. mori can detoxify Pb through excretion and homeostasis.
Field studies published since the 2013 Pb ISA (U.S. EPA. 2013) provide additional evidence for
biodilution in terrestrial food webs. Oil rapeseed (Brassica napus) and insects were collected from 35
agricultural sites in Southwest Poland (Orlowski et al.. 2019). These agricultural sites varied in size,
habitat fragmentation, and percent cover by forests and were characterized by percent arable land,
permanent vegetation, linear woody features, dirt or unpaved roads, and wooded areas. Brassica napus
and the insect community (grouped into guilds: pollinators, consumer/herbivores, saprovores, predators,
and parasitoids) were analyzed for Pb and other trace elements. The concentration of Pb in Brassica
napus (mean: approximately 2 mg Pb/kg) was higher than those in all insects examined (range: 0.77 to
2.31 mg Pb/ kg), and Pb concentration generally decreased with increasing trophic level, suggesting Pb is
diluted in this food web. As the size of the field area increased, the Pb concentration in pollinators
decreased, suggesting that even under low Pb levels, larger areas with more diversified landscapes could
reduce Pb body burden for pollinators.
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The presence of other heavy metals in the soil, specifically Cd in the soil, can affect the uptake
and trophic transfer of Pb. In an agricultural system in Pakistan (Aslam et al.. 2015). alfalfa (Medicago
sativa) seeds were grown in control, Pb (0 mg Pb/kg, 200 mg Pb/kg or 400 mg Pb/kg), Cd (0 mg Cd/kg,
4 mg Cd/kg or 8 mg Cd/kg) or Pb and Cd-enriched soil (200 mg/kb Pb + 4 mg/kg Cd and
400 mg/kb Pb + 8 mg/kb Cd). Soils were treated with Pb(NO;,)2 and Cd(NC>3)2 salts, resulting in
1.45 ± 0.23 mg/kb Pb (mean ± S.E.) for control, 112.0 ± 2.43 mg Pb/kg for 200 mg Pb/kg and
237.4 ± 2.79 for 400 mg Pb/kg at the end of the experiment for Pb-treated soils. Rabbits (Oryctolagus
cunicuius) were placed in chambers and fed with metal-treatedM. sativa for 10 days. Soil, M. sativa root
and shoot, and O. cuniculus blood and fecal Pb and Cd concentrations increased with increasing
concentrations of metal treatment. Medicago sativa BAF in the roots increased with increasing Pb
concentration and in combined treatments with Cd relative to Pb exposure alone. Specifically, the Pb
BAF associated with the 200 mg Pb/kg treatment was 0.87, while the BAF resulting from the
400 mg Pb/kg + 8 mg Cd/kg treatment was 0.96. Conversely, Pb contents in the shoots and leaves ofM
sativa showed higher BAF in the Pb treatments relative to the combined Pb + Cd treatments. Only a small
portion of Pb was transferred to the shoots, as all BAFs were below a threshold of 1.0. Although not
explicitly tested, O. cuniculus blood and feces Pb levels were similar between Pb only and Pb + Cd
treatments (e.g., fecal concentration in 200 mg Pb/kg treatment: 3.86 ± 0.73 mg Pb/kg [mean ± S.E.], 200
mg/kb Pb + 4 mg/kg Cd treatment: 2.89 ± 0.67 mg Pb/kg), suggesting Pb uptake and accumulation is not
influenced by the presence of Cd in O. cuniculus. Combined, this study suggests that although Pb
bioaccumulation is higher in M. sativa roots and lower in the shoots in the combined Pb + Cd treatment
relative to Pb exposure alone, it does not affect the uptake of Pb by herbivores such as O. cuniculus.
Although many observational studies examining BAFs across multiple trophic levels have found
evidence for biodilution of Pb, some studies have observed bioaccumulation. For example, soil samples
(0-15 cm), berseem plants (Trifolium alexandrinum), aphids (Sitobion avenae), grasshopper (Aiolopus
thalassinus) and ladybird beetle larvae (Coccinella septempunctata) were collected from five agricultural
sites in Punjab, Pakistan and analyzed for accumulation of Pb, Cd and Zn. In this study, Pb was not
significantly correlated with any other soil physicochemical variables or metals (percent sand, percent silt,
percent clay, soil OM, CEC, Zn, Cd, or pH). Pb concentrations in the soil were low and similar among all
sites (3.08 ± 0.53 mg Pb/kg, mean ± S.D.). BAFs were greater than 1.0 for Trifolium alexandrinum (BAF
for soil - berseem: 2.26 ± 0.42), Sitobion avenae (BAF for berseem - aphids: 1.40 ± 0.41), Aiolopus
thalassinus (BAF for berseem - grasshoppers: 14.64 ± 3.42). and Coccinella septempunctata (BAF for
aphid - beetle: 2.94 ± 1.31). Overall, this system does exhibit bioaccumulation of Pb, but the
concentrations of Pb in soil were very low. There was no significant correlation between Pb soil
concentration and T. alexandrinum Pb concentration, between S. avenae and T. alexandrinum, between T.
alexandrinum and A. thalassinus and between C. septempunctata and S. avenae.
In summary, Pb generally shows patterns of biodilution through terrestrial food webs; however,
some observational studies have shown bioaccumulation of Pb. Furthermore, the rate at which Pb
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biodilutes or accumulates in food webs depends on the presence of cadmium, the sensitivity of the
organism to Pb exposure and ecosystem type.
11.2.3. Environmental Concentrations of Pb in Terrestrial Biota and
Ecosystems in the United States at Different Locations and Over Time
Studies that present long-term trends of Pb concentrations observed in terrestrial ecosystems are
summarized in this section. National and regional studies that summarize Pb concentrations in soils and
biota on decadal timescales are included.
11.2.3.1. Pb in Soils
Pb concentrations in soils vary across the United States due to a variety of anthropogenic and
natural factors. In general, areas with higher population density and intensity of industrial activity have
higher soil Pb concentrations relative to rural areas. This pattern was observed in the following studies of
national and regional soil Pb concentrations.
A regional survey of forest floor soils sampled in the northeastern United States provides a time
series of Pb concentrations from 1980 to 2011. The region has a large amount of urban and industrial
development associated with high historical anthropogenic Pb emissions. Soils were sampled at 25 sites
in 1980 and sampled again at 16 of those sites in 1990, 2002 and 2011. Sites were located across
northeastern states including Pennsylvania, New York, Connecticut, Massachusetts, Vermont, and New
Hampshire. Across all sites, mean soil Pb concentrations decreased from 151 ± 29 (SE) mg Pb/kg in 1980
to 68 ± 13 (SE) mg Pb/kg in 2011 (Richardson et al.. 2014b) (summarized in Table 11-1). The authors
explained the observed reduction in forest floor Pb concentrations by the dilution effect of added organic
material containing less Pb than in older forest floor organic soil as well as by the leaching of Pb from
upper soil horizons into the underlying soil. Isotopic analysis of Pb samples indicated that gasoline was
the dominant source of the measured soil Pb and that it persisted in forest floor soils until at least 2011,
and likely later. In another analysis of the data set of 1980-2011 northeastern U.S. forest floor soils, Pb
concentrations were estimated to decline 2.0 ± 0.3% per year (Richardson et al.. 2014a).
A 2019 survey of peri-urban soil Pb from 54 sampling sites in southern California counties
including Los Angeles, Orange, San Bernardino, and Riverside found that soil Pb was elevated relative to
the southwestern U.S. region, but lower than concentrations found at contaminated sites near point
sources of Pb, with a mean of 23.9 ± 13.8 mg Pb/kg (Mackowiak et al.. 2021) (summarized in Table
11-1). The mean is considerably lower than the forest floor mean observed in the Richardson et al.
(2014b) surveys and the results of this study are illustrative of the regional variance in U.S. soil Pb
concentrations. Foliage samples from eight shrub and tree species collocated with soil samples were
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collected from the sampling sites of Mackowiak et al. (2021). No correlation was identified between
foliar bioaccumulation and soil Pb concentrations in the study.
Measuring the ratio of Pb concentrations between different soil horizons can provide information
on the relative contribution of anthropogenic Pb to total Pb observed in the soil. In the recent NASGLP
soil survey of the conterminous United States Smith etal. (2013a) (summarized in Section 11.1.3 and
Table 11-1), samples were collected from multiple soil horizons. Stratified sampling enabled the
comparison of Pb concentrations from bedrock to those in upper-horizon soil. In areas with historic
depositional input of Pb, the concentration of Pb observed in upper-horizon soils was often higher than
that in the bedrock. Figure 11-4 C. shows the ratio of A-horizon to C-horizon Pb concentrations mapped
in Woodruff et al. (2015). using inverse-distance weighting methods derived from the NASGLP survey
(Smith et al.. 2013a). This map displays areas with increased concentrations of Pb in A-horizon soils
relative to lower horizons, hinting at the lasting effect of depositional Pb pollution. The mapped ratio of
A-horizon to C-horizon soils from Woodruff et al. (2015) may serve as an indicator for soil in areas
where historical Pb deposition may have a relatively higher effect on people and ecosystems. Patterns of
elevated A- to C-horizon soil Pb concentrations in Figure 11-4 C. are conspicuous in areas with historical
anthropogenic sources of Pb. This pattern is observed in the northeastern United States, with a historically
high population density and intensity of industrial development. Likewise, mapping highlights former Pb
smelting and mining sites, for instance in areas near smelters in Everett and Tacoma, Washington or the
Doe Run smelter in Herculaneum, Missouri (the last Pb smelter in the United States, which closed in
2013). Areas near mining sites, including near Leadville, Colorado, Cooke City, Montana, and northern
Utah, also have a high ratio of A- to C-horizon Pb. Woodruff et al. (2015) emphasized that no known
natural geological process would otherwise explain elevated A-horizon soils relative to the underlying
layers.
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D. Population density (per sq. mile) by county
Urban Areas
C. Lead {Pb) - Ratio of A Horizon/C Horizon
Source: Woodruff et at. (2015)
Figure 11-4 Maps of Pb sampled from A-horizon (A.) and C-horizon (B.) soils,
the ratio of Pb observed in A-horizon to C-horizon soils (C.) and a
map of U.S. population density (D.).
2 Recent national and regional surveys of soil Pb document the spatial and temporal patterns of
3 residual pollution from decades of Pb emissions. Data made available from the NASGLP provide the
4 most comprehensive information on the distribution of Pb across the conterminous United States (Smith
5 et al.. 2013a). Regional studies of soil Pb provide valuable information on temporal trends and relate
6 observed soil Pb concentrations to Pb in biota collocated with soil sampling locations. Elevated upper soil
7 horizon Pb concentrations relative to the underlying soil with greater substratum content observed across
8 the conterminous U.S. in (Woodruff et al.. 2015) and over four decades in the northeast in (Richardson et
9 al.. 2014b) demonstrate the persistence of historical Pb contamination in U.S. soils.
B. Lead (Pb) - C Horizon
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11.2.3.2. Pb in Tree Rings
Dendrochronology can be used to reconstruct historical trends of Pb in air pollution as tree rings
record an annual record of ambient environmental conditions across a tree's lifespan, although radial
transport of Pb within the tree may reduce the precision of historical Pb concentrations reconstructed from
tree rings. Because trees primarily uptake Pb through their roots, there may be a 10-15-year delay in tree-
ring Pb compared with air Pb concentrations as Pb deposition leaches through the soil and is absorbed by
the tree (U.S. EPA. 20131
Several studies conducted after the 2013 Pb ISA report temporal trends in Pb as observed in tree
rings, three from Canada and one from Mexico. A study of white spruce trees (Picea glauca) located in
the Northern Athabasca Oil Sands Region of western Canada near oil sands mining operations
reconstructed Pb concentrations from 1878 to 2009. Tree-ring records of Pb concentrations increased
beginning in 1922, peaked in 1968-1973, then decreased until 2009 (Dinis et al.. 2016). In eastern
Canada, a study reconstructed Pb trends from 1880 to 2007 in red spruce (Picea rubens), beech (Fagus
grandifolia), white pine (Pinus strobus), and white cedar (Thuja occidentalis). The beech trees located in
both Montreal and Georgian Bay exhibited a decline in concentrations after a 1970-1985 peak. The
authors attribute the lack of an observed temporal trend in Pb concentrations in white pine to the radial
mobility of Pb within the tree (Doucct et al.. 2012). Another study of tree-ring Pb concentrations in white
cedar in Quebec dated concentrations from 1850-2010 and recorded increased concentrations from 1950—
2000 near a Pb smelter. The increasing trend at a control site further from the smelter was delayed to
1990-2010. Concentrations across most sites in this study decreased from 2000-2010 (Arteau et al..
2020). In contrast to the trends observed in the Canadian studies, a study of Prosopis julifora tree rings
dated from 1903 to 2007 located near a copper smelter in San Luis Potosi, Mexico found increasing Pb
concentrations from 1990-2007 (Beramendi-Orosco et al.. 2013).
Although trends in reconstructed Pb concentrations varied across tree species and regions, studies
identified atemporal pattern of Pb that increased after 1850-1900 and, in some cases, peaked in 1970-
1985, then decreased afterward. Tree-ring studies with temporal patterns in exception to this pattern were
conducted near persisting industrial point sources of Pb pollution.
11.2.4. Effects of Pb in Terrestrial Systems
This section focuses on studies of the biological effects of Pb on terrestrial biota published since
the 2013 Pb ISA. First, new information on factors that affect biological sensitivity to Pb is discussed,
followed by subsections on effects on vegetation, microbes, invertebrates, and vertebrates. The biological
effects of Pb in the 2013 Pb ISA and in this appendix are generally presented in increasing order of the
complexity of biological organization, from suborganismal responses (i.e., enzyme activities, changes in
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blood variables) to endpoints relevant to the population level and higher (growth, reproduction, and
survival), up to effects on ecological communities and ecosystems.
11.2.4.1. Effects on Terrestrial Microbes
Several field and laboratory studies have examined the relationship between soil Pb concentration
and microbial community structure and processes. Cell viability of bacteria grown in Pb-contaminated
media was unaffected, and bacteria were able to take up Pb in studies reported in the 1977 AQCD (U.S.
EPA. 1977). Furthermore, in other studies reported in the 1977 AQCD (U.S. EPA. 1977). 1986 AQCD
(U.S. EPA. 1986). and the 2013 Pb ISA (U.S. EPA. 2013). soil Pb concentration was correlated with
decreases in the diversity and function of soil microorganisms. New studies since the 2013 Pb ISA added
a gradient of Pb to the soil and showed negative relationships between Pb concentrations and bacterial
abundance. Most new studies since the 2013 Pb ISA were observational and leveraged natural
environmental gradients of pollutants. In these cases, Pb was not the sole contaminant in the soil,
contributing some uncertainty to their interpretation. Observational field studies showed mixed
associations between soil Pb concentration and microbial abundance and diversity metrics. Additionally,
there has been substantial research on how Pb affects the interactions between microbes and their hosts,
specifically, plants and mycorrhizal associations (Section 11.2.4.2).
Pb contamination slightly affected microbial diversity and significantly affected the abundance of
certain bacteria phyla and genera in an agricultural system (An et al.. 2018). Soil in an agricultural field in
China was supplemented with nominal concentrations of 0, 175, or 350 mg Pb/kg using Pb(NO?h and
permitted to age for 3 months while maintaining soil moisture. After 3 months, soil physicochemical
variables and bacterial community structure were analyzed. Available Pb and total Pb concentration in the
soil varied with Pb treatment level (available Pb in the control (mean ± S.D.): 3.97 ± 0.08 mg Pb/kg.
150 mg Pb/kg treatment: 126.6 ± 4.98 and 350 mg Pb/kg treatment: 254.46 ± 7.13). Total and available
Pb concentrations were highly correlated. Some soil physicochemical variables differed between the
control and Pb-spiked soils; soil OM was lower in Pb-spiked soils compared with the control, while soil
moisture was the lowest in the 150 mg Pb/kg treatment. Soil pH. available P. available K, and available N
were similar among all treatments. Pb exposure marginally affected microbial Operational Taxonomic
Unit (OTU) richness and diversity, as well as the abundance-based coverage estimator (ACE), Chao and
Shannon's diversity indices were highest in the 175 mg Pb/kg treatment compared with the control and
the 350 mg Pb/kg treatment (statistics not reported, error bars do overlap). The abundances of certain
genera were affected by Pb treatment; Bacillus, Lactobacillus, and Truepera abundances were negatively
correlated with Pb concentration, while Streptococcus and Arhtorbacter were highest under the low Pb
treatment. Bosea and Aquicella increased in abundance with Pb treatment. Total Pb concentration was
correlated with the abundance of Planctomycetes and Gemmatimonadetes and marginally correlated with
Nitrospirae.
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Microbial enzyme activity was significantly negatively affected in soils collected from a research
station in northwestern Iran, exposed to nominal concentrations of 0, 100, 200, 300, 400, or 500 mg Pb/kg
using aqueous Pb nitrate and incubated for 2 weeks (Shirzadeh et al.. 2022). After 3, 15, 30, 90 and
180 days, microbial enzyme activities and microbial indices, including acid and alkaline
phosphomonoesterase, nitrate reductase, urease, soil microbial biomass carbon, soil basal respiration were
characterized. Nominal Pb concentration, incubation time and the interaction between Pb concentration
and incubation time significantly affected all enzyme activities and microbial indices. In general, higher
concentrations of Pb and longer incubation times resulted in a commensurate reduction in enzyme
activities and microbial indices.
The root nodule allocation by the actinobacteria Frankia on Alder (Alnus glutonisa) was
unaffected by Pb treatment, while Frankia microbial respiration was significantly affected by Pb
treatment (Belanger et al.. 2015). The authors suggested that large difference between the maximum
tolerable concentration (MTC), the highest metal concentration when Frankia has 95% of its relative
respiration capacity (<0.01 mM) and the minimum inhibitory concentration (MIC), when under 5% of
relative respiration capacity occurs (10.0 mM), may be due to sequestration or binding of Pb by Frankia,
which has been shown to occur with other heavy metals.
Bacteria and archaeal abundance and diversity have been found to be affected by soil Pb
concentration in several observational studies. Beattie et al. (2018) examined the relationship between Pb
and other soil heavy metals as well as bacterial and archaeal communities in Oklahoma. Picher, an
abandoned mining town, is located near the Picher mine field (PMF), which was declared a U.S. EPA
Superfund Site in 1983 (Tar Creek Superfund Site). Soil samples were analyzed for trace metals and soil
physicochemical properties (Pb, Al, Ar, B, Cd, Cr, cobalt [Co], Cu, Fe, Mg, Mn, molybdenum [Mo], Ni,
K, sodium [Na], tellurium [Te], titanium [Ti], tungsten [W], vanadium [V], Zn, soil pH and soil moisture)
and soil bacterial and archaeal abundance and diversity using 16S rRNA gene copies. Pb soil
concentration was 76.39 ± 1.37 mg Pb/kg (mean ± S. E.) and ranged from 3.0 mg/kb Pb to 1115.2 mg/kg
(Beattie et al.. 2017). Bacterial abundance (16S rRNA gene copies) was found to be negatively correlated
with soil Pb concentration, while archaeal abundance and the bacteria: archaea ratio were not. In addition
to soil Pb concentration, bacterial copy numbers were significantly correlated with Cd, Zn and Mg. Out of
four metals tested (Pb, Al, Cd and Zn), Pb was the only metal to significantly affect microbial diversity.
Shannon-Wiener diversity and Simpson's evenness indices were negatively correlated with Pb
concentration, while the Simpson diversity index was positively correlated, and the Shannon evenness
index was not correlated with Pb concentration. The authors suggested that these conflicting results might
be due to how the indices were calculated or the presence of an outlier. Given that the other metals
analyzed (Al, Cd, Zn) were not correlated with microbial diversity, the authors suggested that the
microbial community had already reached a stable equilibrium with long-term heavy-metal exposure.
Using CCA to determine the relationship of Pb, Cd, Zn and Al with OTU abundance, 1150 OTUs were
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found to be significantly correlated with Pb. A total of 2,591 OTUs out of 27,082 were significantly
correlated with one of the four metals (Al, Cd, Pb or Zn), and 60% of these OTUs correlated with two or
more metals while 28% correlated with all four metals. Finally, distance-based linear modeling and
redundancy analysis were used to determine which environmental factors best explained variation in the
soil microbial community. Soil Pb explained 6.96% of the variance in community structure, with only Al
and Zn explaining more (Al = 7.99%, Zn = 7.64%).
Long-term exposure to Pb and other heavy metals influence microbial community structure, as
heavy-metal-tolerant fungi have been isolated in forested areas in the United States (Torres-Cruz ct al..
2018). Fungi were isolated from soil collected from N-fertilized and unfertilized plots in Duke Forest,
North Carolina. Fungi tolerant to Pb were isolated from the rest of the fungal community by adding
diluted soil to malt extract agar supplemented with antibiotics and Pb stock solutions (100 or 500 ppm
Pb(NC>3)2). Fungal isolates were identified using OTUs and used in phylogenetic analyses and next
generation sequencing was conducted to determine the abundance of heavy-metal-tolerant taxa. The
number of isolated OTUs tolerant to Pb were higher compared with the number of isolates tolerant to
other heavy-metal stock solutions analyzed in this study, including Al, Cr, Fe, Ni, Cu, Cd and Zn, and the
largest number of isolates were obtained from Pb (30% of all isolates) followed by Zn (14% of isolates).
The genera Trichoderma, Penicillium, Umbelopsis, Pochonia, and Saitozyma, all have isolates tolerant to
Pb stress. The most common taxa, Trichoderma and Penicillium, were detected in all metal-enriched
samples, and the authors hypothesized this gives them a competitive advantage across a wide range of
polluted conditions
Other field studies have found mixed relationships between soil Pb concentration and bacterial
abundance and community structure. For example, Vetrovskv and Baldrian (2015) examined the
relationship between bacteria and actinobacterial biomass and diversity and soil heavy-metal content (Pb,
Cd, Cu, and Zn) across sites ranging in distance from a polymetallic smelter in Pribram, Czech Republic.
Pb soil concentrations ranged from 160.5 ± 3.9 mg Pb/kg (mean ± S.E.) to 1713.5 ± 123.4 mg Pb/kg at
the most contaminated site. Pb concentration in the soil was significantly correlated with Cd, Cu and Zn,
but not oxidizable C, total N content, C/N, and pH. Bacterial biomass, actinobacteria biomass and the
ratio of actinobacteria:bacteria were not significantly correlated with Pb concentration. Finally, the
Shannon-Wiener diversity index increased with increasing heavy-metal contamination.
Although abundance and diversity indices are commonly reported in observational studies
examining the relationship between Pb, other soil metals and microbial communities, some studies have
reported additional effects including average cell wall color development (AWCD) or average carbon
source utilization, microbial growth rate and enzyme activities. These effects can act as surrogates for
microbial activity and diversity. Specifically, Boshoff et al. (2014) used BIOLOG® EcoPlates™ to assess
microbial capacity to metabolize a variety of carbon substrates in two grassland sites that varied in their
distance from an active metal refinery in Antwerp, Belgium. Average carbon utilization AWCD, the
number and variety of utilized substrates (functional richness (R') and the functional diversity (H')) were
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analyzed. Unlike pH, OC, particle size distribution, Cd, Ni and Zn concentration in the soil, Pb
concentration differed significantly between the soils of the two sites, ranging from 147.10 mg Pb/kg to
1373 mg Pb/kg across all subplots. Additionally, soil moisture, temperature, As and Cu differed between
the two grassland sites. Overall, pseudototal Pb and Cu concentration, which was measured by adding
hydrochloric acid and nitric acid to the samples (as well as As and Cu) was negatively correlated with
AWCD, R' and H'; however, when an analysis of covariance was performed to understand the effect of
metal pollution on microbial responses, Pb was not a significant factor driving variation for AWCD, R' or
H', while sampling site and As concentration were significant predictors.
In many observational field studies, total Pb soil concentration is often used when analyzing soil
microbial communities; however, some studies attempt to determine bioavailable Pb in addition to total
soil Pb. The relationship between total and bioavailable concentrations of heavy metals (Pb, Zn, Cu, Cd),
soil physicochemical properties (pH, total N, available P, available K and OM) and soil microbial
communities was explored from soil collected near an abandoned ore-dressing plant in Hezhang County,
China (Wang et al.. 201 Sa). Total soil Pb concentrations ranged from 67.4 ±1.6 mg Pb/kg (mean ± S.D.,
n = 3) to 759.3 ± 11.4 mg Pb/kg, while bioavailable Pb, measured as 0.1 M HCl-extractable Pb (HCl-Pb)
ranged from 33.0 ±1.9 mg Pb/kg to 681.0 ± 33.9 mg/kb Pb. In this study, neither total soil Pb nor HCl-Pb
was correlated with microbial enzyme including fluorescein diacetate hydrolysis activity (FDA), an
indicator of soil microbial activity and urease activity. Additionally, Pb was not significantly correlated
with any microcalorimetric parameters examined; however, when bioavailable Pb (HCl-Pb) was used
instead of total Pb, the direction of these trends changed. Pb and HCl-Pb showed mixed relationships with
bacterial abundances. For example, Thiobacillus, Anaerolineaceae, and Xanthobacteraceae abundances
were significantly positively correlated with HCl-Pb, HCl-Cu, and Cu, while uncultured Acidmicrobiales
showed significant negative correlation with Pb and HCl-Zn.
Previous exposure to pollution in soil may affect the sensitivity of microbial communities in the
rhizosphere to Pb stress (Zhang et al.. 2019b). Ferns (Athyrium wardii) were collected from either a site
exposed to mining (mining ecotype or ME) or a reference site (nonmining ecotype [NME]) in Sichuan
Province, China. Collected A wardii were then grown in uncontaminated soil for several generations and
subsequently exposed to one of five experimental Pb levels: 0, 200, 400, 600 or 800 mg Pb/kg (aqueous
Pb(NC>3)2). After 50 days, soil Pb concentration, soil respiration, microbial biomass carbon (MBC),
aboveground and belowground biomass, soil physicochemical characteristics (total and available N and P,
pH, and OM), and heavy metals were analyzed. Total and available Pb in the rhizosphere increased
significantly with experimental Pb exposure, while OM, TN, available N, available P, available K, and
pH were similar across all Pb treatments. Total Pb was 9.74 ± 0.11, 210.27 ± 0.41, 412.24 ± 0.60,
607.17 ± 0.65 and 811.74 ± 0.44 mg Pb/kg (mean ± S.D.), and available Pb was 2.15 ± 0.24,
72.23 ± 0.28, 166.30 ± 0.38, 242.94 ± 0.19 and 382.17 ± 0.60 mg Pb/kg, respectively. The rhizosphere of
A. wardii ME had significantly higher concentrations (12-4.8 times) of Pb compared with that of the
NME. Microbial activity, characterized through soil respiration and MBC, was reduced under increasing
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Pb concentration for both ecotypes; however, the microbial community in the rhizosphere of NME
experienced a greater reduction in MBC when exposed to high Pb treatments (400-800 mg Pb/kg) than
ME plants (NME 28.4-68.2% versus ME: 21.2-60.9% less MBC than control). Additionally, the MBC of
soils in the rhizosphere of the NME was significantly lower than that of ME for A. wardii exposed to Pb.
Finally, the soil metabolic quotient or soil qCC>2 increased with increasing Pb exposure; however, plant
ecotype did not affect soil qCC>2. The authors suggested that in general, the microbial community in the
rhizosphere of the ME was more adapted to Pb stress than the community in the rhizosphere of the NME,
as soil respiration and MBC are less affected by Pb exposure.
Since the 2013 Pb ISA (U.S. EPA. 2013). additional observational studies, many of which were
natural environmental gradient studies, have linked microbial community structure (e.g., abundance,
diversity) and function (e.g., enzyme activities, respiration rates). Many studies found mixed (negative,
positive, and null) relationships between total or bioavailable Pb soil concentration and the abundance of
bacterial and fungal taxa (Zappelini et al.. 2015). diversity (Aleksova et al.. 2020; Kerfahi et al.. 2020;
Golebiewski et al.. 2014; Tipavno et al.. 2012). microbial C and N (Zeng et al.. 2020). and respiration and
nitrification (Smolders et al.. 2015). Unfortunately, it is difficult to disentangle the effects of Pb exposure
on microbial communities from the effects of other soil contaminants using environmental gradients, as
other heavy metals and soil physicochemical proprieties are significantly correlated with soil Pb
concentration, and many of these factors also influence microbial processes.
11.2.4.2. Effects on Terrestrial Plants and Lichen
In terrestrial plants, Pb is known to induce oxidative stress and impair plant growth, root
elongation, seed germination, transpiration, chlorophyll production, lamellar organization in the
chloroplast, and cell division. However, the extent of these effects varies and depends on the Pb
concentration tested, the duration of exposure, the intensity of plant stress and co-stressors, the stage of
plant development, and the particular organs studied. Plants have developed various mitigations when
exposed to toxic metal exposures including selective metal uptake, excretion, complexation by specific
ligands, and compartmentalization. At sufficiently high Pb exposure, the plant's antioxidant capacity is
exceeded, and peroxidation of lipids and DNA damage follows, eventually leading to accelerated
senescence and potentially, death. In the 2013 Pb ISA, the body of evidence was sufficient to conclude
there are causal relationships between Pb exposure and both plant physiological stress and reduced plant
growth, and inadequate to infer causal relationships between Pb exposure and both plant survival and
plant reproduction (U.S. EPA. 2013).
Previous AQCDs recognized declines in photosynthesis and damage to mitosis as effects of Pb
toxicity in plants (U.S. EPA. 2006a. 1986. 1977). The 2013 Pb ISA added additional experimental studies
showing photosynthesis impairment in plants exposed to Pb, and studies of damage to photosystem II due
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to alteration of chlorophyll structure, as well as decreases in chlorophyll content in plants, lichens, and
mosses. Recent studies have continued to demonstrate decreases in photosynthetic performance due to Pb
exposure (Alkhatib et al.. 2019; Silva et al.. 2017a; Rodriguez et al.. 2015) as well as documented damage
to chlorophyll structure caused by Pb (Tokarz et al.. 2020; Alkhatib et al.. 2019; Rodriguez et al.. 2015).
A substantial amount of evidence of oxidative stress in response to Pb exposure has also been produced
and documented in the 2013 Pb ISA and previous AQCDs. Monocot, dicot, and bryophytic taxa grown in
Pb-contaminated soil or in experimentally spiked soil all responded to increasing exposure with increased
antioxidant activity. Recent studies continue to confirm increased antioxidant activity in plants in
response to Pb stress (Kaur et al.. 2015; Reis et al.. 2015; Rossato et al.. 2012). as well as the genotoxic
effects of Pb exposure (Silva etal.. 2017b). albeit at concentrations that greatly exceed Pb measured in
soils (Table 11-1). Finally, studies of the effects of Pb on terrestrial plants published since the last ISA
continue to support the previous known findings of declines in plant growth under controlled exposures of
Pb (Muradoglu et al.. 2016; Kaur et al.. 2012; Rossato et al.. 2012).
Although Pb exposure is associated with various responses in plant and lichen species, most
effects seen in terrestrial plants occur at exposures that are generally at higher environmental
concentrations than those outside of the boundaries set for consideration in this ISA (Section 11.1.1).
Additionally, while studies find that exposure to Pb has effects on terrestrial plants that could, depending
on a number of factors, then contribute to community- or ecosystem-scale effects, the exposure methods
typically used make it difficult to compare these effects to what might occur under the uncontrolled
conditions encountered in natural environments. Overall, these experiments demonstrate the effects of Pb
exposure in terrestrial plants and the underlying physiological and biochemical mechanisms, but strong
uncertainties remain regarding the natural concentrations at which these effects would be observed.
One novel area of research is the existence of sex-dependent differences in the effects of Pb in
poplar (Populus spp.) trees. In a study of sexual differences in Populus cathayana exposed to Pb in soil or
applied to the leaves, singly and in combination with drought conditions, Han et al. (2013) found
significantly different effects between male and female trees. Male trees exhibited a greater ability to
bioconcentrate Pb in the root systems, a higher heavy-metal tolerance and photosynthesis plasticity, and
less-damaged cell ultrastructure. When Pb was applied to the leaf alone and in both combined treatments,
there were significant effects on dry mass production, photosynthetic activity, long-term water use
efficiency, potential quantum yield of photosystem II and cellular ultrastructure, and greater effects were
observed in females than in males. Drought worsened Pb stress in both sexes, however the effects were
larger on female trees. A second study examined sex-dependent responses to Pb stress in the congeneric
Populus deltoides (Xu et al.. 2016b). Pb-induced negative effects on P. deltoides root growth were sex-
related and branch order-specific. Compared with plants in control conditions, Pb decreased total root
length, total surface area, root diameter and biomass, and the effects were significantly greater in female
trees than in males. This agrees with the findings of Han et al. (2013) that female poplar trees exhibit
greater Pb sensitivity. Xu et al. (2016b) found that males of P. deltoides could sequester Pb in the roots of
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lower orders and suppress transportation of Pb to high-order roots, which may partially explain the greater
Pb tolerance in males when evaluating tree physiological variables.
Recent studies have also examined the protective effects of certain plant nutrients as well as the
influence of mycorrhizal inoculation on the effects of Pb in terrestrial plants. In a hydroponic experiment
with two different ecotypes of Elshotzia argyi (one from an agricultural site and one from an abandoned
Pb mine in China), plants were exposed to 50 (j,M Pb with normal Zn levels (0.5 (j,M) and high Zn
(20 (j,M) for 12 days (Islam et al.. 2011). Application of Pb with normal Zn had negative effects on the
overall growth and antioxidant capacity of both ecotypes; however, the effects were more pronounced in
agriculturally sourced plants. The addition of high Zn improved the growth and antioxidant capacity of
both ecotypes under Pb stress. Finally, a study using Pb exposures on Torreya grandis seedlings (0, 700
and 1400 mg Pb2+/kg) examined the possible protective effects of the addition of 1040 mg/kg Mg2 (Shen
et al.. 2016). The addition of Mg2+ improved the growth of the Pb-stressed seedlings, increased
chlorophyll content, enhanced chloroplast development and improved both the photosynthetic rate and
maximum quantum efficiency in Pb-stressed plants. In addition, Mg2+ addition increased root growth and
oxidative activity and protected the root ultrastructure. These studies showed that some mineral nutrients,
when added beyond the minimal plant requirements, can increase plant tolerance of Pb stress. This is
particularly true of Mg addition. Ling and Hong (2009) hypothesized that Pb2+ may replace either Mg2+ or
Ca2+ in chlorophyll or the oxygen-evolving center, inhibiting photosystem II function through an
alteration of chlorophyll structure.
Mycorrhizal inoculation also appears to protect terrestrial plants from the effects of Pb. One study
examined the effects of AMF (Funneliformis mosseae) on the growth and Pb uptake of Sophora viciifolia
(Xu et al.. 2016a). As expected, the AMF altered root growth and architecture (increasing root length,
fork number, tip number, surface area and volume), and these effects are also present under high Pb stress
(1000 jxg/g). Examining roots under transmission electron microscope and X-ray spectroscopy revealed
that Pb was deposited not only in plant cells but also the cell walls and vacuoles of the AMF intracellular
hyphae, meaning that AMF uptake some of the Pb, alleviating the effects on the plant. Whether the
protective effect of mycorrhizae is species-dependent or not is unknown.
In summary, recent studies have continued to demonstrate various deleterious physiological
effects of Pb exposure, particularly oxidative stress, though uncertainties remain regarding the
environmental concentrations at which these effects would be observed. Additionally, recent studies have
examined the protective effects of mycorrhizae in some plants and of some plant nutrients when added in
excess of plants' minimal requirement. There is still very little evidence addressing the relationship
between Pb exposure and plant survival and reproduction, especially at exposures to concentrations of
interest for this ISA.
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11.2.4.3. Effects on Terrestrial Invertebrates
For terrestrial invertebrates, exposure to Pb generally increases mortality, decreases growth, and
can have detrimental effects on behavior as summarized in previous EPA reviews of this metal. In studies
from the 2006 AQCD, Pb caused antioxidant effects, reductions in survival and growth, as well as
decreased fecundity in soil invertebrates (U.S. EPA. 2006a'). In the 2013 Pb ISA, there was also evidence
for neurobehavioral aberrations and, in some cases, decreasing fecundity via changes in the endocrine
system (U.S. EPA. 2013). Second-generation effects were reported in some invertebrate species. Recent
literature expands the evidence base for suborganism-level and organism-level endpoints and further
supports effects on physiological endpoints in additional invertebrate groups, as well as multigenerational
effects of Pb exposure. In addition, recent literature provides new information on the effects of Pb on
organisms not included in the 2013 Pb ISA such as honeybees. Similarly, while soil nematodes are
aquatic organisms—living in the water-filled pore spaces between particles and in water films on soil
particles—they are included in the terrestrial section since they are exposed to soil Pb concentrations.
Accordingly, adherence to aquatic concentration cutoffs was not strict when effects were examined in
laboratory conditions.
11.2.4.3.1. Suborganism-Level Response
In the 2013 Pb ISA, the body of evidence was sufficient to conclude there is a likely causal
relationship between Pb exposure and suborganism physiological level responses in terrestrial
invertebrates (U.S. EPA. 2013). Changes in enzyme activities and oxidative stress markers were reported
in terrestrial invertebrates, including earthworms, snails, and nematodes. Additional studies published
since the 2013 Pb ISA, primarily in earthworms and snails, provide additional supporting evidence for
perturbation of biomarkers of physiological stress associated with Pb exposure.
Available studies in earthworms have assessed a suite of physiological responses including
protein and lipid content following Pb exposure. In field-collected earthworms (Aporrectodea caliginosa)
from metal-polluted soils across northern France, protein content in earthworm was negatively correlated
with easily extractable Pb (CaCh extractable), and stepwise model selection further correlated protein
content positively with soil clay content (Beaumelle ct al.. 2014). Lipid content was also negatively
correlated with Pb and was positively correlated with silt content. Glycogen was not related to any metal
or soil parameter measured. Total Pb soil concentration varied from 19.6 to 491 mg Pb/kg. It is important
to note that Pb did not occur alone in these soils and is an example of natural pollution conditions. The
authors suggested that energy responses to Pb may be due to demands for mediating oxidative stress
mechanisms or regulation.
Several studies with the earthworm E.fetida assessed changes in biomarkers of physiological
stress following exposure to Pb. In adult E.fetida exposed via soil (40, 250, 500, 1000, 2500 mg Pb/kg,
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nominal values) for 4-weeks followed by a 4-week recovery period, MDA was higher in Pb-exposed
earthworms during both the exposure and recovery periods (Zaltauskaitc et al.. 2020). MDA was
positively correlated with soil Pb exposure, and while MDA concentrations were lower during the
recovery period compared with the exposure period, the levels were still higher than control levels at the
end of the recovery period (1.2-1.9 times higher). While MDA levels did decrease in Pb-exposed worms
during the recovery period, the lack of complete recovery of MDA levels shows worms are not able to
recover from Pb-induced oxidative stress within 4 weeks and that either a longer recovery period is
needed or MDA response to Pb has a delayed effect. Juvenile E.fetida earthworms exposed to Pb had
higher levels of MDA, which increased by 25-54% as soil Pb increased (40, 250, 500, 1000,
2500 mg Pb/kg, nominal values) (Zaltauskaite and Sodiene. 2014). In another study, E. fetida exposed to
5 mg Pb/kg of Pb had lower protein content than control worms but there was no difference at the 50 and
500 mg Pb/kg exposure levels (nominal values) (Wu et al.. 2012a). Cellulase activity, however, was
higher across all Pb exposure levels compared with control. DNA damage in coelomocytes (phagocytic
leukocytes) was measured by changes in olive tail moments, tail length, tail DNA content, and tail
moment using a comet assay. There was no effect of Pb on olive tail moments or tail length. Tail
moments increased but only in the 50 mg Pb/kg treatment, as did tail DNA contents. The authors
concluded since cellulase activity is involved in the breakdown of cellulose, an increase in cellulase
activity suggests Pb may increase E. fetida's ability to degrade plant matter within the soil profile. Pb
exposure at 50 mg Pb/kg appeared to lead to more DNA damage of coelomocytes but not at
500 mg Pb/kg, indicating more research is needed to elucidate the effect of Pb exposure on the earthworm
immune system via DNA damage.
For snails, after 7 days of exposure to Pb via diet, AChE activity in the digestive gland of the
green garden snail (Cantareus apertus) decreased with increasing Pb exposure (nominal values reported)
(Mleiki et al.. 2015). Activity was 200 (imol/nm/mg in control snails, approximately 75 (.unol/nm/mg at
25 mg Pb/kg exposure and about 25 (.unol/nm/mg at 2500 mg Pb/kg Pb exposure. After 60 days of
exposure, the activity level was lower across all groups but followed the same decreasing pattern with
increasing exposure. AChE activity in the foot also followed a similar pattern to the digestive gland, with
decreasing activity at day 7 with increasing exposure. After 60 days, differences across treatments were
not significant in the foot. Overall, Pb caused a decrease in AChE activity in both the foot and digestive
gland, but the effect was stronger in the short term compared with the long term. In another snail study,
metal concentrations in soil, stinging nettle (Urtica dioica), and the digestive gland of Cepaea nemoralis
snails were assessed in relation to the pollution source (metal smelter in Belgium) with various
physiological biomarkers also measured (Boshoff et al.. 2015). Soil Pb concentrations varied from
approximately 50 mg/kg to 1300 mg/kg and generally decreased with increasing distance from the
pollution source. Pb in leaves followed the same general pattern. European land snails prefer nettle leaves
as a food source, and Pb concentrations in the digestive gland followed the same pattern as those in soil
and leaves each week of the experiment, with the pattern becoming more pronounced over time with far
greater concentrations at the pollution source location (orders of magnitude greater than other sites).
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Metal concentration in plants was positively correlated with soil concentrations, and concentrations in the
snail digestive gland were positively correlated with plant concentrations. Protein, glycogen, GST, and
total energy levels measured within the digestive gland showed no clear pattern in relation to Pb and
instead depended on interactions between the specific site, exposure time, and different heavy metals.
There were also no correlational changes in shell morphology.
Physiological stress response linked to Pb exposure was reported in a few additional terrestrial
invertebrates. Overall, gut enzyme activities, with the exception of alpha-glucosidase, were higher in
honeybees (A. mellifera) within urban-located hives in Nigeria compared with wild beehives.
Carbohydrases (amylase and cellulase) were higher than lipase and proteinase across both nesting
habitats. However, there was no difference in Pb concentration in bees between habitats, and differences
in enzyme activities showed no direct correlation to Pb specifically (Lawal ct al.. 2014). In another study,
honeybees in a laboratory setting were fed a sucrose solution with Pb concentrations of 10, 1, 0.1, and
0 mg Pb/L over a 48-hour period. GST enzyme activity and gene expression were examined, along with
AChE activity. No effect of Pb was observed at any exposure concentration on GST activity or gene
expression after 48 hours. AChE activity was lower at 0.1 mg Pb/L and higher at 10 mg Pb/L
concentrations (Nikolic et al.. 2019). In atrophic study examining Pb uptake by mulberry trees (M alba)
and subsequent transfer to silkworms (B. mori), Pb content in silkworms and silkworm excretions (feces
and silk) increased with increasing Pb treatment (0, 200, 400, and 800 mg Pb/kg soil treatments, nominal
values) across lifestages (larvae and moth). Additionally, metallothionein was higher in the midgut in all
Pb treatments compared with control larvae and was higher in the 800 mg/kg treatment compared with the
200 and 400 mg Pb/kg treatments. Metallothionein was also higher in silk-glands and body fat in the 400
and 800 mg Pb/kg treatments (Zhou et al.. 2015).
11.2.4.3.2. Organism-Level Response
In the 2013 Pb ISA, the body of evidence was sufficient to conclude there is a likely causal
relationship between Pb exposure and neurobehavioral responses in terrestrial invertebrates (U.S. EPA.
2013) (and see Table 11-2 of this appendix). Evidence was primarily from feeding studies in snails and
altered behaviors in nematodes (Caenorhabditis elegans). Several new studies have assessed behavior
modification following Pb exposure in soil organisms and flying insects; most were conducted at nominal
Pb concentrations.
Additional studies in nematodes lend further support to Pb neurotoxicity in these organisms. In a
behavioral food preference and food-finding lab study using agar plates, nematodes (C. elegans) avoided
contaminated food and chose uncontaminated food spots at 1 mg Pb/L, but at 129 mg Pb/L (50% lethal
concentration; LC50), Pb contamination interfered with food-finding ability, and there was no difference
in movement toward either contaminated or uncontaminated food (Montciro et al.. 2014). Another study
using C. elegans found that feeding activity decreased as Pb concentration increased. EC50 for feeding
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behavior was approximately 15 mg Pb/L (54 (iM). Pb also increased damage to the dopaminergic neurons
(Tang et al.. 2019). The study also examined the effects of Cd and Mn, the effects when Pb was mixed
with either metal, or the effects of a treatment containing all three metals. The effects on C. elegans
feeding behavior were greater than the additive effect in binary Pb mixtures at /a < 0.85 (fraction of
organism system affected) but less-than-additive at/a > 0.9. The ternary combination had greater-than-
additive effects at /a < 0.75 and less-than-additive effects at /a > 0.8.
New studies in honeybees suggest Pb exposure alters feeding and foraging behaviors. Soil Pb
contamination (approximately 47.3 mg Pb/kg) did not change the number of honeybee, bumblebee, or
megachilid visits to sunflowers but soil contamination did change the foraging behavior of bees (Sivakoff
and Gardiner. 2017). Bumblebees visited uncontaminated grown sunflowers 5.4 times, honeybees 3.7
times and megachilidae 3.6 times longer than sunflowers grown in contaminated soils. Structural equation
modeling analysis shows a direct negative effect of Pb soil contamination on bee visit duration for
bumblebees and honeybees but direct effects of floral traits or indirect effects of Pb on floral traits were
not significant, suggesting Pb contamination directly explains bee visit duration when floral traits are held
constant. In a behavioral lab experiment, A. mellifera were exposed to a range of Pb concentrations (0.07,
0.66, 6.61, 661 mg Pb/kg, nominal values) in a sucrose solution to examine the effect of Pb contamination
on feeding behavior. Only at the highest Pb concentration did bees reduce sucrose solution intake. By
measuring neuron response to sucrose in antennal gustatory sensilla, the authors determined this response
was due not to detection of the Pb but rather due to a decrease in sucrose perception when Pb was added
to the solutioiiT Furthermore, bees readily ingested the Pb-contaminated solution within a range of 0.075
to 0.75 mg Pb/kg, which the authors reported as comparable to concentrations found in flowers (1.1 to
1.735 mg Pb/kg) (Monchanin et al.. 2022). In another behavioral honeybee experiment, the effects of Pb
(0.07 and 0.66 mg Pb/kg, nominal values) on bee cognitive flexibility were tested. Bees exposed to
0.66 mg Pb/kg contaminated food over 70 days showed less flexibility in response to changing flower
rewards. This response was positively correlated with bee body Pb concentration. Furthermore, higher Pb
exposure during the larval state correlated with lower body weight and head size (Monchanin et al..
2021).
A behavioral experiment examined whether there was a difference in foraging behavior between
cabbage white butterflies (Pieris rapae) reared on a Pb-contaminated diet versus those raised on an
uncontaminated diet (Philips et al.. 2017). Larvae were fed either a 4 mg Pb/kg (nominal values) or
control (approximately 0.17 mg Pb/kg) diet. Behavioral testing following Pb exposure involved yellow
sponges soaked in honey (rewarding) or water-soaked blue sponges (nonrewarding). Butterflies reared on
Pb as larvae were more likely as adults to interact with sponges (approximately twice as many adults
interacted with the sponges compared with control-reared butterflies). Of the butterflies that did interact
with the sponges, there was no difference between treatment groups in the proportion that completed five
consecutive landings on the rewarding sponge. There was also no difference in the duration it took for
butterflies to complete the test (time taken to land five times in a row on yellow sponges). The authors
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suggested this species may already have adapted to low levels of Pb in their diets because brassicas
(natural food source for larvae of P. rapae) mature quickly and are often found in disturbed locations
where Pb may be present. Therefore, the 4 mg Pb/kg concentration may not have been high enough to
induce a different response between treatments in the laboratory-exposed butterflies.
In the 2013 Pb ISA, the body of evidence was sufficient to conclude there is a likely causal
relationship between Pb exposure and growth endpoints in terrestrial invertebrates (U.S. EPA. 2013) (see
Table 11-2 of this appendix). Evidence in the 2013 Pb ISA was primarily from concentration-dependent
inhibition of growth in earthworms raised in Pb-amended soil, and, to a more limited extent, for reduced
growth in snails (dietary studies) and nematodes. New evidence continues to show growth related effects
in invertebrate soil organisms.
Additional studies in earthworms since the 2013 Pb ISA continue to support findings of Pb on
growth. Zaltauskaite et al. (2020) examined the effects of Pb exposure on earthworm (E. fetida) weight,
growth, and recovery postexposure. During 4 weeks of soil exposure (40, 250, 500, 1000, 2500 mg Pb/kg,
nominal values), no effect on weight loss was found, but Pb decreased growth rate with a difference of
15.8-40% lower fresh weight compared with control worms. Following exposure, earthworms were given
a 4-week recovery period with no Pb exposure. While earthworms recovered some weight, they did not
reach equal weights compared with non-exposure worms (11-17.6% lower than control at end of
recovery period). Fresh weight was negatively correlated with increasing Pb soil concentration during
both the exposure and recovery periods. Growth and recovery rate varied with concentration, with
earthworms exposed to 40 mg Pb/kg having the greatest growth rate compared with other Pb
concentrations. Earthworms grew slower during the recovery period compared with the exposure period
except for those exposed to 2500 mg/kg, which showed equal growth rates during exposure and recovery.
MDA was also positively correlated with Pb levels. During recovery, MDA concentrations were lower
but did not reach the same levels as control worms. Weight response to Pb exposure and recovery
suggests Pb inhibits earthworm growth and may have a short-term legacy or lag effect as recovery did not
reach 100% within the same time frame. Increased MDA concentration is indicative of oxidative stress,
which may explain the reduced growth since MDA concentrations were still comparatively high after the
recovery period. Another earthworm study by Zaltauskaite and Sodiene (2014) examined juvenile
earthworm growth and time to maturation across nominal soil Pb concentrations of 40, 250, 500, 1000,
2500 mg Pb/kg. There was no overall effect on weight loss, but juveniles exposed to Pb were smaller than
control worms. The EC50 for juvenile growth increased with increasing time of exposure—at 3 weeks, the
EC50 was approximately 100 mg Pb/kg but after 14 weeks the EC50 for reduced weight was 179 mg Pb/kg.
The time of maximum growth in the 40 mg Pb/kg exposure group was during the 8-10-week period,
while maximum growth was delayed in higher Pb treatments. Pb significantly lengthened the time to
sexual maturation. The minimum time to maturation was 9 weeks for the control and Pb treatment groups,
and the minimum weight at this development point was 0.182 g in the 40 mg Pb/kg treatment group.
Since increasing Pb concentrations reduced the growth rate, the time needed to reach the minimum
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maturation size would increase with increasing Pb; therefore, the time needed at 250 mg Pb/kg would be
16 weeks. The total number of earthworms that reached maturity by the end of the experiment was
negatively correlated with Pb concentrations, with only 5-7% of worms reaching maturity in the
250 mg Pb/kg treatment group.
Adding to the evidence for growth effects in snails from the 2013 Pb ISA, studies on green
garden snail (Cantareus apertus) bioaccumulation and growth in response to increasing Pb dietary
concentrations (25, 100, and 2500 mg Pb/kg, nominal values) over 1 week and 8 weeks of exposure found
the wet weight of snails increased with time across all Pb treatments, and the effect was dose-dependent
in Pb-treated snails (Mlciki et al.. 2016). The weight of snails was significantly lower than the weight of
control snails by week 2 in the high Pb-treatment group, by week 3 for medium Pb-treatment snails and
by week 7 for snails in the low Pb-treatment group. The cumulative growth rate followed a similar pattern
but was lower by week 1 for the high Pb-treatment snails, by week 3 for medium treatment and by week 7
for low Pb treatment. Overall, dietary Pb decreased growth in green garden snails, with a lowest observed
effect concentration (LOEC) of approximately 25 mg Pb/kg food within several weeks. A trophic snail
study found soil Pb levels varied from approximately 6 mg Pb/kg to 52 mg Pb/kg across a gradient of
polluted sites in Romania (Nica et al.. 2012). Shell height was negatively correlated with Pb in nettle
leaves (food source), and relative shell height was positively correlated with snail hepatopancreas Pb
levels. Pb in soil was also correlated with other metals (Zn and Cd). Heavy metals are known to
accumulate in snail shells and can often lead to changes in shell size and geometry.
In a study reviewed in the 2013 Pb ISA, body size in nematodes decreased with increasing Pb
concentration in growth medium, albeit at high concentrations (0.5, 16, and 41 mg Pb/L) (Wang and
Yang. 2007). A more recent study conducted at lower concentrations of 0.05 and 0.1 mg Pb/L (50 and
100 |ig Pb/L, nominal values) in aqueous solution showed Pb had a stimulatory effect on growth at
0.05 mg Pb/L and no stimulatory or inhibitory effect at 0.1 mg Pb/L (Montciro et al.. 2014).
The growth effects of Pb reported for earthworms, snails and nematodes are augmented by
studies in a few additional terrestrial invertebrates. In a generational study with tobacco cutworms
(Spodoptera litura) reared on artificial diets with increasing Pb concentration, both Pb and generation
effects were observed on relative growth rate, pupation rate, and eclosion rate (Shu et al.. 2015). First-
generation pupae experienced no effects of Pb stress on pupation rate or relative growth rate. Eclosion
rates did decrease in the 100 and 500 mg Pb/kg treatments groups (nominal values) (eclosion rates were
51.48% and 28.89%, compared with approximately 70% for all other treatments). Fifth generation larvae
showed significantly lower eclosion and pupation rates at 25 and 50 mg Pb/kg compared with
12.5 mg Pb/kg and control treatments. The relative growth rate of fifth generation pupae declined as well
for the 25 and 50 mg Pb/kg treatments. Differences between generations occurred at the 50 mg Pb/kg
treatment, with 50 mg Pb/kg having stronger negative effects in the fifth generation compared with the
first. There was no effect of Pb (4 mg Pb/kg) on cabbage white butterfly (Pieris rapae) development time
or body size regardless of Pb concentration or butterfly sex (Philips et al.. 2017). Kenig et al. (2013)
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reared fruit flies (Drosophila subobscura) in the lab for eight generations at low and high Pb exposure
(10 (ig Pb/mL and 100 |ig Pb/mL, nominal values) from two wild-caught populations with a difference in
Pb exposure history (298.6 mg Pb/kg and 25.7 mg Pb/kg soil). Flies from the population originally
collected from the site with high pollution levels exhibited a decrease in development time over
generations reared at control (no Pb) lab conditions, a decrease in development time when reared at low
Pb-exposure lab conditions and an increase when reared at high Pb-exposure conditions. Flies from the
low historic contamination site exhibited an increase in development time at control conditions, a
decrease at low Pb exposure, and a decrease at high exposure. Across all levels of Pb exposure in the lab,
there were population, generation, and population x generation effects on fruit fly development time.
Overall, the flies from the high Pb-exposure contamination group had faster development time across
both lab exposure Pb concentrations compared with the low historic contamination population responses.
The authors suggest this response in development time in the high historic exposure population may be an
ancestral adaptation response to allow for growth and reproduction to occur before Pb toxicity occurs.
In the 2013 Pb ISA, the body of evidence was sufficient to conclude there is a causal relationship
between Pb exposure and reproduction in terrestrial invertebrates (U.S. EPA. 2013) (see Table 11-2 of
this appendix). Reproduction endpoints examined in the 2013 Pb ISA included brood size and hatching
success. Additional studies in soil invertebrates published since the 2013 Pb ISA continue to report Pb
effects on reproduction and development, adding to the evidence base for this endpoint. Monteiro et al.
(2014) found Pb had a variable effect on number of nematode C. elegans offspring depending on the
concentration tested. An increase in offspring was observed at 0.01, 0.05, and 0.1 mg Pb/L (10, 50, and
100 |ig Pb/L, nominal values), a decrease at 1 mg Pb/L (1000 |ig Pb/L) and no difference from the control
at 0.5 mg Pb/L (500 |ig Pb/L).
Pb exposure (40, 250, 500, 1000, 2500 mg Pb/kg, nominal values) significantly lengthened time
to sexual maturation for juvenile E.fetida earthworms (Zaltauskaite and Sodiene. 2014). The minimum
time to maturation was 9 weeks for the control and Pb treatment groups, and the minimum weight at this
development point was 0.182 g in the 40 mg Pb/kg treatment group. Since increasing Pb concentrations
reduced growth rate, the time needed to reach the minimum maturation size would increase with
increasing Pb; therefore, the time needed at 250 mg/kg would be 16 weeks. The total number of
earthworms that reached maturity by the end of the experiment was negatively correlated with Pb
concentrations, with only 5-7% of worms reaching maturity in the 250 mg/kg treatment group. In
addition, cocoons were only found at the lowest treatment of 40 mg Pb/kg, and the number of cocoons
was less than half of the number of cocoons produced in control soils.
In a multigeneration vinegar fruit fly (D. melanogaster) study, females that were reared under no
Pb conditions preferentially mated with control males (60% of the time) over males reared in Pb
conditions (108 mg Pb/kg) (Peterson et al.. 2017). In the same study, Pb-reared females preferentially
mated with Pb-reared males over control males (65% of the time). Second-generation females did not
show a significant preference for either second-generation male group (Pb-reared mother or control-
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reared mother). Males across treatments showed no mate preference, and second-generation male body Pb
content was not related to parental Pb content. Despite the behavioral response of females in mate
preference, a principal component analysis of male and female pheromones showed no significant
difference between either male or female treatment groups. Furthermore, there was no difference in
multiple male courtship song variables. While the mechanisms for mate preference remain unclear, there
appears to be no generational effect on fitness. There was no difference between Pb treatments in the
parental generation on either parental or second-generation responses in dry body weight, fecundity, or
time to reach either 50% or 80% mortality. Pb accumulates in fruit fly bodies and this accumulation
appears to influence female but not male mate choice but does not lead to any differences in ability,
success, or fecundity of the flies or their offspring. Another study with D. melanogaster observed that
vinegar fruit flies accumulate Pb linearly with Pb exposure concentration and that the number of eggs laid
on Pb-treated media varied with Pb treatment (Peterson et al.. 2020). Control-reared females laid fewer
eggs on Pb-contaminated media than Pb-reared females at both approximately 109 and 217 mg Pb/kg
(250 and 500 (iM; nominal values, PbAc). However, females reared on the highest Pb treatment of
approximately 434 mg Pb/kg (1000 (iM) laid fewer eggs than the other Pb treatment females. These
results suggested females reared in a Pb-free environment avoid laying eggs in Pb-contaminated areas
whereas females raised in a Pb-contaminated environment did not show this preference for egg site. The
authors suggested this may be due to a loss of this specific avoidance behavior due to developmental
exposure or possibly due to changes in microbial composition. The microbial composition influences
oviposition site selection, with females choosing a site with a composition more similar to the one in
which they grew. Pb acetate was used as the source of Pb contamination in this study. Pb acetate may
directly change the microbial community, which could also explain why Pb-reared females did not
discriminate in laying their eggs in a Pb-contaminated site.
Kenig et al. (2013) isolated Drosophila subobscura adults from wild populations collected at two
sites with different Pb contamination histories (high pollution site 298.6 mg Pb/kg soil average and low
pollution site of 25.7 mg Pb/kg). Gravid females from both populations were used to establish separate
population breeding lines. Flies were then reared for multiple generations on either a control substrate (no
Pb contamination), a low Pb contamination substrate (10 (ig Pb/mL, nominal values) and a higher Pb
contamination substrate (100 |ig Pb/mL, nominal values). Reproduction response variables were
measured at the F2, F5, and F8 generations for each of the two population lines. Both populations reared
under control conditions in the laboratory across eight generations exhibited an increase in the number of
eggs laid between the F2 and F5 generation. This was followed by a decrease in egg production by the
F8 generation but only for the population with a lower historic Pb exposure. Under low Pb-exposure lab
conditions, both populations showed the same pattern of increasing number of eggs from F2 to F5
followed by a decrease in production to F8, though this pattern was less pronounced for the low historic
exposure population. Under high exposure conditions, both populations saw egg production decrease by
the F8 generation. Egg viability for the high historic exposure population decreased from F2 to F5/F8
under control conditions, and the low exposure population saw an increase from F2 to F5 followed by a
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decrease to F2 viability levels by generation F8. Under low exposure conditions, both populations
followed the same pattern they showed under control conditions. Under high Pb lab conditions, neither
population showed a change in egg viability across generations but the egg viability of the population
from low historic exposure conditions had overall lower egg viability than the population that experienced
historically high exposure. Individuals from the historic high exposure showed higher viability and
fecundity when exposed to higher Pb concentrations in all generations compared with those from the
historically low exposure population, exhibiting higher tolerance to heavy-metal exposure.
Mazzei et al. (2013) examined isopod Armadillidium granulatum reproductive response to metal
contamination of food. According to the authors, isopod heavy-metal concentration factors vary widely
across species as does their breeding patterns. In this study, Pb concentration (100, 500, 1000 mg Pb/kg,
nominal values) in food led to an alteration of reproductive patterns in A. granulatum. Increasing
concentrations led to a delayed onset in breeding season while also reducing the duration of the season.
Breeding season onset did not differ between control and 100 mg Pb/kg treatments. Breeding season
occurred 1 week later in the 500 mg/kg treatment group and 6 weeks later in the 1000 mg Pb/kg treatment
group. The length of the breeding season decreased from 79 days (control) to 59 (500 mg Pb/kg) and 46
(1000 mg Pb/kg) days. There was no effect of Pb on incubation period (approximately 23 days), and the
percent gravid rate of females increased from 97.2% (control) and 95.8% (100 mg Pb/kg) to 100% for
higher Pb treatments. However, while gravid rate increased, brood number declined (from 1.22 to 1).
Lastly, the number of juveniles for each brood increased with 500 mg Pb/kg treatment. Overall,
contamination at 100 mg Pb/kg did not influence any reproductive endpoint examined for A granulatum
but higher levels led to changes in breeding seasonality and the number of juveniles.
In the 2013 Pb ISA, the body of evidence was sufficient to conclude there is a causal relationship
between Pb exposure and survival in terrestrial invertebrates (U.S. EPA. 2013) (see Table 11-2 of this
appendix). Additional evidence continues to show Pb effects on mortality in some terrestrial
invertebrates, while others appear to be unaffected. In a laboratory study examining green garden snail
(Cantareus apertus) response to increasing Pb dietary concentrations (25, 100, 2500 mg Pb/kg, nominal
values) over a period of 1 to 8 weeks, cumulative mortality was greater in all Pb treatments than the
control after 6 weeks of exposure, with the high treatment having significantly greater mortality after
1 week (Mleiki et al.. 2016). At the end of the experiment, cumulative mortality was below 30% for all
treatments. An observational study of C. apertus exposed to multiple metal-polluted soils with Pb
concentrations ranging from 28.1 to 4574 mg Pb/kg found only 6.5% of snails died after 28 days of
exposure (Paugct et al.. 2013b). Studies in the 2006 Pb AQCD found earthworm LC50 for 14 and 28-day
exposure fell within a range of 2400-5800 mg Pb/kg. A study reported in the 2013 Pb ISA evaluated E.
fetida earthworms exposed to field-collected soils with Pb concentrations up to 390 mg Pb/kg and found
no effect on earthworm survival (Delistratv and Yokel. 2011). In support, juvenile E. fetida earthworms
exposed to a range of Pb concentrations (40, 250, 500, 1000, 2500 mg/kg, nominal values) over 14 weeks
found mortality increased, but only in the 500-2500 mg Pb/kg treatments, with mortality reaching 90% in
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the highest treatment (Zaltauskaitc and Sodiene. 2014). Juvenile mortality increased with the time of
exposure in these treatment groups, with an LC50 of 911 mg Pb/kg for 14 weeks of Pb exposure. Juvenile
mortality did reach 10% by week 3 for the 40 and 250 mg Pb/kg treatments but did not increase any
further over time. However, in another earthworm exposure experiment using adults of E. fetida, across
only 4 weeks of exposure (40, 250, 500, 1000, 2500 mg Pb/kg, nominal values), there was no significant
effect on survival (Zaltauskaitc et al.. 2020). In cabbage white butterflies (P. rapae) raised from eggs
from wild-caught females, no effect on survival was observed in a laboratory study with a diet of
4 mg Pb/kg (Philips et al.. 2017).
In terrestrial invertebrates, literature since the 2013 Pb ISA provides additional support on the
effects of Pb exposure on organismal and suborganismal responses including a decrease in survival and
reduced growth and fecundity. Recently published studies on physiological responses to Pb included
decreases in protein and lipid content and increases in MDA in earthworms. AChE activity decreased in
response to Pb in snails and honeybees while protein, glycogen, other enzymes, and GST responses were
variable depending on modifying site factors or species examined. There are several new studies
quantifying behavioral changes to Pb exposure in bees. Soil Pb contamination altered foraging behavior,
and at high levels (above 600 mg Pb/kg), also altered sucrose intake. However, at low concentrations
(0.66 mg Pb/kg), honeybees showed lower flexibility in response to changing flower rewards, suggesting
Pb may lead to lower nectar and pollen supply and subsequently slower colony development or winter
survival. New literature on growth endpoints suggests Pb can have lasting effects even postexposure on
earthworms. Growth, eclosion, and pupation rates of the common cutworm were all lower under Pb
exposure, and fruit fly development time increased within eight generations in populations with historic
Pb pollution exposure. In addition to previously assessed endpoints of Pb on brood size and hatching
success, new literature shows Pb exposure slows time to maturation in earthworms, delays onset to and
duration of breeding season in isopods and influences mate selection in fruit flies. While the literature
since the 2013 Pb ISA has primarily provided additional support on previously examined organisms and
endpoints, there has been new information on new organisms as well as on modifying factors on organism
response including habitat, exposure history, seasonality, and duration of effects.
11.2.4.4. Effects on Terrestrial Vertebrates
In observational and experimental studies, commonly observed effects of Pb on terrestrial
vertebrates include decreased survival, reproduction, and growth, as well as effects on development and
behavior (U.S. EPA. 2006a). The 2013 Pb ISA (U.S. EPA. 2013) also provided evidence for Pb effects on
hormones and other biochemical variables (U.S. EPA. 2013). Recent studies provide additional support to
suborganism-level and organism-level endpoints and expand on the effects on hematological and
physiological endpoints.
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11.2.4.4.1. Suborganism-Level Response
In the 2013 Pb ISA, the body of evidence was sufficient to conclude there is a causal relationship
between Pb exposure and hematological effects in terrestrial vertebrates (U.S. EPA. 2013) (see Table
11-2 of this appendix). Since the 2013 Pb ISA, numerous new studies have continued to support the
connection between Pb exposure and hematological effects. The relationship between Pb concentrations
and aminolevulinic acid dehydratase (ALAD) activity has been explored in the literature, across a broad
assortment of different vertebrate species including songbirds (Bever et al.. 2013). house sparrows (Cid et
al.. 20IS). Japanese quail (Bever et al.. 2014). griffon vultures (Espi'n et al.. 2015). eagle owls (Espi'n et
al.. 2015). common ravens (Herring et al.. 2018). turkey vultures (Herring et al.. 2018). Canada geese
(van der Merwe et al.. 2011). mallards (Binkowski and Sawicka-Kapusta. 2015). coots (Binkowski and
Sawicka-Kapusta. 2015). giant toads (Ilizaliturri-Hernandez et al.. 2013). cattle (Rodrigucz-Estival et al..
2012). and sheep (Rodriguez-Estival et al.. 2012).
Bever et al. (2013) investigated blood, liver, and kidney concentrations of Zn, Cu, Pb, and Cd and
ALAD activity in northern cardinals (Cardinalis cardinalis) and American robins (Turdus migratorius)
living in Pb-contaminated mining sites in southeast Missouri. Birds from contaminated locations had
ALAD activity levels that were decreased by between 58 and 82% compared with those from
noncontaminated locations. Another field study that examined the relationship between Pb and ALAD
activity found similar results in griffon vultures (Gyps fulvus) and eagle owls (Bubo bubo) (Espi'n et al..
2015). Blood samples were taken from birds near an industrial area (electric power plants, explosives, and
ship-building factories) and a historic Pb-Zn mine. The study found a significant negative relationship
between blood Pb levels and ALAD activity in griffon vultures and in eagle owls, with ALAD inhibition
of up to 94% and 79%, respectively.
Herring et al. (2018) examined the effects of Pb exposure on ALAD activity in two species of
free-living scavengers in the Pacific Northwest: common ravens (Corvus corax) and turkey vultures
(Cathartes aura). The authors speculated that environmental Pb exposure in these species was most likely
associated with a variety of sources including hunting, Pb-based paint, soil, and sediment Pb, and mining
and smelting activities. Both species exhibited decreased ALAD activity (mean = 5.9 ± 1.4 SE) in birds
with blood Pb concentrations greater than 0.2 jxg/g (the subclinical toxicity benchmark) when compared
with birds with blood Pb concentrations below this benchmark (mean = 9.9 ± 0.6 SE).
Binkowski and Sawicka-Kapusta (2015) is another field study that examined the relationship
between blood Pb levels and ALAD activity in free-living birds published since the 2013 Pb ISA. This
study investigated free-living mallards (Anas platyrhynchos) and Eurasian coots (Fulica atra) in Poland.
In both species, there was a significant negative correlation between Pb concentrations in blood and
ALAD activity. The authors suggested that Pb exposure mainly occurred through Pb shot, van der Merwe
et al. (2011) also found evidence of a relationship between Pb concentrations and ALAD inhibition in
waterfowl. Geese from the tri-state mining district of Kansas, Oklahoma, and Missouri and multiple
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different metal concentrations were measured (silver [Ag], As, barium [Ba], Cd, Co, Cr, Cu, Fe, Mg, Mn,
Mo, Ni, Pb, Se, Ti, V, Zn). This study found that ALAD activity was inversely correlated with tissue Pb
concentrations in all tissue except muscle.
Multiple laboratory studies have examined this relationship. Cid et al. (2018) exposed house
sparrows (Passer domesticus) to sublethal oral doses of Pb acetate solution (1.3, 3.5, 5.5, 7.0, 14.0 jxg/g
animal/day) for 5 days. This resulted in a gradual decrease in ALAD activity between 3.5 and 7.0 |ig Pb/g
animal/day, with the 7.0 and 14.0 |ig Pb/g animal/day doses producing greater a-ALAD activity inhibition
(82% less activity than control group). This study also examined the effects of Pb exposure in drinking
water for 15 or 30 days. Inhibition of ALAD activity was similar between the two groups, with an
approximately 35% decrease when comparing the mean value of both treatment groups and the controls.
Bever et al. (2014) studied the effect of Pb-contaminated soil on captive Japanese quail (Coturnix
japonica) to examine the relationship between Pb exposure and hematological effects and to determine
benchmark doses associated with different percentages of ALAD reduction. Quail were fed experimental
diets containing 0% to 12% contaminated soil by weight (0.12 to 382 mg Pb/kg, dry weight) for 6 weeks.
All quail groups exposed to Pb-contaminated soil had a significantly lower mean ALAD activity than the
control group. ALAD activity also decreased with increasing dosage, with control quail having the
highest amount of activity and the 12% contaminated soil group having the lowest. The benchmark doses
of Pb associated with a 50% reduction in ALAD activity were 0.62 mg Pb/kg in the blood, dry weight,
and 27 mg Pb/kg in the diet.
Although there is limited new evidence on the effects of Pb on ALAD activity in other terrestrial
vertebrates since the 2013 Pb ISA (U.S. EPA. 2013). two nonbird studies examined this relationship.
Rodriguez-Estival et al. (2012). investigated this relationship in both cattle and sheep from livestock
farms in Spain. Blood Pb level was found to be negatively correlated with ALAD reaction ratio in both
cattle and sheep. Blood Pb level also had a negative effect on ALAD activity. Ilizaliturri-Hernandez et al.
(2013) examined the relationship between blood Pb levels and ALAD inhibition in giant toads (Rhinella
marina) in Veracruz, Mexico. Blood Pb levels ranged from 10.8 to 70.6 (ig/dL and were significantly
higher in industrial sites. Toads at industrial sites also had a 78% decrease in ALAD activity when
compared with those at rural sites. Examining the relationship between blood Pb levels and ALAD, a
strong inverse relationship was identified. The authors stated that Pb exposure was most likely from
pollution released into the air and water by chemical and petrochemical companies in the area.
In the 2013 Pb ISA, the body of evidence was sufficient to conclude there is a likely causal
relationship between Pb exposure and physiological stress for terrestrial vertebrates(U.S. EPA. 2013) (see
Table 11-2 of this appendix). Since then, multiple new studies have added to this evidence base. Many
different factors are included in physiological stress, including oxidative stress, corticosterone (CORT)
levels, and immune response, all of which are discussed here.
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Two different studies investigated CORT levels in response to Pb exposure. Meillere et al. (2016)
evaluated the relationship between feather Pb levels and feather CORT levels in wild common blackbirds
(Turdus merula) along an urbanization gradient. Male adult blackbirds were found to have an average
feather Pb concentration of 1.00 ± 0.76 jxg/g, dry weight, which was positively correlated with the degree
of urbanization. Feather CORT levels were found to be significantly and positively related to both the
degree of urbanization and feather Pb levels. Herring et al. (2018) also investigated CORT levels in birds.
Examining the relationship between fecal CORT levels (FCORT) and blood Pb levels in common ravens
(Corvus corax), it was found that blood Pb significantly affected FCORT levels only when there was
simultaneous exposure to mercury (Hg). FCORT was either not related or negatively correlated with
blood Pb when blood Hg concentrations were below 0.2 jxg/g, wet weight. Above this blood Hg
concentration, the FCORT response increased with increasing blood Pb concentrations.
Another aspect of physiological stress that has been linked to Pb exposure is oxidative stress.
Espin et al. (2014) assessed oxidative stress related to Pb in the Eurasian eagle owl (Bubo bubo). One
study in three different subareas in Murcia, southeastern Spain (rural, industrial, and mining areas)
evaluated the relationship between Pb exposure and oxidative stress biomarkers in blood. Glutathione
peroxidase (GPx) activity had a significant inverse correlation with Pb concentrations. Catalase (CAT)
activity was inversely related to Pb concentration as well. Both GPx and CAT are antioxidant enzymes
that catalyze the breakdown of free radicals and indirectly support the antioxidant defense system. Espin
et al. (2016) also examined these oxidative stress biomarkers in relation to blood Pb concentrations with
different results. In two different gull species, Audouin's gull (Ichthyaetus audouinii) and slender-billed
gulls (Chroicocephalus genei), total glutathione (GSH) content, antioxidant enzymes activities (GPx,
superoxide dismutase (SOD), CAT, GST), and lipid peroxidation (thiobarbituric acid reactive substances)
were analyzed to determine whether blood Pb concentrations had any effect on these oxidative stress
biomarkers. The only significant linear regression on Pb was the positive effect of Pb on GSH levels in
Audoin's gulls. The authors speculated that this could reflect the necessity to up-regulate GSH to balance
increased oxidative stress caused by metals. A laboratory study of female Japanese quail (Coturnix
japonica) also examined these effects, as well as other effects including liver histology and lipid
metabolism (Kou et al.. 2020). Quail were fed one of five experimental concentrations of Pb solution (0,
50, 250, 500 and 1000 ppm) for 49 days. Pb exposure of 250, 500, and 1000 ppm induced severe
histopathological damages (liver lipid vacuoles and accumulation, hepatic cytoplasmic hyalinization and
vacuolization, hepatocyte necrosis, hepatic sinusoid congestion). It also led to a significant decrease in
GPx, SOD, and CAT activities in the liver.
Immune response has also been linked to Pb exposure, for example, in the following two studies.
Vermeulen et al. (2015) examined the effects of Pb exposure on the innate immunity of great tit (Parus
major) nestlings in populations along a metal pollution gradient. Average Pb concentration in red blood
cells was significantly higher in the populations closest to the pollution source than the farthest
population. There were significant differences in lysis scores among the populations, with lysis varying
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inversely to Pb concentrations. Farsang et al. (2017) used the ratio of heterophils to lymphocytes (H/L
ratio) in mute swans (Cygnus olor) to determine physiological stress levels. A higher H/L ratio indicates a
higher immune response, thus higher physiological stress. Mean blood Pb concentration was 0.239 jxg/g
(range: 0.028-0.675 jxg/g). H/L ratio was found to increase with blood Pb level, indicating that birds with
higher blood Pb levels had higher physiological stress.
11.2.4.4.2. Organism-Level Response
In the 2013 Pb ISA, the body of evidence was sufficient to conclude there is a causal relationship
between Pb exposure and reproduction and developmental endpoints in terrestrial vertebrates (U.S. EPA.
2013) (see Table 11-2 of this appendix). Since the 2006 AQCD (U.S. EPA. 2006a) and 2013 Pb ISA
(U.S. EPA. 2013). several field studies have examined the relationship between Pb exposure and
reproduction. Fritsch et al. (2019) found that the lifetime breeding success of free-living female European
blackbirds (Turdus merula) in Northwest Poland decreased with increasing levels of Pb in tail feathers
(average tail feather Pb = 6.7 |ig Pb/g dry weight). This same study also examined the relationship
between breeding success, lifespan, and Pb exposure. In birds with the greatest exposure and highest
breeding success, there is likely a trade-off between breeding effort and survival, as their lifespans tended
to decrease as Pb exposure increased. Chatelain et al. (2016) also studied how Pb exposure affected
reproduction. Adult feral pigeons (Columba livid) were dosed with one of four exposure treatments: Pb
only (1 ppm Pb acetate in tap water), Zn only (10 ppm ZnSC>4 in tap water), Pb and Zn (1 ppm Pb
acetate +10 ppm Zn sulfate in tap water), or control (tap water with no metal addition) every other day
for 2 weeks. One-day old nestlings of parents exposed to Pb (Pb and Pb +Zn groups) weighed
significantly less than the nestlings from other treatments (control and Zn groups) (mean 14.94 ± 0.72 and
17.20 ± 0.67 g, respectively). Additionally, eggs from parents exposed to Pb had significantly thinner
eggshells than those from the other groups (mean: 0.47 ± 0.00 and 0.49 ± 0.01 mm respectively).
While Fritsch et al. (2019) examined reproduction at the organism level, Hargitai et al. (2016)
examined suborganismal level responses to Pb exposure in relation to reproduction. Hargitai et al. (2016)
found that in great tit (Parus major) eggs from both woodland and urban habitats in the Pilis Mountains
of Hungary, egg yolk lutein and retinol levels were negatively related to the concentrations of Pb in the
eggshell. Lutein and retinol are both important antioxidants related to embryo viability in birds.
In the 2013 Pb ISA, the body of evidence was sufficient to conclude there is a likely causal
relationship between Pb exposure and neurobehavioral effects in terrestrial vertebrates (U.S. EPA. 2013)
(see Table 11-2 of this appendix). Several additional studies in birds have since been published that assess
Pb effects on behavioral endpoints in birds. One new study of the neurobehavioral effects of Pb-exposure
evaluated the relationship between the behavior of free-living Northern mockingbirds (Mimus
polyglottos) and the soil Pb concentrations in their habitats in New Orleans, LA (Mcclelland et al.. 2019).
Birds living in neighborhoods with high soil Pb concentrations had higher Pb concentrations in their
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blood and feathers than those from the neighborhood with low soil Pb concentrations. This study used
simulated territory intrusions to examine the level of aggression displayed by individuals from different
neighborhoods. Birds from the high Pb neighborhoods exhibited a more aggressive response to simulated
intrusions than birds from the low Pb neighborhood.
Another study of the effects of Pb exposure on behavior examined how early-life dietary Pb
exposure in great tits (Parus major) affected both physical and neurological development (Ruuskancn ct
al.. 2015). Wild birds in selected nests were given an oral dose of Pb acetate in distilled water (4 jxg/g
body weight for high exposure and 1 jxg/g body weight for low exposure) every day for 12 days, starting
at 3 days after hatching. At 15 days old, the birds were brought into captivity and kept there for the
remainder of the experiment to assess their development after Pb exposure. Early-life Pb exposure was
found to have no effect on activity, exploration, neophobia, or success in learning and spatial memory
tasks.
Commonly observed effects of Pb on terrestrial vertebrates include decreased survival,
reproduction, and growth, as well as effects on development and behavior (U.S. EPA. 2006a). The 2013
Pb ISA (U.S. EPA. 2013) also provided evidence for Pb effects on hormones and other biochemical
variables. New studies have expanded upon the relationship between Pb exposure and a-ALAD activity
by adding more species of birds, amphibians, and mammals to the evidence base. More evidence of
oxidative stress has been gathered, as well as evidence of effects on CORT levels and immunity in birds.
Literature since the 2013 Pb ISA continues to add to evidence relating to reproductive effects at both the
organism and suborganism levels including effects on lifetime breeding success and some specific
secondary sexual traits. New studies of behavioral effects included increased aggression in mockingbirds.
11.2.5. Exposure and Response of Terrestrial Species
As previously reported in the (U.S. EPA. 1977). the 1986 Pb AQCD (U.S. EPA. 1986). the 2006
Pb AQCD (U.S. EPA. 2006a) and the 2013 Pb ISA (U.S. EPA. 2013). a large number of experimental
studies have exposed a wide variety of terrestrial organisms to gradients of Pb exposures and reported a
broad assortment of responses, including growth, reproduction, survival, antioxidant levels and markers
of oxidative stress. More than 80 such additional experimental studies conducted since the 2013 Pb ISA
were identified. Organisms subjected to these exposure-response experiments have included various wild
plants including reeds and ferns, cultivated crops, microbes, lichens, fungi including mycorrhizae,
bacteria, nematodes, worms, collembolans, beetles, spiders, rodents, and birds. The 2006 AQCD and
2013 Pb ISA (U.S. EPA. 2013. 2006a) reported that variation in exposure is generally associated with
commensurate variation in growth, reproduction, survival, antioxidant activity and more. Such coupling
of exposure and response is considered a strong indicator of causality (U.S. EPA. 2015). and exposure-
response studies with Pb thus continue to provide evidence supporting the causality of Pb for the effects
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they investigate, as highlighted in the sections of this appendix dedicated to specific groups of terrestrial
organisms.
With very few exceptions, experimental exposure-response studies of terrestrial organisms
generate multiple level of exposure through addition of various soluble salts of Pb to the culture medium
(natural or artificial soil or hydroponic solution) or to food in the case of some animals. This makes it
possible to create a gradient that is easy to quantify and manipulate and is isolated from confounding,
nuisance and interacting variables. In principle, these attributes are desirable, as they allow for a more
accurate measurement and modeling of exposure-response relationships. They may introduce limits on
the scope of inference, but can nonetheless lead to credible, accurate predictive estimates of response,
within an acceptable range of natural conditions wherein factors other than exposure are left to vary
freely. However, in the particular case of terrestrial organisms and estimates of their response that are
obtained through experiments in which exposure is accomplished using salts of Pb, this may not be the
case. These experiments are informative for establishing causality, but not for deriving accurate predictive
estimates of response under natural conditions.
Section 11.2.2.1 discussed environmental variables that have a strong impact on bioavailability in
soils. They include pH, CEC, salinity, aging, OM, soil type and the presence of other metals. The use of
soluble salts of Pb brings pH, CEC, salinity, and aging into ranges far removed from those found in
natural environments following exposure to Pb emissions. Predicted effects derived from those
experiments cannot be expected to be accurate in environmental conditions, not only because the
experimental conditions of pH, CEC, salinity and aging diverge too far from those present in the
environment, but, more intractably, because in both the experiments themselves and in the environments
in which a prediction is attempted, the measurement of Pb concentration may sharply diverge from the
concentration actually affecting the organism. These difficulties were discussed in the 2006 Pb AQCD
(U.S. EPA. 2006a') and the 2013 Pb ISA (U.S. EPA. 2013). as well as in studies explicitly designed to
clarify these issues, such as Smolders et al. (2009). Chevns et al. (2012) or Davton et al. (2006). In 2009,
following extensive toxicity testing with both spiked soils and contaminated field soils, Smolders et al.
(2009) concluded that "despite all of the efforts made, a large proportion of the difference between the
toxicity observed in field-contaminated soils and that in laboratory-amended soils remains unexplained."
Smolders et al. (2009. p.) further demonstrated that not only are the effects of pH, for example, more
complex than previously thought, but pH, CEC, DOM, Fe and Mn oxides, aging and soil type are all
powerful modifiers of Pb toxicity to soil-dwelling organisms. Underscoring the complexity of modifying
effects, Chevns et al. (2012). for instance, showed that in tomato and barley plants, soil type is a major
modifier of toxicity, but that once pH is controlled, toxicity can be mediated by the nutrient deficiencies
that stem from reactions of Pb with essential nutrients in the soil solution, whereby the apparent effects of
Pb are caused by nutrient deficiencies from Pb robbing the plants of P, for example, by forming Pb
phosphates.
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The following more recent studies have continued to untangle the respective roles of the various
factors that complicate predictive estimation of the effects of Pb in terrestrial organisms from exposure-
response studies (Table 11-3). With enough knowledge of the effects of these factors on the exposure-
response relationship, it could, in principle, become possible to use some of those experiments to generate
useful estimates of concentrations associated with responses of interest from experiments. Experimental
procedures might be adjusted, for example by aging or leaching soils prior to exposing organisms, or the
modeling of the relationship itself might be modified, for example by adding correction coefficients to the
exposure.
Among other questions, Zhang and Van Gestel (2019b) investigated the effects of 18 months of
aging, form of Pb and percolation (leaching) on the toxicity of Pb to the worm Enchytraeus crypticus in
natural standard soil spiked with nine levels of Pb between 0 and 3200 mg Pb/kg dry soil using Pb(NO;,):
and between 0 and 1000 mg Pb/kg dry soil using PbO. Among the complex interactions between these
variables, they found that while leaching dramatically decreased porewater concentration of Pb in fresh
and aged soils, and more so for Pb(NC>3)2 than for PbO, it did not affect Pb uptake, which was greater for
the more soluble form (Pb(NO;,)2). LC50 and LC10, estimated from logistic regression on all nine levels
was higher following leaching for Pb(NC>3)2 but not for PbO. The authors concluded that generally, the
effect of percolation on the toxicity of Pb-spiked soils was dependent on the chemical form used for
spiking as well as on aging, and porewater Pb concentration could not explain Pb toxicity. For survival,
leaching decreased the toxicity of Pb(NO;,): but did not affect the toxicity of PbO. For effects on
reproduction, leaching had a greater influence in freshly spiked soils than in aged soils. This suggests that
manipulating or accounting for aging and form of Pb might be useful in generating effect predictions in
natural environments from spiking experiments, but that manipulating leaching may not be.
The same authors also included variation in the length of the aging period in another exposure-
response experiment (Zhang and Van Gestel. 2019a'). Using the same materials and methods as in Zhang
and Van Gestel (2019b). they incubated the soil samples for five periods from 0 to 18 months, after
spiking and before exposure of the worms. Toxicity increased with aging when soils were spiked with
PbO but not with Pb(NOs)2, as did availability when estimated via CaCh extraction. This may conflict
with (Smolders et al.. 2015). who found that lethality declined with five years of aging, but in outdoors
conditions that included leaching by rain rather than laboratory incubation. Including aging in the
translation from experiment to field thus appears warranted, but not without also including the form of Pb
and leaching.
Finally, Zhang et al. (2019a) investigated the effects of soil properties toxicity to Enchytraeus
crypticus using the same materials and methods as Zhang and Van Gestel (2019b') and six standard
natural soils, using Pb(NO;,): but not PbO treatments, and not varying aging or percolation. The soils
varied in OM content, pH, CEC, water-holding capacity, dissolved OC, and composition. Soil type had
very large effects on survival of earthworms in the presence of Pb even though no effect was observed on
the internal Pb concentration of worms, with effects ranging from no survival at the mid range of Pb
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concentration, to complete survival even at the highest concentration. However, soil type had only weak
effects on survival when exposure was measured as porewater Pb and no effect on survival when
measured as CaCh-extractable Pb. Similarly strong effects of soil type were seen on the exposure-
response relationship of Pb concentration and earthworm reproduction. However, the same weak effects
of Pb as for survival were observed for reproduction when using porewater Pb concentrations, and no
effects were observed when using CaCh-extractable Pb. Furthermore, measuring exposure as CaCh-
extractable Pb resulted in accurate and precise predictions of responses regardless of soil type. In
contradiction with other studies cited above, such as Chevns et al. (2012) or Smolders et al. (2009). the
authors suggested that despite soil type having a strong effect on toxicity when exposure is measured as
simple soil concentration using CaCh-extractable Pb as a metric of exposure may be sufficient when
estimating the effects of Pb on worms, since using that metric supported accurate and precise prediction
of earthworm responses regardless of soil type, and the exposure-response relationship was then
insensitive to soil type.
Romero-Freire et al. (2015) assessed the respective influence of soil properties in laboratory
toxicological assays, with the same aim of making experimental exposure-response studies with spiked
soils usable for environmental risk assessment. Seven natural soils of varying pH, conductivity, texture,
OC, water-holding capacity, CEC, specific area, carbonate content and metal oxides were spiked with five
levels ofPb(N03)2 and incubated for 4 weeks. The authors observed that pH and CaCC>3 content were the
soil properties with the highest influence on Pb extractability and interacted strongly with total Pb
concentration, with extractability most affected at higher concentrations of Pb. However, they also found
that retention via organic complexation kept most of the Pb from being bioavailable and that texture
(silt/sand/clay proportions) and Fe and Mn oxides also had major effects on extractability. In three tests of
toxicity—one with lettuce seeds, one with a strain of marine bacterium, and one measuring microbial soil
respiration—soil type strongly modified overall toxicity in all tested organisms and the relative effects of
each concentration of Pb (in other words, the slope of the response curve). In addition, the magnitude of
these modifying effects differed among the three tests. The authors did not attempt to partition the effects
of every soil property beyond the most salient effects on extractability noted above. They concluded that
soil properties in the particular locations and land use where risk is to be assessed must be taken into
consideration when conducting risk assessment, including at minimum, pH, OM and carbonate.
Many variables distinguish natural soils from each other with regard to influence on Pb toxicity,
as enumerated in the experiments cited here. As noted by Romero-Freire etal. (2015). Zhang et al.
(2019a). Smolders et al. (2009) and others, given practical limitations on the number of soils that can be
included in one experiment, it is not possible to definitively separate the effect of each of the variables
that define soil type, let alone quantify their interactions. It is possible however to separate some variables
that affect the exposure-response more strongly from those that have little or no influence, and it may be
possible to identify measures of exposure under which the exposure-response relationship is insensitive to
soil type, but nonetheless support accurate and precise estimation of toxic effects.
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Another study of the factors that contribute most strongly to differences between responses
occurring in natural environments and those observed in Pb-spiking experiments was conducted by
Smolders et al. (2015). The study was aimed at assessing the relative magnitude of the effects of salinity,
acidification, and aging on the toxicity of Pb to invertebrates, plants, and microbes. Samples of three
natural soils were spiked with seven levels of Pb ranging from 0 to 8,000 mg Pb/kg as Pb(NC>3)2 and as
PbCk Some samples were used unleached and imaged, some were leached and pH-corrected, and some
were leached, pH-corrected and aged for five years, a much longer period than in most aging studies.
Tomato and barley seedlings were grown in all nine treatments, and biomass was measured after 21 days.
Nitrification and soil respiration were measured to assess microbial activity, and the reproduction of the
worm E. fetida and the collembolan F. Candida was likewise measured for the nine treatments. Relative to
the unaged, unleached treatment, the increase in ECio with leaching and pH correction, aging or leaching,
pH correction and aging, showed very wide variation between endpoints. All endpoints demonstrated
strong toxicity relative to controls at all levels of added Pb in all three unaged, unleached soils. The EC50
for all endpoints increased with leaching and pH correction except for earthworm reproduction in one
soil, again with wide variation among endpoints. Finally, aging for five years combined with leaching and
pH correction increased EC50 to such a degree for all endpoints that its value could not be estimated for
any of them. Earthworm reproduction was the endpoint for which EC50 increased the least. Smolders et al.
(2015) attempted to identify which variables among total Pb concentration, porewater Pb, Pb2+ ionic
activity, pH and porewater ionic strength were most strongly correlated with endpoints. Overall,
porewater ionic strength was the variable most strongly correlated with toxicity. Based on this correlation,
the authors suggest that increased salinity, i.e., salt stress compounding true Pb toxicity in freshly spiked
soils, is likely the greatest modifying factor of toxicity. They found the effect of pH to be inconclusive
due to limitations of their experimental protocol, and perhaps surprisingly, caution about giving too much
weight to the effects of aging despite its seemingly large effect. They re-emphasized the limitations of the
experimental protocol, specifically the leaching that preceded aging. For plants, they noted a deficiency of
P, with both increased Pb concentration and aging as the more direct factor explaining the effects on plant
growth. The authors concluded that regardless of the mechanisms behind their observations, this study
offered "... a strong confirmation that acute dosing of soluble Pb2+ salts does not appear to be an
appropriate model for environmental sources of Pb where Pb gradually enters soils via atmospheric
deposition as PbO, PbS, and PbSC>4..."
In 2021, Ports et al. (2021) proposed two corrections to the results of exposure-response
experiments conducted with addition of soluble salts of Pb to soil and used them to derive some examples
of ecological soil standards. They suggested first that a single correction factor can be applied to the
toxicity results of fresh, i.e., unleached, unaged, spiking experiments to adequately convert the results to
the values that would have been observed following leaching and aging. They further proposed to
demonstrate that this conversion generates values that correspond to the toxicity levels that would be
observed in corresponding hypothetical field conditions. The second correction was intended to adjust
differences in toxicity that arise from differing soil properties. Although as referenced previously,
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multiple properties of soils have been shown to affect Pb toxicity in both spiking experiments and field
conditions, the authors argued that adjusting for CEC is sufficient. The authors demonstrated the
derivation of predicted no-effect concentrations (PNEC) according to the European REACH Regulation
Parliament and Council (2006). using the two corrections above and data that conformed to the REACH
requirements. In contrast with Eco-SSL values, none of the derived standards were lower than
background soil Pb concentration.
A few methodological developments in analyzing and using Pb exposure-response experiments
have also been explored since the 2013 Pb ISA (U.S. EPA. 2013). although they may not be of immediate
applicability to risk assessment or standard setting. Zhang and Van Gestel (2017) used one standard
natural soil spiked with seven levels of Pb(NC>3)2 between 0 and 3200 mg/kg soil to study the
toxicokinetics and toxicodynamics of uptake, elimination, and survival in the worm Encytraeus crypticus.
Uptake and toxicity were measured at seven time intervals and elimination at six. The measurement and
statistical modeling of the time course of uptake, elimination and survival demonstrated that accumulation
and toxicity were dependent on exposure duration, and that once the time course of exposure was taken
into consideration, the internal concentration of Pb in worms may be a better predictor of survival than
soil concentration. Using the model organism C. elegans exposed to five levels of Pb between 0 and
2000 ppm as Pb acetate, Sudama etal. (2013) combined chromatographic metabolite profiling and
principal component analysis to show that changes in the purine pathway and its metabolites can be
detected after exposure to extremely low concentrations of Pb.
Finally, the applicability of Species Sensitivity Distribution analysis was investigated by Ding et
al. (2016) using 21 natural soils spiked with four levels of Pb between 0 and 350 mg/kg soil as Pb(NC>3)2
and 12 cultivars each of carrot (Daucus carota), radish (Raphanus sativus), and potato (Solarium
tuberosum), to show that Species Sensitivity Distribution analysis could be a reliable approach to
determining safety thresholds, as long as the threshold values are derived from experiments designed for
that purpose. However, exposure was from soluble salt, and the safety thresholds the authors investigated
were for the safety of human consumers of vegetables grown in heavily polluted sites. They therefore
measured only accumulation in the plants and not the effects on the plants themselves.
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Table 11-3 Studies of factors that affect the interpretability of exposure-response experiments in terrestrial
biota, since the 2013 Pb ISA.
Organism
Experimental conditions
Pb
concentrations
Study
factors
other than
Pb
exposure
Effects of
Pb
Effect
concentration
Effects of
additional
study factors
Reference
Barley
(Hordeum
vulgare)
Tomato
(Lycopersicon
esculentum)
Form of
Pb:
PbCh
Medium:
Six topsoils
from five
European
countries
Hydroponic
system
Exposure
method:
Salt mixed
with soil
Salt in
hydroponic
solution
jdH:
Soils 7.4, 6.5, 6.7, 5.7, 5.2, 4.7 (pH
CaCh (0.01 M) Adjusted with CaO)
Hydroponics 6.1
CEC:
Soils 14.7, 27.1, 8.7, 4.2, 7.6, 41.7
(cmolc/kg soil)
Hydroponics N/A
PC:
Soils 14, 31, 10, 15, 21, 310 (gC/kg
soil)
Hydroponics N/A
Aqinq/leachinq:
Soils. Leached by immersion and
draining after 1 wk incubation. Three
1-wk periods of moist incubation
separated by 1-wk periods of dry
storage and one dry storage period
of up to 20 wk
Soils Measured:
6 levels * 6
soils = 36
values between
47 and
12,700 mg/kg,
plus 1
control x 6 soils
with background
Pb between 4.7
and
135 mg Pb/kg
soil
Hydroponics 1,
3.2, 10, 32, 100,
320 mM
Soil
(location of
origin)
Soil P
content 44,
48, 67, 89,
90,
121 mg P/kg
soil
Hydroponics
Gradually
increasing P
supply for
17 days to
maintain
growth rate
and avoid
precipitation
AND
7 levels of P
supply
based on P
content in
plant tissue
(0.10-
0.32% P in
plant tissue)
Tomato
growth:
decreasing
with
increasing
Pb in all soils
Barley
growth: no
effect of Pb
in three soils,
decreasing
with
increasing
Pb in three
soils
Tomato shoot
dry weight
NOEC for six
soils:
4,400, 750,
<250, 440, 260,
1,100 mg Pb/kg
soil
ECso: 6,000,
6,500, 2,200,
2,700, 1,600,
5,400 mg Pb/kg
soil
Barley shoot dry
weight NOEC for
six soils: >7,200,
>5,000, 2,000,
>3,400, 260,
1,100 mg Pb/kg
soil
ECso: >7,200,
>5,000, 4,900,
>3,400, 1,900,
8,300 mg Pb/kg
soil
Strong
interaction
effect of soil
type and Pb
concentration
on growth
P content in
plants was
strongly
influenced by
Pb and
explained the
effect of Pb
across soils and
in hydroponic
experiment
Chevns et
al. (2012)
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Organism
Experimental conditions
Pb
concentrations
Study
factors
other than
Pb
exposure
Effects of
Pb
Effect
concentration
Effects of
additional Reference
study factors
Hydroponics N/A
Potworm
(Enchytraeus
crypticus)
Form of
Pb:
Pb(N03)2
PbO
Medium:
LUFA2.2
standard
soil
Exposure
method:
Soil spiked
with
powdered
salt
£tL
Nominal pH 5.49
CEC:
9.10 cmolc/kg
PC:
not reported
Aaina/leachina:
Spiked soils were aged for 0, 3,
and 18 mo. No leaching
6, 12
Nominal Aging and
concentrations chemical
of: form of Pb
Pb(N03)2
0, 50, 100, 200,
400, 600, 800,
1600 and
3200 mg Pb/kg
dry soil
PbO
0, 78, 156, 312,
625, 1250,
2500, 5000 and
10000 mg Pb/kg
dry soil
E. crypticus
mortality
increased
with
increasing
Pb soil
concentration
Pb(N03)2:
CaCI2
extractable Pb
0, 3, 6, 12, 18-
mo LCso = 2.18,
3.06, 2.49, 2.28,
1.72 mg Pb/kg
ECso = 0.149,
0.125, 0.090,
0.103,
0.093 mg Pb/kg
Pore water Pb
0, 3, 6, 12, 18-
mo
LCso = 0.247,
0.346, 0.328,
0.366,
0.583 mg Pb/L
ECso = 0.020,
0.016, 0.019,
0.015,
0.046 mg Pb/L
The dose- (Zhang
response and Van
curves and Gestel.
toxicity values 2019a)
(LCso and ECso)
based on total
Pb
concentrations
differed widely
between the
two forms of Pb
Pb(N03)2 was
more toxic than
PbO in freshly
spiked soils, but
the toxicity of
PbO increased
with aging,
while the
toxicity of
Pb(N03)2
remained
constant
Internal Pb
0, 3, 6, 12, 18-
mo LCso = 76.2,
76.4, 77.1, 73.4,
76.8 mg Pb/kg
dry body weight
ECso = 22.2,
24.7, 30.0, 31.5,
CaCh-
extraction
provided the
best estimate of
Pb toxicity and
bioaccumulation
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Organism
Experimental conditions
Pb
concentrations
Study
factors
other than
Pb
exposure
Effects of
Pb
Effect
concentration
Effects of
additional
study factors
Reference
20.1 mg Pb/kg
dry body weight
PbO:
CaCI2
extractable Pb
0, 3, 6, 12, 18-
mo
LCso = 3.02,
3.15, 2.36, 2.66,
2.45 mg Pb/kg
ECso = 0.170,
0.135, 0.098,
0.138,
0.101 mg Pb/kg
Pore water Pb
0, 3, 6, 12, 18-
mo
LCso = 0.262,
0.312, 0.286,
0.302,
0.391 mg Pb/L
ECso = 0.025,
0.048, 0.050,
0.023,
0.048 mg Pb/L
Internal Pb
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Organism
Experimental conditions
Pb
concentrations
Study
factors
other than
Pb
exposure
Effects of
Pb
Effect
concentration
Effects of
additional
study factors
Reference
0, 3, 6, 12, 18-
mo LCso = 78.0,
77.7, 74.4, 78.4,
78.7 mg Pb/kg
dry body weight
ECso = 23.7,
18.1, 19.8, 16.9,
12.0 mg Pb/kg
dry body weight
Potworm
Form of
jdH:
Nominal
Percolation,
E. Crypticus
PbfNOab:
When exposure
(Zhana
(Enchytraeus
Pb:
Aged Soil:
concentrations
chemical
mortality
CaCb-
was measured
and Van
crypticus)
Pb(N03)2
of:
form of Pb
increased
extractable Pb
as total soil Pb,
Gestel,
and aging.
with
aged,
aged+leached,
aging increased
2019b)
PbO
p H pw. 5.61
Pb(NOs)
increasing
Pb soil
toxicity for both
forms of Pb and
Medium:
pHcace: 5.14
0, 50, 100, 200,
400, 600, 800,
concentration
freshly spiked
and freshly
spiked+leached.
leaching had no
meaningful
effect. However,
LUFA2.2
standard
soil
Freshy Spiked:
1600 and
all effects of
3200 mg Pb/kg
LCso = 1.72,
form, aging or
pHpw: 5.93
dry soil
2.42, 2.07 and
2.78 mg Pb/kg
leaching
disappeared
Exposure
method:
pHcace: 5.65
PbO
ECso = 0.093,
when exposure
was measured
as CaCh-
Soil spiked
with
powdered
salt.
CEC:
not reported
0, 78, 156, 312,
625, 1250,
2500, 5000 and
0.173, 0.044 and
0.109 mg Pb/kg
extractable Pb
OC:
1000 mg Pb/kg
dry soil
Pore water Pb
aged,
not reported
aged+leached,
freshly spiked
and freshly
Aaina/leachina:
spiked+leached.
LCso = 0.583,
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Organism
Experimental conditions
Pb
concentrations
Study
factors
other than
Pb
exposure
Effects of
Pb
Effect
concentration
Effects of
additional
study factors
Reference
One soil form was spiked and then
aged for 18 mo, while the other soil
form was used without aging as
freshly spiked soil
Half of each set of soils were
leached with deionized water equal
to two times the base moisture
content
0.201, 0.686 and
0.148 mg Pb/L
ECso = 0.046,
0.063, 0.012 and
0.033 mg Pb/L
Internal Pb
aged+leached,
freshly spiked
and freshly
spiked+leached.
LCso = 76.8,
84.4, 77.3 and
83.6 mg Pb/kg
dry body weight
ECso = 20.1,
22.1, 25.5 and
32.7 mg Pb/kg
dry body weight
PbO:
CaCI2
extractable Pb
aged,
aged+leached,
freshly spiked
and freshly
spiked+leached
LCso = 2.45,
2.01, 2.79 and
2.16 mg Pb/kg
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Organism
Experimental conditions
Pb
concentrations
Study
factors
other than
Pb
exposure
Effects of
Pb
Effect
concentration
Effects of
additional
study factors
Reference
ECso = 0.101,
0.160, 0.123 and
0.168 mg Pb/kg
Pore water Pb
aged,
aged+leached,
freshly spiked
and freshly
spiked+leached.
LCso = 0.391,
0.233, 0.197 and
0.097 mg Pb/L
ECso = 0.048,
0.043, 0.047 and
0.031 mg Pb/L
Internal Pb
aged,
aged+leached,
freshly spiked
and freshly
spiked+leached.
LCso = 78.7,
76.4, 83.3 and
84.5 mg Pb/kg
dry body weight
ECso = 12.0,
14.5, 41.1 and
38.6 mg Pb/kg
dry body weight
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Organism
Experimental conditions
Pb
concentrations
Study
factors
other than
Pb
exposure
Effects of
Pb
Effect
concentration
Effects of
additional
study factors
Reference
Potworm
(Enchytraeus
crypticus)
Form of
Pb:
Pb(N03)2
Medium:
LUFA2.2
standard
soil
Exposure
method:
Soil spiked
with
aqueous
salt solution
jdH:
Nominal pH of 5.49
CEC:
9.10 cmolc/kg
PC:
not reported
Aqinq/leachinq:
After 14-d exposure in spiked soils,
the surviving E. crypticus were
transferred to clean soil for the 14-d
elimination phase
Nominal
concentrations
of 0, 100, 200,
400, 800, 1600
and
3200 mg Pb/kg
dry soil
Measured
Concentrations
of 16, 114, 202,
391, 793, 1,601
and
3,585 mg Pb/kg
dry soil
Exposure
duration
Toxicity was
dependent
on both the
concentration
and duration
of exposure.
Pb toxicity
developed
more slowly
than uptake,
with final
LCso not yet
reached after
21 d
Days 4, 7, 10, 14 Strong
and 21
Total
(Zhang
and Van
concentration in
soil:
LCso = 2,336,
2278, 1,220, 756
and
558 mg Pb/kg
dry soil
Internal
concentration:
LCso = >287,
>270, 161, 76.6
and
76.4 mg Pb/kg
dry body weight
interaction
effect of Gestel.
duration and Pb 2017)
concentration
on mortality
Potworm
(Enchytraeus
criticus)
Form of
Pb:
Pb(N03)2
Medium:
Five
standard
soils (LUFA
standard
soil 2.1, 2.2,
2.3, 2.4,
5 M) and
one soil
from a
soccer field
EhL
4.86,
5.66, 5.38, 6.87, 6.99, 6.85
CEC:
2.23, 7.59, 4.04, 20.1, 10.1 and
20.0 cmolc/kg
PC:
DOC
45.7, 61.7, 34.4, 72.0, 51.2 and
189 mg/L
Aqinq/leachinq:
Nominal
concentrations
of 0, 100, 200,
400, 600, 800,
1200, 1600,
2400 and
3200 mg Pb/kg
dry soil
Soil type,
soil
properties:
OM, DOC,
PH, CEC,
water-
holding
capacity,
composition
Reproductive
toxicity and
mortality
increased
with Pb
concentration
in soil
LUFA standard
soil 2.1, 2.2, 2.3,
2.4, 5 M and
soccer field,
respectively.
Total Pb:
LCso = 246,
1,192, 655,
3,125, 2,875 and
>3,092 mg Pb/kg
dry soil
ECso = 81.4,
238, 205, 948,
Correlation of
single soil
properties with
endpoints,
followed by
simple
regression,
followed by
stepwise
multiple
regression
suggested that
pHcaci2 was the
best
explanatory
factor for LCso
(Zhang et
al.. 2019a)
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Organism
Experimental conditions
Pb
concentrations
Study
factors
other than
Pb
exposure
Effects of
Pb
Effect
concentration
Effects of
additional
study factors
Reference
in the Soil equilibrated for 14-d.
Netherlands
Exposure
method:
Soils spiked
with
aqueous
solution
1,008 and
991 mg Pb/kg
dry soil
CaCI2
extractable Pb:
LCso = 2.35,
2.11, 1.86, 1.64,
2.11 and
>1.39 mg Pb/kg
dry soil
ECso = 0.329,
0.193, 0.107,
0.180, 0.241 and
0.115 mg Pb/kg
dry soil
values based
on total Pb
concentration
The differences
between soil
toxicity were not
present when
exposure was
measured as
CaCh-
extractable Pb
concentration
Porewater Pb:
LCso = 0.308,
1.25, 0.335,
0.334, 0.933 and
>0.754 mg Pb/L
ECso = 0.044,
0.127, 0.117,
0.169, 0.046 and
0.105 mg Pb/L
Internal Pb:
LCso = 95.7,
83.0, 87.0, 84.3,
81.7 and
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Organism
Experimental conditions
Pb
concentrations
Study
factors
other than
Pb
exposure
Effects of
Pb
Effect
concentration
Effects of
additional
study factors
Reference
>47.7 mg Pb/kg
dry body weight
Tomato Form of pH:
(Lycopersicon Pb: 6 1-74
esculentum) pbCh
Barley
(Hordeum
vulgare)
Collembola
(Folsomia
Candida)
Earthworm
(Eisenia
fetida)
CEC:
Pb(NC>3)2 8.2-27.1 cmolc/kg soil
Medium:
Soils
gathered
from
topsoils in
Spain, the
United
Kingdom
and Belgium
Exposure
method:
Soil spiked
with salt
PC:
10-43 g C/kg soil
Aqinq/leachinq:
Soils were given three different
treatments. Treatment A: freshly
spiked. Treatment B: leached and
pH-corrected. Treatment C: leached,
pH-corrected and aged for 5 yr
ECso = 13.6,
34.1, 26.0, 39.9,
27.2 and
32.6 mg Pb/kg
dry body weight
Nominal
Leaching
All effects
ECsos calculated
Strong
Smolders
concentrations
combined
increased
for tomato
interaction
et al.
of, 250, 500,
with pH
with
growth, barley
effect of
(2015)
1,000, 2,000,
correction,
increasing
growth,
leaching, aging
4,000 and
aging
Pb in freshly
nitrification rate,
and Pb
8,000 mg Pb/kg
combined
spiked
nitrification 28-d,
concentration
with
(unaged,
respiration, E.
on all
leaching
unleached)
Fetida
responses.
and pH
soils
reproduction and
Leaching
correction
F. Candida
combined with
reproduction in
pH correction
each soil,
decreased
respectively.
toxicity for all
Spain:
Freshly spiked
ECso = 2,900,
2,380, 3,240,
7,190, 8,720,
480 and
712 mg Pb/kg
soil
Leached and
pH-corrected
ECso = 6,370,
7,190, 2,200,
effects. Aging
following
leaching and pH
correction
further
decreased
toxicity for most
effects but not
all. Authors
suggest
decreased ionic
strength (salt
stress) and
changes in pH
are the main
drivers of
External Review Draft
11-91
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-------
Organism
Experimental conditions
Pb
concentrations
Study
factors
other than
Pb
exposure
Effects of
Pb
Effect
concentration
Effects of
additional Reference
study factors
7,120, 12,300,
1,182 and n.s.
mg Pb/kg soil
decreasing
toxicity
Aged 5 yr
ECso = 12,600,
n.s, n.s, n.s,
7,020, 1,270 and
n.s. mg Pb/kg
soil
United Kingdom:
Freshly spiked
ECso = 6,140,
6,750, 2,820,
1,750, 9,970,
2,400 and
4,530 mg Pb/kg
soil
Leached and
pH-corrected
ECso = 6,420,
5,020, 4,920,
n.s., 6,160,
1,700 and
5,020 mg Pb/kg
soil
Aged 5 yr
ECso = n.s., n.s.,
n.s., n.s., n.s.,
3280 and n.s.
mg Pb/kg soil
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Organism
Experimental conditions
Pb
concentrations
Study
factors
other than
Pb
exposure
Effects of
Pb
Effect
concentration
Effects of
additional
study factors
Reference
Belgium:
(no test for E.
fetida)
Freshly spiked
ECso = 1,240,
1,710, 1,470,
1,410, 1,680 and
1,710 mg Pb/kg
soil
Leached and
pH-corrected
ECso = 1,430,
4,580, 1,640,
2,820, 8,150 and
2,700 mg Pb/kg
soil
Aged 5 yr
ECso = 4480,
n.s., n.s., n.s.,
n.s. and n.s.
mg Pb/kg soil
Lettuce
Form of
Reported for soils H1-H7
Nominal
Soil type
All effects
Reported for
Strong
Romero-
(Lactuca
Pb:
respectively
concentrations
(location of
increased
soils H1-H7,
interaction
Freire et
sativa)
Pb(N03)2
of 500, 1000,
origin)
with
respectively
effect of soil
al. (2015)
jdH:
2000, 4000 and
increasing
type and Pb
8000 mg Pb/kg
Pb in all soils
L. sativa'.
concentration
Bacterium
Medium:
7.96, 8.67, 8.79, 6.74, 7.20, 5.87 and
soil
on all
(Vibrio
Seven soils
7.03
EC10 = 499,
responses.
fischeri)
representing
1,363, 254,
Authors suggest
the main
CEC:
1,097, 3,452,
that the main
498 and
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Study
Organism
Experimental conditions
p. factors
concentrations otheprbthan
exposure
Effects of
Pb
Effect
concentration
Effects of
additional Reference
study factors
soil groups
21.4, 9.83, 2.94, 9.91, 25.9, 3.83 and
344 mg Pb/kg
soil properties
in Spain
15.5 cmolc/kg
soil
that affected
toxicity were
pH, carbonate
content and OC
Exposure
OC:
V. fischerr.
method:
5.43, 0.42, 0.38, 0.61, 8.22, 0.49 and
EC10 = >8,000,
Spiked with
0.66%
5,337, 2,901,
aqueous
386, 2,473, 8
solution.
Aaina/leachina:
Soils were incubated for 4 wk after
and
744 mg Pb/kg
spiking
Soil Respiration:
EC10 = >8,000,
3,128, 5,951, 90,
>8,000, 122 and
45 mg Pb/kg
CaCI2 = calcium chloride; CaO = calcium oxide; CEC = cation exchange capacity; DOC = dissolved organic carbon; EC50 = 50% effect concentration; LC50 = 50% lethal concentration;
LUFA = Landwirtschaftliche Untersuchungs- und Forschungsanstalt; mo = months; N/A = not available; NOEC = no observed effect concentration; n.s. = nonsignificant. OC = organic
carbon; OM = organic matter; P = lead; Pb = lead; Pb(N03)2 = lead nitrate; PbCI2 = lead chloride; PbO = lead(ll) oxide; pHpw = pH of porewater; pHCaci2 = pH via calcium chloride;
wk = weeks; yr = years
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11.2.6. Terrestrial-Community and Ecosystem Effects
In the 2013 Pb IS A the body of evidence was sufficient to conclude there is a likely causal
relationship between Pb exposure and terrestrial-community and ecosystem effects (U.S. EPA. 2013). In
the 2006 Pb AQCD (U.S. EPA. 2006a'). terrestrial ecosystems near stationary Pb sources exhibited
decreased species diversity, changes in floral and faunal composition, and a reduction in vegetation
fitness. In the 2013 Pb ISA (U.S. EPA. 2013). a study reported decreased population growth of
earthworms. Additional studies in the 2013 Pb ISA examined how the presence of AMF or earthworms
affect plant Pb uptake and fitness. Recent evidence of the effects of Pb at the community and ecosystem
levels include several studies of the relationship between Pb soil concentration and species interactions
and invertebrate community structure. Specifically, studies conducted since the 2013 Pb ISA have
reported that Pb affects plant-insect interactions and is correlated with invertebrate community structure.
In an experimental study, Jiang et al. (2020) demonstrated trophic transfer of Pb can affect the
chemical defenses of larch seedlings (Larix olgensis) against an economically important pest, the Asian
gypsy moth (Lymantria dispar), in China. Larch seedlings were enriched with Pb at 0, 500, or
1500 mg Pb/kg. Second instar L. dispar larvae raised from field-collected egg masses were placed onZ.
olgensis seedlings for 7 days. Pb content in L. dispar larvae were significantly higher than L. olgensis
needles for the 500 mg Pb/kg and 1500 mg Pb/kg treatments, and Pb bioaccumulated in this experiment,
as the transfer coefficients were 0.97 for the 0 mg Pb/kg treatment, 5.43 for the 500 mg Pb/kg treatment
and 6.03 for the 1500 mg Pb/kg treatment. Pb treatment reduced L. olgensis total biomass (40.36%
reduction in the 1500 mg Pb/kg compared with control) and L. dispar larval weights (by 34.44-52.05%)
and survival rates (by 30.91-59.28%) in a dose-dependent manner compared with the control.
Antioxidants (peroxidase and SOD) of L. olgensis increased under 500 mg Pb/kg treatment and were
reduced under 1500 mg Pb/kg. Phytochemical defenses, protease inhibitors (trypsin inhibitor and
chymotrypsin inhibitor) and the secondary metabolites (total phenolic acids) were significantly increased
under the low dose of Pb (500 mg Pb/kg) compared with the control, while all phytochemical defense
chemicals, including condensed tannins, decreased significantly under high Pb stress (1500 mg Pb/kg).
Lymantria. dispar fed with L. olgensis seedlings had higher antioxidase activities in the fourth instar
(SOD and CAT), while nonenzymatic antioxidants were significantly decreased (glutathione content and
ascorbic acid content), suggesting that the reduction of antioxidants might lead to the oxidative stress
experienced by L. dispar larvae. Finally, MDA content increased with Pb exposure.
Heavy-metal concentration along a pollution gradient in Romania affected soil mite (Acari:
Mesostigmata) community structure (Manu et al.. 2019; Manu et al.. 2017). Manu et al. (2017) examined
soil mite communities in relation to soil metal content and physicochemical properties in 12 grasslands.
Some heavy metals (Pb, As, Cu and Zn) influenced the soil mite community in highly polluted sites,
while altitude and soil humidity played larger roles in less polluted sites. Pb soil concentration ranged
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from 28.21 ± 4.62 mg/kb Pb to 421.12 ± 71.62 mg/kb Pb. The sites with the highest Pb were closest to the
pollution source. Canonical correspondence analysis (CCA) determined that heavy metals (Cu, Zn and
Pb) as well as the C/N ratio, humidity, total N, altitude, and slope were the strongest determinants of
species composition, and Pb soil concentration showed association with the abundance of Zercon
berlesei. In another study, Manu et al. (2019) collected soil from a pollution gradient surrounding the
Certej ore deposit and characterized heavy-metal concentration and soil mite communities. Pb
concentrations ranged from 153.68 to 292.35 mg Pb/kg across five sites (mean concentration). The
relationship between mite abundance and heavy metals was examined using CCA, and the first axis
accounted for 50.67% of the variation in mite community and was highly correlated with Pb
(correlation = 0.81), Cu, As and Mn. The abundance of Arctoseius cetratus showed the strongest
relationship with Pb.
Potworm (Enchytraeidae) diversity, but not herbaceous plant diversity, was negatively correlated
with soil Pb concentration across 41 sites near a Zn-Pb mining site in South Poland (Kapusta and
Sobczvk. 2015). Pb soil concentration varied across sites, ranging from 300 ± 300 mg Pb/kg
(mean ± S.D.) to 9,600 ± 14,100 mg Pb/kg at sites closer to the smelter, and water-soluble Pb showed a
similar pattern, with higher Pb concentrations found closer to the smelter site (range:
0.103 ± 0.068 mg Pb/kg to 0.477 ± 0.212 mg Pb/kg Pb). Pb concentration was positively correlated with
silt content, OC, total Cd, total Zn, exchangeable Cd, water-soluble Cd, and water-soluble Zn and
negatively correlated with distance from the smelter. Water-soluble Pb was positively correlated with
distance from the smelter, OC, and water-soluble Zn. Total Pb was significantly negatively correlated
with Enchytraeid species richness, genus richness, and density in 2010, but not density in 2009, while
water-soluble Pb showed no significant relationships with species richness, genus richness, or density in
2009 or 2010. Plant community species richness and herbaceous cover showed no correlation with total
Pb in the soil or water-soluble Pb.
The abundance of insects on Pb-contaminated kale (Brassica oleracea L. var. acephala) was
higher than control B. oleracea plants in a field experiment in Brazil (Moralcs-Silva et al.. 2022).
Brassica oleracea plants were grown in control soil (background Pb concentration: 25.9 mg Pb/kg) or in
soil spiked with Pb(NC>3)2 to nominal concentrations of 144, 360, or 600 mg Pb/kg and exposed to natural
insect populations. Lepidoptera and their associated parasitoids, as well as aphids and their predators and
parasitoids, were collected from plants. At the end of the experiment, plant biomass was unaffected by Pb
soil contamination, while plants exposed to 600 mg Pb/kg had significantly higher concentrations of Pb in
the leaves compared with plants in the control, 144, and 360 mg Pb/kg treatments. Brassica oleracea
plants in the control treatment had significantly higher abundance of insects compared with the
contaminated plants, regardless of Pb level.
Longer-lived nematodes with lower fecundity are most affected by experimental Pb exposure
(Park et al.. 2016). Tomatoes (Lycopersicon esculentum) were grown in pots of soil collected from an
agricultural field in Korea and exposed to Pb via irrigation. Measured Pb concentrations of the soil were
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16.97 ± 0.24 mg Pb/kg (mean ± S.D) for the control soil, 15.19 ± 0.55 mg Pb/kg, 15.54 ± 0.42 mg Pb/kg,
18.08 ± 0.67 mg Pb/kg and 34.98 ± 2.57 mg Pb/kg. Soil nematode communities were characterized before
L. esculentum were planted and after 18 weeks of growth. Nematode community structure was analyzed
using a variety of metrics, from trophic guilds to maturity indices to the abundance of colonizers and
persister (cp-1 = colonizer to cp-5 = persister). Pearson's correlation coefficients between Pb and
nematode community indices were largely nonsignificant, except for the negative relationship between Pb
and the richness of cp-3 as well as the maturity index and the positive relationship between Pb and the
abundance of fungivores as well as the abundance of cp-2. There was a significant decrease in nematode
abundance in omnivores-predators (OP) and cp-4 at the highest concentrations of Pb. Nematode richness
decreased at higher concentrations of Pb, particularly for OP, cp-4, and cp-5. The authors suggested that
these groups are likely most sensitive to environmental stress, as they have longer-life cycles and lower
reproduction rates.
In another nematode study, the diversity and abundance of nematode communities were
correlated with soil Pb concentration near a ferroalloy manufacturer in North Slovakia (Salamun ct al..
2011). Soil samples near the factory and downwind of the factory were analyzed for heavy metals,
including Pb. The total Pb concentration ranged from 0.815 ± 0.471 mg Pb/kg to 1.766 ± 0.082 mg Pb/kg
(mean ± S.D). Soil Pb concentration was positively correlated with the abundance of certain trophic
guilds and ecological indices of nematodes, specifically, predators, root-fungal feeders, and maturity
index (MI2-5). Maturity index (2-5) is used as a measure of functional diversity, which incorporates the
abundance of r and /^-strategists in a community. Pb was not significantly correlated with any other
trophic group or ecological index (bacterial feeders, fungal feeders, omnivores, plant feeders, maturity
index, plant-parasite index, genera richness, Shannon-Weaver index, Simpson index or abundance). In a
follow-up study, Salamun et al. (2012) examined nematode community structure in relation to the total
element concentration of Pb, Zn, Cu Cr, Ca, and As, in another region of Slovakia using an HNO3
extraction and mobilization fraction Na2EDTA extraction. Unlike Salamun et al. (2011). in which Pb was
positively correlated with certain trophic groups, total soil Pb concentration was negatively correlated
with the abundance of omnivorous nematodes, MI2-5, structure index and genera richness.
Since the 2013 Pb ISA (U.S. EPA. 2013). several studies have found evidence that Pb affects
species interactions, including chemical defenses (Jiang et al.. 2020) and pollinator foraging behavior
(Xun et al.. 2018). Additionally, several studies found negative relationships between Pb concentration
along a pollution gradient and aspects of the invertebrate community structure, specifically in soil mites
(Manu et al.. 2019; Manu et al.. 2017). potworms (Kapustaand Sobczvk. 2015). insect communities on
kale (Morales-Silva et al.. 2022). and nematodes (Salamun etal.. 2011).
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11.3
Freshwater Ecosystems
11.3.1. Summary of New Information on Effects of Pb in Freshwater
Ecosystems and Causality Determination Update Since the 2013 Pb ISA
Recent evidence further supports the findings of the previous Pb AQCDs and 2013 Pb ISA that
waterborne Pb is toxic to freshwater plants, invertebrates, and vertebrates, with toxicity varying with
species and lifestage, duration of exposure, form of Pb, and water quality characteristics (U.S. EPA. 2013.
2006a. 1986. 1977). The majority of the available studies of Pb exposures in freshwater biota are
laboratory toxicity tests on single species in which an organism is exposed to a known concentration of
Pb, and the effect on a specific endpoint is evaluated. These studies provide evidence for a temporal
sequence between Pb exposure and an effect, an aspect important in judging causality. Concentration-
response data from freshwater organisms indicate that there is a gradient of response to increasing Pb
concentration and that some effects in sensitive species are observed at or near the upper limit of Pb
concentrations quantified in U.S. surface waters (Table 11-1). New evidence for freshwater biota (Table
11-5) continue to support the existing causality determinations from the 2013 Pb ISA summarized in
Table 11-4 of this document. In most cases, new evidence expands somewhat the evidence for endpoints
that were already established as causal in the 2013 Pb ISA. Some studies have reported effects at lower
effect concentration than in the 2013 Pb ISA. There are no changes to existing causality
determinations for freshwater biota or ecosystems from the 2013 Pb ISA (Table 11-4).
For physiological stress endpoints in freshwater plants, invertebrates, and vertebrates, new
evidence continues to support the likely to be causal determination from the 2013 Pb ISA. A small subset
of studies that report molecular or cellular perturbations of Pb concurrently assess an effect on
reproduction, growth, or survival. Few studies were identified since the 2013 Pb ISA that quantified
ALAD response in freshwater invertebrates or vertebrates; hence there is not sufficient evidence to
warrant a reconsideration of any of the causality relationships for the hematological effects of Pb.
Neurobehavioral effects of Pb were concluded to have a likely to be causal relationship for Pb
exposure for freshwater invertebrates and vertebrates in the 2013 Pb ISA. For invertebrates, a few new
studies in amphipods, bivalves and gastropods further support the 2013 finding of a likely to be causal
relationship between Pb exposure and neurobehavioral endpoints (Section 11.3.3). Effects on locomotion
were observed in adult amphipods, G. fossarum, following Pb sublethal exposure (analytically verified
concentrations were 2.1 and 2.7 (ig Pb/L in two separate studies, one conducted for 24 hours, another
conducted for 5 days) (Lebrun and Gismondi. 2020; Lebrun et al.. 2017). Alteration of neurotransmitter
(AChE) activity was reported for two freshwater bivalve species including P. corrugata, in which AChE
activity was significantly induced at 26 |ig Pb/L in 21-day aqueous exposure. Impaired foot movement
was also observed in this species at a similar concentration (Brahma and Gupta. 2020). AChE activity was
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significantly induced in the freshwater snail B. aeruginosa during 28-day exposure to Pb-spiked sediment
(29.7 mg Pb/kg dry weight) (Liu et al.. 201%).
The 2013 conclusion of a likely to be causal relationship between Pb exposure and
neurobehavioral effects in freshwater vertebrates is bolstered in this current ISA by multiple studies with
zebrafish (D. rerio) as an animal model for human health effects including developmental and
neurological changes associated with Pb exposure (Section 11.3.4.4). Effects on behavioral endpoints
such as locomotion and social interactions in larval zebrafish were reported at lower effect concentrations
than studies in the 2013 Pb ISA, with some effects reported at < 20 |ig Pb/L; a subset of these studies
analytically verified Pb in the exposure water (Kataba et al.. 2020; Zhao et al.. 2020; Wang et al.. 2018b;
Zhu et al.. 2016). Neurological responses of fish to Pb exposure were first reported in the 1986 Pb AQCD
(U.S. EPA. 1986). The likely to be causal determination in the 2013 Pb ISA was based primarily on
altered behaviors, such as reduced locomotion and prey capture ability, observed in fish following Pb
exposure. These included a decrease in zebrafish larval startle response to mechanosensory and visual
stimuli following nominal exposure to Pb (2.0 and 6.0 |ig Pb/L) (Rice etal.. 2011). and reduced prey
capture in assays with 10-day old fathead minnows born from adult fish exposed to 120 |ig Pb/L for
300 days then subsequently tested in a breeding assay for 21 days (Mageretal.. 2010). In another study in
the 2013 Pb ISA with fathead minnows, swimming performance measured as critical aerobic swim speed
was significantly impaired in minnows in 24-hour acute (139 (ig Pb/L) and chronic 33 to 57-day
(143 |ig Pb/L) exposures; however, no significant difference in swim speed was observed in chronic
exposures to 33 |ig Pb/L (Mager and Grosell. 2011). The evidence in the 2013 Pb ISA and previous
AQCDs also included effects on molecular targets; however, these experiments were typically conducted
at Pb concentrations that greatly exceeds environmental concentrations.
In the 2013 Pb ISA, there was a conclusion of a causal relationship between Pb exposure and
reduced survival in both freshwater invertebrates and vertebrates. Newly available evidence continues to
support these causal determinations. For invertebrates, several studies provide further characterization for
known effects on survival in a few sensitive species of freshwater invertebrates at <20 |ig Pb/L (Section
11.3.5). In the gastropod L. stagnalis, survival was significantly decreased at 8.4 |ig Pb/L after 21-day
exposure to the end of a 56-day full life cycle assessment (Munlcv et al.. 2013). In a chronic 42-day
bioassay with the amphipod H. azteca, the EC20 for survival was similar under two different experimental
diets administered concurrently (LC20 =15 fxg Pb/L and LC20 =13 jxg Pb/L) (Besser et al.. 2016). For
freshwater vertebrates, studies in fish provided the basis for causality determination in the 2013 Pb ISA
(Section 11.3.5). Additional fish bioassays conducted in varying water chemistry conditions report effects
on survival at Pb concentrations similar to those reported in the 2013 Pb ISA. For larval zebrafish (D.
rerio), 96-hour LC50 values varied with water hardness; LC50 = 52.9 |ig Pb/L in soft water and
LC50 = >590 |ig Pb/L in hard water (Alsop and Wood. 2011). Several studies considered the role of Pb
and other trace metals on the decline of the white sturgeon in U.S. waters, and one study examined
endpoints in westslope cutthroat trout. In 96-hour acute toxicity assays conducted with two lifestages of
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white sturgeon (A. transmontanus), the lowest 96-hour LC50 was 111 |ig Pb/L for 8 dph larvae (Vardv ct
al.. 2014).
For growth effects in freshwater organisms associated with Pb exposure, recent studies continue
to support the findings in the 2013 Pb ISA. There was a likely to be causal relationship between Pb
exposure and reduced plant growth concluded in the 2013 Pb ISA. Most primary producers experience
EC50 values for growth at concentrations that greatly exceed Pb concentrations typically found in U.S.
surface waters. One new study reported growth rates in three commonly tested algal species (P.
subcapitata, C. kesslerii, and C. reinhardtii) at lower effect concentrations than previously reported. P.
subcapitata was the most sensitive in 72-hour bioassays, with an EC50 = 83.9 |ig Pb/L,
EC20 = 45.7 |ig Pb/L and EC10 = 32.0 |ig Pb/L based on filtered Pb concentration. Varying the pH resulted
in greater sensitivity (Dc Schamphelaere et al.. 2014). In the 2013 Pb ISA, there was a causal relationship
concluded to exist between Pb exposure and reduced growth in invertebrates. Since then, additional
studies have supported previous findings of Pb effects on the growth of snails (L. stagnalis) in the
low |ig Pb/L range (Cremazy, 2018, 6708984} (Munlcv et al.. 2013; Brix et al.. 2012; Esbaugh ct al..
2012). Reduction in weight gain and specific growth rate were observed in juvenile Oriental river prawn
(M nipponense) exposed to 25 |ig Pb/L in chronic 60-day trials. No growth effects were observed in
prawns at 12 (ig Pb/L (Ding et al.. 2019). The evidence remains inadequate to infer a causality
relationship for Pb exposure and reduced growth in freshwater vertebrates. One study reported a threshold
of 160 |ig Pb/L for tadpole growth in dark-spotted frogs (P. nigromaculata) (Huang et al.. 2014).
Reproductive and developmental effects were concluded to be causally related to Pb exposure for
freshwater invertebrates in the 2013 Pb ISA. This remains the case in newer studies. Recent evidence
further supports previous observations of Pb effects on reproductive endpoints at low |ig/L concentrations
in sensitive species of gastropods, cladocerans and rotifers, especially under chronic exposure scenarios
(Section 11.3.5) (see Table 11-5). In L. stagnalis, a gastropod known to be sensitive to Pb at low |ig Pb/L
concentration, NOEC<1.0 |ig Pb/L and LOEC =1.0 jxg Pb/L were determined for the number of egg
masses and time until the first egg mass in a 56-day life cycle bioassay (Munlev et al.. 2013). In this
species, the egg capsule and embryo diameter were significantly reduced after 7 days of development at
2.7 (ig Pb/L (the highest concentration in which reproduction was observed in the study). For the
cladoceran C. dubia, 7-day EC20 values for reproduction ranged from 12 to 223 |ig Pb/L in assays
conducted in a variety of natural waters across the United States with different water chemistries; 7-day-
EC50 values ranged from 20 to 573 |ig Pb/L in the same test waters (Esbaugh et al.. 2012). Using the same
sampled waters from across the United States, reproduction (as population growth) was also assessed in
rotifer P. rapida over a 4-day exposure period. Chronic EC20 and EC50 in this species ranged from 3 to
103 |ig Pb/L and from 10 to 154 (ig Pb/L, respectively.
Several studies in fish in which Pb concentration was analytically verified further support the
causal determination reported in the 2013 Pb ISA between Pb and reproductive and developmental effects
for freshwater vertebrates (Section 11.3.4.4). For example, hatching success rates in zebrafish embryos
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were reduced at 4.5, 9.6 and 18.6 |ig Pb/L aqueous exposure; at 72 hpf, the hatching success rates at all
three concentrations were significantly decreased compared with the control, indicating that Pb caused a
hatching delay. This effect persisted until the end of the experiment at 96 hpf (Zhao et al.. 2020).
Endocrine disruption (significant reduction in thyroid hormones T3 and T4) was observed in zebrafish
larvae following exposure to 30 (ig Pb/L, although there was no effect on the hatching success rate (Zhu
et al.. 2014). These studies at analytically verified concentration of Pb are bolstered by additional fish
studies conducted at nominal concentrations (Section 11.3.4.4.1) and several studies in amphibians
(Section 11.3.4.4.3).
In the 2013 Pb ISA, the body of evidence was sufficient to conclude there is a likely to be causal
relationship between Pb exposure and freshwater-community and ecosystem effects, and recent evidence
continues to support this finding (Section 11.3.6). Reductions in species abundance, richness or diversity
associated with the presence of Pb in freshwater habitats are reported in the literature, usually in heavily
contaminated sites where Pb (and other metal) concentrations are higher than typically observed
environmental concentrations. Most evidence is from sediment-associated macroinvertebrate
communities. Observational and experimental studies published since the 2013 Pb ISA continue to show
negative associations between sediment and/or porewater Pb concentration and macroinvertebrate
communities. The evidence is expanded somewhat with studies reporting associations with Pb and
periphyton abundance. Uptake of Pb into aquatic and terrestrial organisms and subsequent effects on
mortality, growth, developmental and reproduction at the organism level can cascade up to ecological
populations and communities and lead to ecosystem-level consequences, and thus provide consistency
and plausibility for causality in ecosystem-level effects. Although the evidence is strong for the effects of
Pb on growth, reproduction, and survival in certain species in experimental settings at or near the range of
Pb concentrations reported in surveys of U.S. freshwater systems, considerable uncertainties exist in
generalizing effects observed at a smaller scale, particular conditions up to predicted effects at the
ecosystem level of biological organization. In many cases, it is difficult to characterize the nature and
magnitude of effects and to quantify relationships between ambient freshwater concentrations of Pb and
ecosystem response due to the presence of multiple stressors, variability in field conditions and
differences in Pb bioavailability at that level of organization.
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Table 11-4 Summary of Pb causality determinations for freshwater plants,
invertebrates, and vertebrates
Level Effect Freshwater
2013 Pb ISA3
2023 PbISA
Community and Ecosystem
Community and Ecosystem Effects
Likely Causal
Likely Causal
Reproductive and Developmental
Effects - Plants
Inadequate
Inadequate
Reproductive and Developmental
Effects - Invertebrates
Causal
Causal
Population-
level
Reproductive and Developmental
Effects -Vertebrates
Causal
Causal
Growth - Plants
Likely Causal
Likely Causal
Endpoints
Organism-level
Growth - Invertebrates
Causal
Causal
Responses
Growth - Vertebrates
Inadequate
Inadequate
Survival - Plants
Inadequate
Inadequate
Survival - Invertebrates
Causal
Causal
Survival - Vertebrates
Causal
Causal
Neurobehavioral Effects -
Invertebrates
Likely Causal
Likely Causal
Neurobehavioral Effects - Vertebrates
Likely Causal
Likely Causal
Hematological Effects - Invertebrates
Likely Causal
Likely Causal
Hematological Effects - Vertebrates
Causal
Causal
Suborganismal
Responses
Physiological Stress - Plants
Likely Causal
Likely Causal
Physiological Stress - Invertebrates
Likely Causal
Likely Causal
Physiological Stress - Vertebrates
Likely Causal
Likely Causal
Conclusions were based on the weight of evidence framework for causal determination in Table II of the 2013 Pb ISA (U.S. EPA.
2013). Ecological effects observed at or near Pb concentrations measured in sediment and water in Table 6-2 of the 2013 Pb ISA
were emphasized and studies generally within one to two orders of magnitude above the reported range of these values were
considered in the body of evidence for freshwater (Section 6.4.12) (U.S. EPA. 2013).
1 Inputs of Pb into freshwater ecosystems include air-related sources and non-air sources
2 (Appendix 1: https://cfpub.epa.gov/ncea/isa/recordisplay.cfm?deid=357282).
3 Atmospherically derived Pb can enter aquatic systems through direct wet or dry deposition and erosional
4 transport or resuspension of Pb from terrestrial systems (Section 11.1.2). Receiving water bodies include
5 lakes (lentic systems) and rivers and streams (lotic systems). Freshwater wetlands, some of which may be
6 inundated occasionally or constantly, also provide habitat for aquatic biota. The focus of this section is on
7 Pb bioavailability, bioaccumulation, and the effects of Pb on freshwater organisms including algae,
8 aquatic plants, microbes, invertebrates, vertebrates, and other biota with an aquatic lifestage (e.g.,
9 amphibians).
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The following sections review the recent literature published since the 2013 Pb ISA on effects of
Pb on freshwater ecosystems. The new evidence is considered along with the ecological findings of
previous Pb assessments. The 2013 Pb ISA developed causality determinations for freshwater biota based
on the weight of evidence for Pb effects on specific endpoints and taxonomic groups (Table 11-4). In the
2013 Pb ISA, the body of evidence was sufficient to conclude that there was a causal relationship between
Pb exposure and reproductive and developmental effects in freshwater invertebrates and vertebrates,
reduced growth and survival of invertebrates, reduced survival of vertebrates, and hematological effects
in vertebrates. Relevant concentrations for causality judgments for the welfare effects of Pb in the 2013
Pb ISA were determined considering Pb concentrations "generally within one or two orders of magnitude
above those which have been observed in the environment and the available evidence for concentrations
at which effects were observed in plants, invertebrates, and vertebrates" (U.S. EPA. 2013). Of these
causal relationships concluded for freshwater ecosystems, effects on reproduction, growth, and survival in
sensitive freshwater invertebrates are well characterized from controlled studies at concentrations at or
near Pb concentrations occasionally encountered in U.S. fresh surface waters. The 2013 Pb ISA
concluded there is a likely to be causal relationship between Pb exposure and physiological stress in
freshwater biota. For hematological effects there was a likely to be causal relationship for freshwater
invertebrates. Effects on neurobehavioral endpoints were likely to be causal for freshwater invertebrates
and vertebrates. Pb effects on plant growth were likely to be causal and were only reported at relatively
high concentrations compared with effects on invertebrates. There was also a likely to be causal
relationship between Pb exposure and community and ecosystem-level effects. For all effects in
freshwater biota, the toxicity of Pb varied with species and lifestage, duration of exposure, form of Pb,
and water quality characteristics. Key uncertainties from the last review for freshwater ecosystems
included the uncertainties associated with generalization of effects observed in controlled laboratory
studies to conditions in streams, rivers, and lakes where many modifying factors affect Pb bioavailability
and toxicity. For example, there is a discrepancy between the sensitivity of aquatic insect taxa in
laboratory studies compared with longer-term field studies. In a meta-analysis of study findings, longer-
term studies suggest that aquatic insect taxa are more sensitive to metals than indicated in acute exposure
scenarios (Brix etal.. 2011). In aquatic ecosystems affected by Pb, exposures are most likely
characterized as low-dose, chronic exposures, whereas the majority of available toxicological data for this
metal is from acute laboratory exposures, typically conducted at higher concentrations. There are
considerable uncertainties associated with generalizing effects observed in controlled studies to effects at
higher levels of biological organization. Furthermore, available studies on community and ecosystem-
level effects are usually from contaminated areas where Pb concentrations are much higher than typically
encountered in the environment and multiple contaminants are present. At the time of the 2013 Pb ISA,
the connection between air concentration of Pb and ecosystem exposure was poorly characterized for
aquatic habitats (U.S. EPA. 2013). Furthermore, the previous review noted that the level at which Pb
elicits a specific effect is difficult to establish in freshwater systems, due to the influence of other
environmental variables (e.g., pH, OM) on both Pb bioavailability and toxicity, and due to substantial
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species differences in Pb sensitivity. Evidence indicated that Pb is bioaccumulated in biota; however, the
sources of Pb in freshwater organisms have only been identified in a few studies, and the relative
contribution of Pb from all sources, including atmospheric deposition, is usually not known.
Studies published since the 2013 Pb ISA that characterize bioavailability and uptake of Pb, and its
effects in freshwater organisms and ecosystems, that identify additional uncertainties, or decrease
uncertainties identified in the prior NAAQS review of this criteria air pollutant are presented throughout
the following sections. Brief summaries of conclusions from the 1977 Pb AQCD (U.S. EPA. 1977). the
1986 Pb AQCD (U.S. EPA. 1986). the 2006 Pb AQCD (U.S. EPA. 2006a) and the 2013 Pb ISA (U.S.
EPA. 2013) are included where appropriate. Recent research on the bioavailability and uptake of Pb into
freshwater organisms including plants, invertebrates and vertebrates is presented in 11.3.2. Information on
environmental concentrations in freshwater biota and ecosystems in the United States at different
locations and over time is presented in Section 11.3.3. Toxicity of Pb to freshwater flora and fauna
including growth, reproductive and developmental effects (Section 11.3.4) are followed with data on the
exposure and response of freshwater organisms (Section 11.3.5). Responses at the community and
ecosystem levels of biological organization are reviewed in Section 11.3.6.
11.3.2. Factors Affecting Bioavailability, Uptake and Bioaccumulation and
Toxicity in Freshwater Biota
Toxicity of Pb to aquatic life varies with the physicochemical properties of surface waters (U.S.
EPA. 2013). Factors affecting the bioavailability and subsequent toxicity of Pb to biota include chemical
factors (primarily water hardness, DOC, pH) and biological factors (e.g., lifestage, development of
tolerance, organism interactions). Water hardness, DOC, and pH can be quantified, are directly related to
the toxic effects and are used in bioavailability models to predict acute and chronic toxicity (Adams et al..
2020) (Section 11.1.6). Biological factors discussed in prior Pb AQCDs or the 2013 Pb ISA that may
influence organism response to Pb exposure include the lifestage of an organism, genetics, and nutrition
(see Section 7.2.3, 2006 AQCD (U.S. EPA. 2006a) and Section 6.4.9, 2013 Pb ISA (U.S. EPA. 2013)).
These factors are more difficult to link quantitatively to toxicity. Often, species differences in
metabolism, sequestration and elimination rates control the relative sensitivity and vulnerability of
exposed organisms. The organism route of exposure also influences Pb toxicity. Uptake of Pb by aquatic
invertebrates and vertebrates may preferentially occur via exposure routes other than direct absorption
from the water column, such as ingestion of contaminated food and water, uptake from sediment
porewater, or incidental ingestion of sediment (U.S. EPA. 2013. 2006a). Fewer studies assess uptake,
bioaccumulation, and subsequent toxicity of Pb via diet than via aqueous exposure. Of the available Pb
feeding studies in freshwater biota, only a few pair the same concentration of waterborne exposure with
dietary exposure to compare the relative importance of dietary versus aqueous uptake pathways (Alsop et
al.. 2016; Deforest and Mever. 2015). Studies published since the 2013 Pb ISA on chemical factors (water
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hardness, DOC, pH, temperature, and other metals) and biological factors discussed in this section further
enhance understanding of Pb uptake and subsequent toxicity in freshwater systems. Biological factors
include those that were well characterized in previous AQCDs and the 2013 Pb ISA, (e.g., lifestage), and
factors not previously considered, such as the role of parasites in modulating Pb bioaccumulation.
11.3.2.1.1. Water Hardness
The role of water hardness (the amount of Ca2+ and Mg2+ ions) in Pb uptake and subsequent
toxicity was reported in previous Pb AQCDs and the 2013 Pb ISA. Furthermore, EPA's existing Pb
AWQC are hardness-based (Section 11.1.7.3) (U.S. EPA. 1985a). Generally, as water hardness increases,
there is less Pb uptake due to competition of Ca2+ and Mg2+ for binding sites. Newer literature has
continued to examine the role of Ca2+ and Mg2+ and other cations commonly present in surface waters
(e.g., K+, Na+) in modulating Pb bioaccumulation and toxicity. For example, in a study of the amphipod
Gammarus pulex exposed 2 days to 10 (ig Pb/L and a range of environmentally relevant cation
concentrations (Na+, Mg2+ or Ca2+), both Na+ and Mg2+ had no significant effect on Pb uptake while
increasing Ca2+ concentrations inhibited Pb uptake (Urien et al.. 2015). In a study reviewed in the 2013
Pb ISA, Ca2+ influenced Pb accumulation and toxicity in the fathead minnow (Pimephales promelas)
during waterborne exposure (Grosell et al.. 2006a). In a newer study in fish, Ca2+, Mg2+ or H+
significantly decreased Pb accumulation and toxicity in zebrafish larvae Danio rerio, while K+ and Na+
showed no effect (Feng et al.. 2018) (see Section 11.3.2 for further discussion of water hardness and Pb
toxicity).
As described in prior AQCDs and the 2013 Pb ISA, the effect of water hardness is variable;
generally, both the acute and chronic toxicity of Pb increase with decreasing water hardness as Pb
becomes more soluble and bioavailable and less Ca2+ and Mg2+ ions are available to compete with Pb for
binding sites. Studies available since the 2013 Pb ISA are also illustrative of the varying influence of
water hardness on the toxicity of Pb. In reproductive toxicity tests with C. dubia, 7-day EC50 was
81.2 (ig Pb/L at 10 mg/L Ca (0.25 mM) and 130 |ig Pb/L at 70 mg/L Ca (1.75 mM), showing that the
daphnids tested in the soft water were more sensitive to Pb toxicity (Nvs et al.. 2014). However, in a
bioassay with the rotifer Brachionus calyciflorus, Ca was not protective in a chronic (48-h) exposure (Nvs
et al.. 2016b). In bioassays with zebrafish larvae, Pb was more toxic in soft water (11.7 mg CaCCh/L)
compared with hard water (141 mg CaCCh/L) (Alsop and Wood. 201IV
11.3.2.1.2. Dissolved Organic Matter and Dissolved Organic Carbon
In studies cited in the 2013 Pb ISA, DOC was shown to have a protective effect on Pb toxicity in
freshwater invertebrates and fish Esbaugh et al. (2011); Mager et al. (201 la); Mager et al. (201 lb), and
newer studies continue to support these observations. Esbaugh et al. (2012) compared the relative
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importance of water chemistry variables including DOC, Ca, and pH in the toxic response of freshwater
cladoceran (Ceriodaphnia dubia), mollusk (Lymnaea stagnalis) and rotifer (Philodina rapida) to a range
of Pb concentrations in bioassays conducted in a variety of natural waters from across North America.
The greatest toxicity to the cladoceran and snail species was observed in low-DOC waters, and toxicity
was found to be correlated with DOC using multilinear regression modeling analysis. This was not the
case in rotifer/', rapida, where toxicity was most closely correlated with Ca and pH, not DOC. In
contrast, in the rotifer B. calyciflorus, high DOC was protective against Pb chronic reproductive toxicity;
however, when expressed as free-ion activity, toxicity increased with increasing fulvic acid concentration
(Nvs et al.. 2016b). The authors suggest that fulvic acid-Pb complexes may also contribute to Pb
bioavailability in B. calyciflorus. Taking metal speciation into consideration, Dong et al. (2014)
calculated the Comparative Toxicity Potential of Pb (described as the ecotoxicological impact associated
with a unit emission of substance to defined ecological receptors via different pathways of exposure). Pb
had the highest Comparative Toxicity Potential in water with low DOC, moderate pH and hardness, and
the lowest Comparative Toxicity Potential in water with moderate DOC, high pH, and hardness. Pb
typically has high affinity to DOC, resulting in a low fate factor (residence time) and bioavailability factor
(fraction of truly dissolved metal within total metal) (Dong et al.. 2014). Additionally, Zhang et al. (2021)
found that modeling Pb and other heavy metals was improved when incorporating total OC and AVS.
Since the 2013 Pb ISA, studies have further elucidated the relationship between the
characteristics of humic substances and Pb bioavailability, such as molecular weight (MW) or other
additional effects associated with solar irradiation. In lake sediments, Pb-humic acid complexes are more
stable when the MW of the humic acid is lower. In particular, humic acids with MW lower than 10 kDa
could increase the biosorption capacity of Pb (Bai et al.. 2019). While Pb-humic acid complexes are
discussed in the 2013 Pb ISA, the study by Kostic et al. (2013) suggests a mechanism for the binding of
Pb to humic acid may be the "acid-like" nature of Pb(II). Pb(II)-ions strong affinity for humic acid may be
explained by its borderline acid properties and by how humic acids behave as weak acid polyelectrolytes.
Humic acids carry a variety of oxygen-containing functional groups such as carboxylic, hydroxyl,
phenolic and carbonyl groups with oxygen as a donor atom, which helps them form strong bonds with
Pb(II). This is also supported by the study by Liu et al. (2022). which found Pb(II) caused greater
quenching (the decrease of fluorescence by the metal addition) in humic-like DOM compared with
protein-like DOM. The finding was likely due to humic-like components complexing with Pb(II) through
carboxyl and hydroxyl (-COOH and -OH) groups, which generally bonds to Pb(II) preferentially over
protein-like DOM that contains significant amounts of the amino group (-NH2).
The bioaccumulation capacity for Pb in algae is influenced by the presence of organic acids. Que
et al. (2020) found that adding organic acids, such as malic acid or citric acid prolonged the adsorption
equilibrium time of the algae-Pb binary system. Citric acid showed a greater bioaccumulation capacity for
Pb in algae than malic acid, due to ternary complex formation. The binding capacity of Pb to OM is also
influenced by solar or UV-B radiation. Pb complexation with representative humic substances (Suwannee
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River humic acid and Suwannee River fulvic acid) decreased with increasing simulated solar radiation
(Spierings et al.. 2011). This may be due to an increase in the relative abundance of the carboxyl groups
in the photoaltered humic substances and from decreased aromaticity (and thus less electronegativity)
with increasing irradiation doses. The presence of Pb2+ can also increase the photodegradation of
microcystin and thus reduce microcystin accumulation in sediments and in certain fish (Dai et al.. 2017).
Reduced amounts of humic acid were adsorped to the freshwater microalga Chlorella kesslerii, which
then reduced Pb bioavailability to the microalgae because the humic substances increase the
bioavailability of Pb to microalgae by adding supplementary binding sites and because Pb uptake by C.
kesslerii is controlled by transport across the biological membrane rather than by diffusion in the medium
(Spierings et al.. 2011). However, there was no correlation with an increase in free Pb ions and algal
intracellular Pb content, likely due to the formation of additional binding sites on the photoaltered humic
acids. In additional tests using Elliott humic acid under simulated solar radiation, free Pb ions were
released from the metal-DOM complex as the irradiation dose increased, and there was a 33% increase in
intracellular Pb concentration in Chlamydomonas reinhardtii at high irradiance (Worms et al.. 2015).
11.3.2.1.3. pH
As described in prior AQCDs and the 2013 Pb ISA, uptake and subsequent toxicity of Pb to
freshwater biota can be affected by pH, either directly or indirectly. Generally, at low pH, there is more
Pb2+ available to bind to the biotic ligand. As pH increases, there is increased formation of Pb organic
(DOC) and inorganic (OH-, CO,2 ) complexes, which decrease Pb bioavailability. Since the 2013 Pb
ISA, several studies have further characterized Pb complexation and adsorption under changing pH
conditions. There are more binding sites for Pb to humic acids at pH 6 than at pH 4, likely due to the
higher content of dissociated functional groups in humic acids at higher pH, and more favorable
electrostatic attraction when binding surfaces become deprotonated at higher pH (Bai et al.. 2019). Xu et
al. (2018) found that the binding dynamics of DOM groups in response to Pb(II) addition were regulated
by both pH and ionic strength. Specifically, at lower pH and ionic strength (e.g., pH 4.7 and ionic strength
0.01 M), as Pb(II) was added, aryl C-H and carboxyl C = O groups gave the fastest response, followed by
polysaccharide C-OH and chromophoric groups at 265 nm (CDOM265). However, when pH was raised
to 6.0, the opposite binding sequence was found, in that the CDOM265 group was bound first, followed
by the polysaccharide C-OH and carboxyl C = O, and finally the aryl C-H groups. Hua et al. (2013) found
that Pb absorption to biofilms was greatest at pH 9, which was 3.5 times greater than that at the minimum
adsorption (pH = 7).
Several studies since the 2013 Pb ISA have tested the effects of changing pH on Pb toxicity to
biota. In the freshwater algal species Pseudokirchneriella subcapitata, as pH increased from 6.0 to 7.6,
the 72-hour EC50 decreased from 72.0 to 20.5 |ig filtered Pb/L (De Schamphelaere et al.. 2014). Further,
Antunes and Kreager (2014) observed greater toxicity (more bioavailability) for common duckweed (L.
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minor) at higher pH; this was due to less H+ and competition at the macrophyte binding sites. The
apparent increase in Pb2+ toxicity at pH >7.0 coinciding with a changing ratio of [Pb2+]/[Pb(OH)+] (due to
the marked increase in [Pb(OH)+]) suggests that Pb(OH)+ also contributed to the toxicological response.
In some freshwater invertebrates, recent studies generally support previous understanding that
higher pH is protective; however, these findings vary by the duration of the toxicity bioassays and by
taxa. In a series of chronic reproductive toxicity tests with daphnia C. dubia conducted at different pH
values, high pH was protective of Pb toxicity. At the lowest pH tested (pH 6.4), the EC50 = 99.8 |ig Pb/L,
while at the highest pH (pH 8.2), the EC50 = 320 |ig Pb/L ("Nvs et al.. 2014). Similarly, decreasing toxicity
of Pb to D. magna with higher pH was observed by Qin et al. (2014); as pH increased from 5.0 to 9.0, the
24 h-LCso increased from 784 |ig Pb/L to 9,473 |ig Pb/L, and the predicted proportion of free Pb2+ ion was
99.75% at pH 5.0 and 2.9% at pH 9.0. High pH was also protective in chronic reproductive toxicity tests
with rotifer B. calyciflorus. Both the population growth rate and population size generally decreased with
increasing pH in bioassays conducted at pH values ranging from 6.4 to 8.2 (Nvs et al.. 2016b). Wang et
al. (2015b) found that for crustaceans, Pb toxicity increased with increasing pH, but for mollusks and
worms, toxicity decreased with increasing pH. For fish, toxicity was least at neutral pH and increased at
lower or higher pH levels. The toxicity of Pb can increase at higher pH when there is less competition
between H+ and metal binding sites on cell-surface ligands. However, there may be higher toxicity at
lower pH due to increased solubility and altered Pb speciation, which can increase Pb bioavailability for
certain animals. Uptake studies in natural environments have also pointed to the importance of pH in
uptake of Pb. A field study conducted in 36 headwater streams in the Lake District of England reported
statistically significant correlations between total dissolved Pb in stream water and body burdens in the
sampled aquatic insect taxa (Leuctra spp., Simuliidae, Rhithrogena spp., Perlodidae) (Dc Jongc et al..
2014). In the streams, H+ ion activity was the overriding factor influencing Pb body burden, while DOC
was not a significant factor.
In fish, the effects of pH on toxicity were variable in studies cited in the 2013 Pb ISA. For
example, lower pH was shown to result in increased sensitivity to Pb in juvenile fathead minnows
following 30-day exposure to Pb at varying concentrations (Groscll et al.. 2006a). Additionally, Birceanu
et al. (2008) determined that fish (specifically rainbow trout) were more susceptible to Pb toxicity in
acidic, soft waters, characteristic of sensitive regions in Canada and Scandinavia. Hence, fish species
endemic to such systems may be more at risk from Pb contamination than fish species in other habitats. In
a study published after the 2013 Pb ISA, Esbaugh et al. (2013) compared three methods used to acidify
laboratory bioassay water on LC50 values in fathead minnow. Pb toxicity varied significantly depending
upon the acidification method used in the experiment. The authors recommended direct acid-base addition
rather than CO2 or 3 -(N-morpholino)propanesulfonic acid buffer. In an approach that linked metal
accumulation with toxicity through a BLM-aided toxicokinetic-toxicodynamic model, Gao et al. (2015)
demonstrated that increasing concentrations of H+ in test media significantly reduced Pb accumulation in
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zebrafish larvae within the exposure duration of >4-72 hours. In the same study, increasing [H+]
significantly decreased the mortality of the larvae at >12-96 hours.
11.3.2.1.4. Water Temperature
In the 2013 Pb ISA, water temperature was noted as a factor affecting the toxicity of Pb to aquatic
organisms, with higher temperatures generally leading to greater response; a few recent studies reported
variable responses to Pb with temperature. Isopods Asellus aquaticus exposed for 10 days to one of two
water temperatures (15 ± 1°C and 20 ± 1°C) and three concentrations of Pb (0.0353 (imol/L, 7.3 |ig Pb/L),
0.353 |imol/L (73 |ig Pb/L) and 0.882 (imol/L (181 |ig Pb/L) exhibited distinct responses at the two
temperature treatments (Van Ginneken et al.. 2019). At 15°C, respiration decreased as Pb concentration in
the isopods increased. In the higher temperature treatment, feeding and respiration rates were higher and
were positively correlated with Pb uptake and accumulation. Park et al. (2020) assessed survival,
malformation and heart rate in zebrafish embryos exposed to three analytically verified concentrations of
Pb (2, 10 and 17 |ig Pb/L) at two temperatures (26°C and 34°C). At 26°C, the survival rate decreased
early in the 7-day exposure at the two highest concentrations, reaching 73% at 10 (ig Pb/L and 57% at
17 fxg Pb/L by the end of the experiment, with no significant effect at 2 |ig Pb/L. At 34°C, the survival
rate decreased significantly in all concentrations and to a greater extent in the highest concentration; at
7 days, embryo survival at 17 |ig Pb/L was 30% that of the control. Malformations such as spinal
curvature were observed in all tested concentrations at both temperatures. At 34°C, heart rate was
significantly decreased at all Pb concentrations, while at 26°C, heart rate was significantly decreased at
the two highest tested concentrations.
11.3.2.1.5. Other Metals
Multiple metals are present simultaneously in aquatic environments and may interact with one another,
influencing Pb uptake and resulting in antagonistic, synergistic, or other toxic effects. Recent advances in
in multimetal research since the 2013 Pb ISA have included development and evaluation of
bioavailability models to predict the toxicity of acute and chronic metal mixtures, of which Pb is one
component (Nvs et al.. 2017; Farley et al.. 2015; Santore and Ryan. 2015). Since the 2013 Pb ISA,
considerable research beyond the scope of this document (Section 11.1.1) has focused on metal mixture
assessment, including how uptake and bioaccumulation are affected in freshwater biota in the presence of
multiple metals. The mechanisms of metal interactions may include competition for the same metal
transporter at the biological membrane or displacement of one metal by another metal on DOM, which
leads to changes in the free metal ion concentration in water (Cremazv et al.. 2019). The effects of metals
on Pb uptake and toxicity vary by metal. In the juvenile freshwater snail L. stagnalis, Ni and Zn had no
effect on Pb uptake, but a small but significant inhibitory effect was observed with Ag (Cremazv et al..
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2019). In the isopod A. aquaticus exposed to Cd and Pb simultaneously, synergistic interactions occurred
with metal uptake as well as on growth rates and mortality rates when compared with single-metal studies
(Van Ginneken et al.. 2015). In juvenile rainbow trout (Oncorhynchus my kiss) uptake studies of binary
mixtures with Pb paired with other metals, Pb uptake into gill tissue was significantly inhibited in a
noncompetitive manner by Ag, Cd and Cu, while Ni and Zn had no effect on Pb uptake (Brix et al..
2017). In another study with juvenile rainbow trout, there was no effect on ionoregulation at a low Pb
concentration of 5.4 |ig Pb/L (26.1 nmol/L) (Clcmow and Wilkie. 2015). However, in combination with
Cd, there was greater-than-additive toxicity, likely due to differences in the underlying the mechanism of
action, with some shared binding sites between the two metals. In 5-day postfertilization zebrafish larvae
exposed nominally to Pb alone (10 (ig Pb/L), Cd alone (5 |ig Pb/L) or Pb + Cd since 4 hours
postfertilization, the respective mean concentrations of Pb and Cd in tissue were statistically significantly
lower in the co-exposure group than in the groups exposed to Pb or Cd alone (Liao et al.. 2021). There
were differences in behavioral outcomes in the three treatment groups; Pb primarily affected locomotor
activity, Cd affected circadian behavioral rhythm and the two compounds in combination were
antagonistic for both locomotor activity and behavioral rhythm. The bioavailability of Pb is also affected
by the formation of complexes with various Fe (oxyhydr)oxides, such as ferrihydrite, schwertmannite,
jarosite, goethite, hematite, and magnetite (Shi et al.. 2021). Fe (oxyhydr)oxides influence the speciation,
partitioning and transport of Pb through adsorption and coprecipitation, and this can vary by acidity,
alkalinity, temperature, and oxic conditions.
11.3.2.1.6. Lifestage
The differential sensitivity of early lifestages of aquatic biota to contaminants is well-established
in the scientific literature, such that national and international entities (e.g., U.S. EPA, Organisation for
Economic Co-operation and Development, European Union) have standardized laboratory toxicity assay
protocols that call for testing with embryo, larval or juvenile organisms to assess effects at the most
sensitive lifestages. Differences in susceptibility to Pb at distinct lifestages for freshwater invertebrates
and fish are discussed in Section 6.4.9.4 of the 2013 Pb ISA. Recent studies conducted with freshwater
organisms reviewed in Sections 11.3.4 and 11.3.5 continue to demonstrate that lifestage is an important
determinant of increased sensitivity to Pb. For example, endangered white sturgeon (Acipenser
transmontanus) were three and a half times more sensitive when exposed to Pb at 8 days posthatch (dph)
than at 40 dph (Vardv et al.. 2014).
11.3.2.1.7. Species Sensitivity
As described in previous EPA reviews of Pb, sensitivity to this metal can vary by several orders
of magnitude across freshwater biota. Pb elicits responses in some species at low (<5 to 10 |ig Pb/L range
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under some water conditions) concentrations while others appear to be unaffected at concentrations
greatly exceeding 1,000 |ig Pb/L. In a study reported in the 2013 Pb ISA, a series of SSD showed the
greatest sensitivity to Pb in crustaceans, followed by cold water fish, and warm water fish and aquatic
insects, which exhibited a similar sensitivity (Brix et al.. 2005). A comparison of cladoceran and copepod
freshwater species curves generated by Wong et al. (2009) indicated that cladoceran species, as a group,
were more sensitive to the toxic effects of Pb than were copepods, with respective hazardous
concentration values for 5% of the species of 35 and 77 |ig Pb/L. Following the 2013 Pb ISA, Deforest et
al. (2017) used acute and chronic toxicity data across a range of freshwater species and genera, taking into
account the differences in sensitivity to Pb, to propose updated aquatic life AWQC for Pb (Section
11.3.5).
Some uncertainty is associated with the extrapolation of toxicity values generated from
laboratory-based single-metal acute exposure assays to chronic exposure to multiple metals and other
contaminants in field studies. (Brixetal.. 2011) provided examples of acute laboratory exposures with
aquatic insects that suggested the insects are relatively insensitive to metals, in contrast to field studies
that report sensitivity. The authors conducted a meta-analysis of laboratory and field studies that generally
supported the finding of greater sensitivity of aquatic insects in chronic exposure field conditions.
However, the majority of available field studies involve multimetal exposures. The authors speculated
there could be a difference in the mechanism of toxicity between acute exposure and chronic exposure in
aquatic insects or that dietary metal exposure is another important contributing factor to toxicity in these
organisms.
11.3.2.1.8. Development of Tolerance
Tolerance to prolonged Pb exposure may develop over time in some organisms as they
physiologically adapt and survive under low variations of various environmental stresses, including Pb.
Evidence for genetic selection in the natural environment has been observed in some aquatic populations
exposed to metals in studies, as reviewed in the 2006 AQCD. Fewer laboratory-based assays have
examined the development of Pb tolerance. In a study reviewed in the 2013 Pb ISA, multigenerational
exposure to Pb appears to confer some degree of metal tolerance to Chironomus plumosus larvae;
however, metal-tolerant larvae were significantly smaller than larvae reared under clean conditions
(Vcdamanikam and Shazilli. 2008). In a more recent multigenerational test with D. magna exposed to an
analytically verified concentration of 50 |ig Pb/L, the LC50 ( = 430 |ig Pb/L at the F0 generation)
increased to 2.1 10 |ig Pb/L in the F9 generation. The LC50 of control organisms in the F9 generation
varied from 430 |ig Pb/L to 890 |ig Pb/L suggesting that the Pb-exposed organisms developed some
tolerance to Pb over time (Arauio et al.. 2019). In a comparative study of adult amphipods Gammarus
fossarum, either freshly collected from the field and exposed to 2.1 |ig Pb/L for 24 hours or chronically
exposed to the same concentration for 10 weeks, there were differences in response to Pb. In the freshly
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collected amphipods, both locomotion and respiration were significantly decreased compared with
unexposed organisms, whereas in the chronically exposed amphipods, no statistically significant response
to these endpoints was observed, suggesting that the compensatory response developed overtime (Lebrun
and Gismondi. 2020). In another study with G. fossarum and the amphipod Gammarus pulex, a history of
metal exposure did not affect Pb bioaccumulation parameters, as accumulation and elimination
parameters were similar between reference and pre-exposed populations collected from field sites and
exposed to Pb in microcosms (IJrien et al.. 2017). Amphipods were exposed to water spiked with an
analytically verified concentration of 10 (ig Pb/L for 7-days, then transferred to mineral water for
depuration for 7 days. The net bioaccumulation of Pb was quantified by subtracting the basal
concentrations of Pb from the total Pb concentration after exposure. There was no interpopulation
variability or difference in the pattern of accumulation or elimination between G. pulex and G. fossarum.
The peak Pb body concentration was slightly higher in pre-exposed populations relative to the reference
populations for both species.
11.3.2.1.9. Seasonality
In the 2013 Pb ISA, several studies reported seasonal alterations in aquatic plant Pb tissue
concentrations, suggesting that species-dependent seasonal physiological changes may control Pb uptake
in aquatic macrophytes (Section 6.4.9.1) (U.S. EPA. 2013). Several studies published since the 2013 Pb
ISA further describe changes in Pb bioavailability linked to season. In a study examining the interacting
effects of macrophytes and season, metal concentration in small fish inhabiting the phytoplankton-
dominated northern zone of Lake Taihu, China was significantly greater in summer than in small fish
collected from the southern zone of the lake characterized by a high density of macrophytes (Zcng ct al..
2012). These differences in metal concentration in small fish collected from the two regions of the lake
disappeared in winter, suggesting that the presence of algae and macrophytes modified trace metal
concentrations during the summer months, resulting in two distinct ecological regions that differed in
their potential for metal exposure. Differences in metal accumulation in larger fish from the two lake
zones varied with season in some tissues, but no significant differences were reported in carnivorous fish.
Chen et al. (2019) quantified seasonal differences in Pb mobility in lake sediments from phytoplankton-
dominated and macrophyte-dominated areas of Lake Taihu. In the phytoplankton-dominated region,
labile and dissolved Pb in sediment was highest in April and July and lowest in October and January. The
opposite pattern was observed for the macrophyte-dominated region. In littoral anoxic sediment, the
periodic drying and rewetting process can increase the bioavailability of Pb to aquatic organisms (Liu et
al.. 2020). Even though high total OC content in the sediment facilitates the formation of anoxic
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conditions, periodic drying oxidizes the sediment and leads to sulfide oxidation, which increases the
mobility and bioavailability of Pb because it is less firmly bound to sediment in these conditions.
11.3.2.1.10. Parasites
The combined n effects of endoparasites and other stressors such as metals modulate uptake and
toxicity to host organisms (Marcoglicsc and Pietrock. 2011). Multiple studies have reported differences in
Pb accumulation between parasitized and nonparasitized organisms. European chub (Squalius cephalus)
showed greater Pb uptake in the gill and bile of nonparasitized host fish compared with parasitized fish,
and the highest metal concentrations were detected in acanthocephalan parasites (Surcs et al.. 2003; Sures
and Siddall. 1999). European perch (Perca fluviatilis) infected with two parasites, the acanthocephalan
(Acanthocephalus lucii) and a tapeworm (Proteocephalus percae) showed greater Pb accumulation in the
muscle, liver, and hard roe than P. fluviatilis parasitized with either A. lucii or P. percae alone (Brazovaet
al.. 2015). The number of P. percae was negatively correlated with Pb concentration in the muscle of P.
fluviatilis. In a recent synthesis of parasite-host studies, Pb was accumulated to a higher degree in
parasites than in tissues of host species, and Pb accumulation in infected hosts was consistently lower
compared with uninfected conspecifics (Sures et al.. 2017). In field-collected European chub infected
with intestinal parasites (Pomphorhynchus laevis or Acanthocephalus anguillae), lower metal
concentrations were found in infected fish in the postspawning period compared with uninfected
individuals, suggesting that the presence of parasites modulate metal accumulation (Marijic et al.. 2014).
The concentrations of Pb were lower in trematode-infected Biomphalaria alexandrina snails compared
with uninfected snails (Mostafa et al.. 2014). These studies suggest that the effects of parasites on host
organisms in the presence of Pb are complex.
11.3.2.1.11. Bioturbation/Association with Sediment
Since the 2013 Pb ISA, several studies have examined how the activities of sediment-associated
benthic invertebrates influence Pb transfer to the water column and subsequent bioavailability to other
aquatic organisms. A statistical Random Forest model that took into account riverine invertebrate
community traits such as feeding strategy, respiration and locomotion to predict metal bioaccumulation
from environmental compartments (water column, sediment, suspended particulate matter) showed that
the strongest predictor of metal bioaccumulation in the organisms was the degree to which taxa live in or
directly on sediment (Peter etal.. 2018). In mesocosms with two (Amphipod, Bivalve) or three
(Amphipod, Bivalve, Oligochaete) sediment-associated species combinations, water, and tissue
concentrations of Pb (and other trace elements primarily associated with organic colloids) increased as the
number of bioturbating organisms present increased (Soledad Andrade et al.. 2020). One set of
experiments Blankson et al. (2017); Blankson and Klerks (2017. 2016a. 2016b) used oligochaete worm
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Lumbricuius variegatus in Pb-spiked mesocosms as a model organism for bioturbation in freshwaters.
First, Blankson and Klerks (2016a) quantified the transfer of Pb from sediment to overlying water from
mesocosms with different numbers of worms. At the end of the 14-day experiment, overlying water Pb
concentration increased with increasing density of L. variegatus. Next, the researchers added water flea
(Daphnia magna) to Pb-spiked sediment microcosms with and without L. variegatus, restricting some D.
magna to the water column (Blankson and Klerks. 2016b). The presence of L. variegatus in the
microcosms significantly increased the amount of Pb transferred from the sediment to the water column
(mean Pb in water column with L. variegatus present: 2.51 |ig Pb/L versus L. variegatus absent:
0.38 |ig Pb/L). The turbidity of the water column when L. variegatus was present was 85% higher than
that in microcosms without L. variegatus. Turbidity was unaffected by the containment of D. magna and
the interaction between these two factors. Microcosms with restricted D. magna and those with free-
swimming D. magna did not affect the concentration of Pb in the water column at the end of the
experiment. Transport profiles in microcosms contaminated with Pb-spiked sediments (137.6, 681.9 or
3396.2 mg Pb/kg dry weight) showed the bioturbation activity of L. variegatus was affected by increasing
Pb concentration; with a decline in bioturbation with worms exposed to 681.9 mg/kg and 3396.2 mg/kg
(Blankson et al.. 2017). In 14-day exposure to Pb-spiked mesocosms (104 mg/kg dry weight) with natural
sediments collected from different sites, the amount of Pb transferred to the water column varied with
sediment characteristics (Blankson and Klerks. 2017). For transfer of Pb to the water column, the most
important variables were silt/clay content and sediment pH; Pb bioaccumulation in the worms was
influenced by OM in the sediments and the pH of the porewater. Specifically, Blankson and Klerks
(2017) found that organic content and sediment porewater pH had a significant negative effect on the
change in worm tissue Pb concentrations, with Pb in worm tissue being 26-fold lower with 5-10% greater
OM content. In addition, Pb concentration in worms was about 50% lower in sediment with 10 and 30%
silt/clay content compared with unmodified sediment (4.58% silt/clay content) (Pb bioaccumulation was
reduced by a factor of 2.6 at an 8-times higher silt/clay content). Overall, bioturbation by oligochaetes
could bring about the transport of Pb from sediments to the water column. This means that the presence of
these bioturbators can enhance Pb availability to organisms in the water column and potentially cause
toxic effects in planktonic and nektonic organisms.
11.3.2.1.12. Intraspecific Interactions
Additional research published since the 2013 Pb ISA provides experimental evidence that
interactions among individuals of the same species may affect sensitivity to metals. The influence of
intraspecific competition on Pb (13 to 236 |ig Pb/L) toxicity was explored by Gust et al. (2016) using
single daphnia exposures conducted concurrently with assays of multiple daphnia (proportionally scaled
assays of 20 D. magna per beaker) and at two different feeding regimens (low-feed ration and high-feed
ration). After 14-day exposure to Pb, the LC50 was threefold higher in assays with single daphnia (232
[156-4810] |ig Pb/L) compared with assays with multiple individuals (68 [63-73] |ig Pb/L) at the lower
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feeding ration. Similar results were obtained with the higher feeding ration experiment with multiple
daphnia per experimental unit (LC50 = 79 (74-84) |ig Pb/L) and the single-animal treatment
(LC50 = 236 |ig Pb/L (no 95% confidence interval could be calculated). Moreover, reproduction (neonate
production) decreased with intraspecific competition at 9 and 14 days in both feeding ration groups
compared with assays with single daphnia where no negative effects on reproduction were observed at
any concentration tested. The authors proposed that individual daphnia modulate their life-history
response in the presence of others of the same species through chemical cues, and this has a modifying
effect on toxicity.
11.3.2.1.13. Predator-Stress and Metal Mixture Effects
Research published since the 2013 Pb ISA tested the effects of multiple stressors on Pb uptake
and toxicity. Predator stress and the presence of other metals affected the accumulation and sensitivity of
the aquatic sowbug (A. aquaticus) to Pb stress (Van Ginneken et al.. 2018). Individual A. aquaticus
collected from a stream in Belgium were placed in a control, 0.0232 |imol/L. (4.8 |ig Pb/L), 0.276 |imol/L
(57 |ig Pb/L) or 3.08 |imol/L (638 |ig Pb/L) solution with two black alder (Alnus glutinosa) leaf discs.
Each Pb treatment and metal mixture (Cu + Pb, Cd + Pb and Cu + Cd + Pb) was crossed with one of two
treatments, either a heterospecific predator cue or conspecific alarm cue. To create the heterospecific
predator cue solution, one damselfly larva (Calopteryx splendens) was placed in a container of water for
72 hours. Next, one adult three-spined stickleback (Gasterosteus aculeatus) and the ninespine stickleback
(Pungitiuspungitius) were placed in water for 24 hours. After removing the predators from the water,
equal parts of stimulus water were mixed to create the heterospecific predator cue. To create the
conspecific alarm cue, one A. aquaticus was homogenized in solution. Either water (control) or predator
or the conspecific alarm cue solution was added to the control and Pb-contaminated containers with A.
aquaticus every day for 10 days. Afterward, A. aquaticus Pb concentration, growth rate, feeding rate,
percent active time, survival and respiration rate were recorded. Overall, there were no significant effects
of either heterospecific or conspecific predator cues on Pb accumulation in A. aquaticus, although
respiration rates did increase when exposed to predator cues. Pb accumulation in the isopods was
positively correlated with Pb free-ion activity. There were no significant effects of predator stress on
isopod body burdens. Metal mixture significantly affected Pb accumulation, as the slope of the
relationship between Pb treatment and Pb body burden decreased when Cu and Cd + Cu were added.
Respiration rates were affected by both Pb exposure and predator stress. Differences in respiration rates
between predator-stress and control treatments were greater when isopods had greater Pb body burdens.
Activity levels decreased as Pb body burden increased, but there was no difference between predator
treatments and the interaction between Pb treatment and predator stress. Growth rate (mg/day) was
negatively correlated with Pb free-ion activity in the water but was not found to vary with predator stress
or body burden. Although Pb body burden did not influence feeding rates (mg/mg/day), the Pb body
concentration of A aquaticus exposed to the Pb + Cd mixture had the greatest effect on feeding rate
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compared with Pb, Pb + Cu and Pb + Cu + Cd. Finally, activity decreased with increasing Pb body
burden, but was unaffected by predator stress and Pb-metal mixtures.
11.3.2.2. Uptake and Bioaccumulation in Freshwater Plants and Algae
Studies on bioavailability of Pb in aquatic plants and algae published since the 2013 Pb ISA
continue to support previous findings that plants tend to sequester larger amounts of Pb in roots as
compared with shoots and that there are species-specific differences in uptake of Pb from water and
sediments, as well as compartmentalization of that sequestered Pb (U.S. EPA. 2013. 2006a'). Further, it
has previously been established that many plants accumulate heavy metals in environments with high
concentrations and are used for phytoremediation at such sites; additional studies on this topic have little
relevance in the current assessment.
Very little new information is available on the bioavailability of Pb in freshwater algae at levels
that are within the concentrations of interest in this ISA (Section 11.1.1). One study contains data on
bioavailability and partitioning between water and sediment correlated with toxic harmful algal blooms
(HABs), which are of concern in many freshwater bodies. This study, conducted in a freshwater reservoir
in Portugal, examined in situ interactions between Pb and Microcystis aeruginosa, a HAB-forming
cyanobacterium found in the United States (Baptista et al.. 2014). The metal content of water and
sediments from both the reservoir and an upstream reference site were monitored monthly for 16 months,
during which timeM aeruginosa bloomed twice, firstly forming a scum, and later with colonies scattered
throughout the reservoir. No correlation was found between Pb in the water column and algal blooms.
When blooms occurred, a significant increase of metal levels in the sediment occurred simultaneously
(average Pb concentration was measured at 43.2 mg/kg); however, quantification of the exchangeable
metal fraction during this algal bloom indicated that this Pb was probably not bioavailable. The authors
speculate shallow water depth would have allowed the cells ofM aeruginosa to deposit upon the
sediments rapidly, and the presence of the cyanobacteria in the sediment might have contributed to an
increase in metal content, meaning that algae may be an important biotic compartment for Pb during such
blooms. In three Scottish lakes receiving varying inputs of metals solely from atmospheric deposition
changes in phytoplankton biomass, cellular Pb and the P content of cells were measured simultaneously.
The results showed that algal bloom events in the lakes diluted the mass-specific Pb in the phytoplankton
(Gormlcv-Gallaghcr et al.. 2016). As total cellular P increased, there was a corresponding increase in
phytoplankton growth, and the concentration of Pb declined.
In freshwater floating macrophytes, there is also very little new information on the bioavailability
of Pb. These life forms are important because their roots dangle in the water column instead of being
buried in substrate, and thus, Pb uptake occurs solely through the interface with the water column. One
U.S. study examined the uptake and distribution of metals by a floating macrophyte, water lettuce (Pistia
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stratiotes L.), in storm water impoundments in Florida (Lu et al.. 2011). Two stormwater impoundment
ponds were divided into two plots, a control without P. stratiotes and one with enough young plants to
initially cover l/20th of the water surface. While the authors stated that water Pb levels were mostly low
(below the Maximum Daily Limits), they did not provide the concentrations. Even at these low
concentrations, reported BCFs of Pb from the water column into plant roots were higher than 104. Lead
was found inside and adsorbed to plant roots, with approximately 60% of Pb within the root tissue.
Another study by Chen et al. (2019) found that submerged macrophytes in lakes can accumulate Pb,
which is absorbed either from the sediments through roots or from the water by leaves.
Though the EPA Framework for Metals Risk Assessment states that the latest scientific data on
bioaccumulation do not currently support the use of BCFs and BAFs when applied as generic threshold
criteria for the hazard potential of metals (U.S. EPA. 2007). such metrics are useful to provide
information about the amount of uptake of metals into plants, compartmentalization into different plant
tissues, and differences between species. In a series of field studies undertaken in Sicily, spanning a
gradient of affected wetlands, Pb concentrations in soil, water, and plant tissues of several wetland species
were quantified (Bonanno et al.. 2018; Bonanno and Cirelli. 2017; Bonanno and Vvmazal. 2017;
Bonanno et al.. 2017; Bonanno. 2013). These studies affirmed that metal uptake is species-specific
despite similar ecology, anatomy, and life form, and that Pb is mainly compartmentalized in root tissue in
freshwater plants.
11.3.2.3. Uptake and Bioaccumulation in Freshwater Invertebrates
This section expands on the findings from the 1986 Pb AQCD (U.S. EPA. 1986). the
2006 Pb AQCD (U.S. EPA. 2006a) and the 2013 Pb ISA (U.S. EPA. 2013) on the bioaccumulation and
sequestration of Pb in freshwater invertebrates. Uptake and subsequent bioaccumulation of Pb varies
greatly between species and across taxa, as characterized in previous EPA reviews of this metal. In
invertebrates, Pb can be bioaccumulated from multiple sources, including the water column, sediment and
dietary exposures, and factors such as the proportion of bioavailable Pb (Section 11.1.6) lifestage, age and
metabolism can affect the accumulation rate. As reviewed by Wang and Rainbow (2008) and supported
by subsequent studies, there are considerable differences between species in the amount of Pb taken up
from the environment and in the levels of Pb retained in the organism.
Uptake studies generally show that aquatic invertebrates accumulate Pb from water in a
concentration-dependent manner and may reach an equilibrium depending on the organism's ability to
eliminate or store Pb. In a study reviewed in the 2013 Pb ISA, the tissue concentration of Pb in adult
Eastern Elliptio mussel (Elliptio complanata) increased for the first 14 days in an aqueous exposure at an
exposure-dependent rate then did not change significantly for the remainder of the 28-day exposure
(Mosher et al.. 2012b) In another study with the same species conducted after the 2013 Pb ISA, Pb was
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measured in hemolymph every 7 days during a 28-day exposure, and distinct patterns of response were
observed with Pb concentration. At the lowest concentrations (< 6 |ig Pb/L), Pb gradually increased in the
hemolymph but did not exceed the exposure concentration, at mid-range concentration (up to
66 |ig Pb/L), the mussels appeared to regulate Pb by day 14, whereas at the highest concentration tested
(251 |ig Pb/L), Pb in hemolymph increased throughout the exposure period (Mosher et al.. 2012a). Pb in
tissue was highly correlated with the exposure concentration at the end of the experiment. The lowest
exposure concentration of 0.9 |ig Pb/L resulted in an average tissue concentration of 1.5 |ig Pb/g dry
weight.
Studies in the 2006 Pb AQCD and 2013 Pb ISA generally showed that the tissue distribution of
Pb in aqueous exposures of freshwater invertebrates is primarily sequestered in the gills, hepatopancreas
and muscle. Recent short-term (3-4-hour) aqueous uptake studies with juvenile snail L. stagnalis showed
no significant difference in Pb accumulation among foot, mantle, digestive tract and remaining soft
tissues, suggesting uptake occurred directly across the skin (Cremazv et al.. 2019). L. stagnalis was
previously identified as one of the aquatic invertebrates most sensitive to Pb exposure (Groscll and Brix.
2009; Grosell et al.. 2006b).
There is some evidence to suggest patterns of tissue distribution differ when uptake of Pb is from
sediment. In 28-day exposure to Pb-spiked sediments (205 ± 9 and 419 ± 16 mg/kg dry mass) the
freshwater bivalve Hyridella australis accumulated Pb in both the low (2.2 ± 0.2 mg/kg dry mass) and
high treatments (4.2 ± 0.1 mg/kg dry mass) in the order labial palps>mantle>gill>visceral mass>muscle
(Marasinghc Wadige et al.. 2014). Labial palps accumulated significantly more Pb than other tissues,
consistent with the sediment-burrowing activities of this species. After 28-days, 83%—91% of the
accumulated Pb in hepatopancreas of the bivalves was in the biologically detoxified fraction, primarily
sequestered in MRG. Concurrently, the relative proportion of Pb sequestered in the metallothionein-like
protein fraction (13% to 32%) decreased with Pb exposure. The biologically active metal fraction
significantly increased with increased Pb exposure, and the highest percentage of Pb was associated with
the mitochondrial fraction.
The 2006 Pb AQCD recognized the potential importance of the dietary uptake pathway as a
source ofPb exposure for invertebrates. Additionally, several studies reviewed in the 2013 Pb ISA
quantified water versus dietary uptake of Pb in aquatic invertebrates (Komiarova and Blust. 2009;
Borgmann et al.. 2007; Besser et al.. 2005). Since the 2013 Pb ISA, the relative importance of dietary
versus aqueous uptake pathways has been further discerned for some biota. Camusso etal. (2012) applied
a biologically based Biodynamic Model to previously published data and additional unpublished data on
uptake of trace metals in L. variegatus from field-collected sediments to assess the main uptake route in
this sediment-dwelling organism. The modeled data suggest that for Pb, both free dissolved concentration
in porewater and dietary uptake contributed to body burden, and the amount of Pb taken up in the gut
appears to be controlled by how tightly Pb is bound to sediment. In D. magna fed under two different
dietary regimens (regular diet = 3/105 Raphidocellis subcapitata algal cells/mL; restricted diet = half
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algae concentration), Pb uptake from water was gradual in individuals with restricted food intake and
faster under regular feeding, suggesting that a portion of Pb uptake occurred via diet (Arauio et al..
2019).Hadii et al. (2016) used a series of microcosms in which the amphipod G. pulex was exposed to Pb
for 6 days in the water column only (0.36, 0.71. 3.62, 6.75 (ig Pb/L) or water column (0.31, 0.57, 3.07,
5.02 (ig Pb/L) with access to food (poplar leaves Poplus nigra pretreated for 1 week in Pb concentrations
ranging from 0.5 to 10 jxg Pb/L). In the water-column-only microcosms, Pb-treated poplar leaves were
present but were enclosed in mesh bags so that the gammarids could not feed. At the end of the study, Pb
was significantly higher in amphipods with access to Pb-contaminated leaves than in amphipods exposed
to Pb via the water column alone. The dietary contribution ranged from 29% to 31% in the tested
concentrations. In an 8-day depuration period, there were no significant differences in elimination
regardless of exposure route.
Few studies have assessed the relationships between Pb speciation, water chemistry and
biouptake in aquatic invertebrates in situ. In aquatic insect taxa (Leuctra spp., Simuliidae, Rhithrogena
spp, Perlodidae) sampled from 36 headwater streams in the Lake District of England, pH was the
prevalent factor influencing Pb uptake, and there were statistically significant correlations between total
dissolved Pb in stream water and insect body burdens (De Jonge et al.. 2014). For prediction of observed
body burdens, Windermere Humic Aqueous Model modeling of stream chemistry and Pb chemical
speciation that took into account competition among cations for uptake in biota resulted in a better model
fit than "metal accumulation as a function of total dissolved metal levels or the free ion alonc'YDc Jonge
et al.. 2014).
11.3.2.4. Uptake and Bioaccumulation in Freshwater Vertebrates
In freshwater vertebrates, Pb uptake in tissues generally increases with increasing concentration
of Pb in exposure media (U.S. EPA. 2013); recent studies continue to support these observations.
Evidence in the 2013 Pb ISA supported the 2006 AQCD conclusions that the gill is a major site of Pb
uptake in fish and that there are species differences in the rate of Pb accumulation and distribution of Pb
within the organism. In dietary studies reviewed in the 2013 Pb ISA, the anterior intestine was identified
as a target of Pb in fish. New uptake studies continue to show distinct patterns of Pb tissue distribution in
water versus dietary exposures. As reviewed in Lee et al. (2019). some studies in fish reported higher
rates of Pb accumulation in gill tissues from waterborne exposure compared with dietary exposure. Pb
typically accumulates in metabolically active organs including kidney, liver, and intestine in both aqueous
and dietary exposure.
In a study designed to investigate the relative influence of waterborne and dietary Pb on
accumulation by rainbow trout (O. mykiss), juvenile trout were exposed to Pb ( 8.5, 20, 60 or
110 jxg Pb/L), for 7 weeks via waterborne Pb only, dietary Pb only in the form of live prey (worms L.
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variegatus pre-exposed for 28-days to the same concentration of Pb as the fish) or simultaneously to
waterborne and dietary Pb (Alsop et al.. 2016). Accumulation of Pb in fish was significantly higher via
the waterborne exposure pathway compared with dietary exposure in all tissues except in the gut, which
accumulated similar amounts of Pb regardless of the exposure route. When fish were exposed to Pb from
both water and their diet, whole-body Pb was reduced up to 61% at 110 |ig Pb/L, and Pb accumulation
was significantly reduced at a threshold of ~50 |ig Pb/L, with significantly lower concentrations in the
liver and carcass but not the gill or gut. The authors noted that Pb may have altered the nutrient quality of
the prey; carbohydrates and lipid levels in the worms were significantly decreased even at the lowest Pb
concentration.
11.3.2.5. Uptake and Bioaccumulation Through Food Web
In the 2006 Pb AQCD (U.S. EPA. 2006a) and the 2013 Pb ISA (U.S. EPA. 2013). transference of
Pb through the food web was generally found to be low, with lower Pb accumulation at higher trophic
levels; however, some studies found bioaccumulation of Pb at higher trophic levels. Recent evidence
supporting little bioaccumulation through freshwater food webs is reviewed here.
In a review published since the 2013 Pb ISA, Cardwell et al. (2013) compiled laboratory and field
studies published prior to the 2013 Pb ISA to examine the transfer of Pb and other heavy metals through
aquatic food webs. The concentrations of Pb decreased with increasing trophic position in food web
studies examining trophic transfer between phytoplankton, cladocera and fish. In most of the field studies
reviewed, no evidence was found for biomagnification of Pb across trophic levels in freshwater systems.
Specifically, 17 studies examined trophic transfer of heavy metals through aquatic lake or stream food
webs; while 10 of these studies found no evidence of Pb biomagnification, one study found possible
evidence, and six studies did not examine Pb or did not present data on Pb. More recent studies are
presented below.
In a high-elevation lake in the Alps, Pastorino et al. (2020b) examined the accumulation of heavy
metals, including Pb, in sediment, chironomids, and fish. Surface sediment, benthic macroinvertebrates,
and fish were sampled from a glacial-origin lake, Dimon Lake, in Northeast Italy. While there is only a
single fish species in this lake, i.e., the European bullhead (Cottus gobio), the benthic macroinvertebrate
community consists of midges (Diptera Chironomidae), worms (Oligochaeta), and leeches (Hirudinea).
The only prey found in the stomachs of C. gobio was Diptera Chironomidae, and therefore only these
specimens were used for trace-element analysis. Surface sediment Pb was 109.6 ±1.2 mg Pb /kg, whole-
body Diptera Chironomidae Pb concentration was 49 ± 0.5 mg Pb/kg, and Pb concentration in C. gobio
was 0.06 ± 0.03 mg Pb/kg in the muscle and 0.03 ± 0.4 mg Pb/kg in liver. The BAF and trophic transfer
factor (TTF) in Diptera Chironomidae and C. gobio muscle and liver samples were less than 1.0 for Pb,
indicating biodilution. The BAF in Diptera Chironomidae was 0.45 and the BAF in fish muscle and liver
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was 0.0005 and 0.003, respectively. The TTF in C. gobio was 0.002 in muscle and 0.007 in liver. In a
similar study, Pastorino et al. (2020a) examined BAFs for all the benthic macroinvertebrates from Dimon
Lake (Chironomidae, Oligochaeta and Hirudinea) and from another nearby lake, Balma Lake
(Chironomidae, Oligochaeta). In this analysis, Dimon Lake surface sediment was 110 ± 1.1 mg Pb/kg
(mean ± S.D.), while Balma Lake had considerably less Pb (41 ± 1.2 mg/kb Pb). In addition to lower Pb
concentration in the surface sediments, Balma Lake had a lower pH (mean ± S.D.; summer: 6.70 ± 0.34;
autumn: 7.64 ± 0.09) than Dimon Lake (summer: 8.77 ± 0.12; autumn: 9.44 ± 0.05). The lower pH was a
result of Balma Lake's granite bedrock whereas Dimon Lake covers volcanic rock and sandstone. No
correlation was found between the sediment trace-element concentrations and the benthic
macroinvertebrates. BAFs were calculated using the mean Pb sediment concentration from each lake
across the summer and the fall. In this study, BAFs for Dimon Lake Chironomidae were similar to results
found in Pastorino et al. (2020b) for Chironomidae at 0.45. Additionally, Dimon Lake BAFs were 0.42
for Oligochaeta and 0.1 for Hirudinea, suggesting biodilution. In Balma Lake, however, BAFs were
above 1.0, suggesting bioaccumulation for the benthic macroinvertebrate community (1.61 for
Chironomidae and 1.66 for Oligochaeta).
Some studies use stable-isotope analysis to characterize trophic position in a food web. Using
stable isotopes, Pb accumulation was found to decrease with increasing trophic level in Korean wetlands
(Kim and Kim. 2016). The Upo wetlands consist of four smaller wetlands (Upo, Mokpo, Sajipo, and
Jokjibul), which have different water inflow sources and consequently abiotic condition and biotic
communities. Sediment and biota (primary producers: water caltrop [Trapa japonica\, primary
consumers: leaf beetle [Galerucella nipponensis] and secondary consumers: water strider [Gerris sp.] and
wolf spider [Arctosa sp.]) were collected and characterized for metal content (Pb, Cd, Cu, and Zn).
Afterward, 813C and 815N isotopes were used to characterize the food web. Sediment Pb concentrations
ranged from approximately 35 to 50 mg Pb/kg and differed significantly among sites. In general, the plant
and leaf beetle had lower 813N and 815N signatures than water striders and wolf spiders. Concentrations
of Pb in the leaves of T. japonica were the highest compared with the other organisms analyzed at all
sites. Pb concentrations in G. nipponensis were significantly lower than those in T. japonica. Pb
accumulation in the secondary consumer Arctosa sp. was lower than Pb accumulation in Gerris sp.
Overall, Pb concentrations decreased significantly as trophic level increased (plant
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2021). The existence of the positive correlations suggested there was no decoupling of Pb concentration
between the aquatic vegetation and insects and terrestrial spiders. Unlike the other metals, Pb may be
retained during the insect metamorphosis phase, and the spiders might be an important link in terrestrial
transfer from aquatic environments. In another study, risk quotients for Pb were calculated for
communities of birds along the Emory River in Tennessee based on Pb concentrations in, as riparian
spiders, which can represent a significant portion of the diet, especially for nestlings (Bcaubicn et al..
2020). Riparian orb-weavers (Tetragnatha elongata), which feed primarily on adult aquatic insects, had
wet-weight Pb concentrations ranging from 0.03 ± 0.003 mg Pb/kg to 0.045 ± 0.045 mg Pb/kg
(mean ± S.D.). Risk quotients for Pb and other heavy metals were calculated for bird species using the
contaminant exposure or the reach-specific spider mean metal concentration, divided by the toxic
threshold for each study reach. Lead chronic risk quotients calculated for the Emory River study area
ranged across species, with the highest risk quotients found for 1 and 12-day Chickadee nestlings (Poecile
spp.) (range: 0.81-1.52; percentage of diet consisting of spiders: 25.0%), Eastern Bluebird 2-day nestlings
(Sialia sialis) (range: 0.81-0 1.21; percentage of diet consisting of spiders: 30.9%), and Red-cockaded
Woodpecker 9-12-day nestlings (Picoides borealis) (range: 0.80-1.20, percentage of diet consisting of
spiders: 60%). All Pb acute risk quotients reported were 0.00. Chronic spider-based avian wildlife values
for adult and nestling birds ranged from 0.03 mg Pb/kg for 1-day nestlings for Poecile spp. to
1.347 mg Pb/kg for Setophaga discolor (prairie warbler) 12-day nestlings.
In another example of aquatic insect transfer of Pb to the surrounding environment, Fletcher et al.
(2022) found that 80-95% of Pb in dragonfly species was shed with emergence. Ten dragonfly species
were collected from a constructed wetland at the Savannah River Site, a National Environmental
Research Park in South Carolina, United States, where materials for nuclear weapons are produced.
Although sediment and freshwater concentrations were not reported in this study, average Pb
concentrations in the shed exuviae of 10 dragonfly species (Brachymesia gravida, Libellula auripennis,
Libellula luctuosa, Orthemis ferruginea, Plathemis lydia, Pachydiplax longipennis, Perithemis tenera,
Pantala flavescens, Pantala hymenaea, and Tramea lacerata) ranged from 2.94-10.7 mg Pb/kg, which
was significantly higher than Pb concentrations in the tenerals, or the freshly molted adult insect
(< 0.4 mg Pb/kg), suggesting that Pb in the exuviae was 17-96 times higher than the concentrations in the
teneral.
New observational studies and literature reviews since the 2013 Pb ISA (U.S. EPA. 2013)
generally confirm that many freshwater food webs exhibit reduced accumulation of Pb in higher trophic
levels (Pastorino et al.. 2020b: Kim and Kim. 2016: Cardwell et al.. 2013). although one study reported
the bioaccumulation of Pb (Pastorino et al.. 2020a). Additional studies demonstrated that Pb can transfer
between aquatic food webs and terrestrial ecosystems via aquatic insect emergence and predation by and
of riparian spiders (Fletcher et al.. 2022: Kraus et al.. 2021: Beaubien et al.. 2020).
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11.3.3. Environmental Concentrations of Pb in Freshwater Biota and
Ecosystems in the United States at Different Locations and Over Time
Few U.S. studies have examined national or regional-scale trends of Pb in freshwater biota. The
1986 AQCD reported the results of Lowe etal. (1985). a nation-wide survey of metal concentrations in
fish from 1979 to 1981. At 112 monitoring stations, they found an average (geometric mean) of
0.19 |ig Pb/g wet weight for the period 1978 to 1979 and 0.17 |ig Pb/g wet weight for 1980 to 1981 (U.S.
EPA. 1986). In the 2006 AQCD, a representative median and a range of Pb concentrations were reported
in surface waters (median 0.50 |ig Pb/L, range 0.04 to 30 |ig Pb/L), sediments (median 28 mg Pb/kg dry
weight, range 0.5 to 12,000 mg Pb/kg dry weight) and fish tissues (geometric mean 0.54 mg Pb/kg dry
weight, range 0.08 to 23 mg Pb/kg dry weight [whole body]) in the United States based on a synthesis of
National Ambient Water Quality Assessment data (U.S. EPA. 2006b). The 2013 Pb ISA reported survey
results from the Western Area Contaminants Assessment Project (2002-2007), which included the
concentration of Pb in fish tissue (0.0033 [fillet] to 0.97 [liver] mg Pb/kg [dry weight]) from a set of
national parks in the western United States (NPS. 2011; Landers et al.. 2008). No recent studies
examining spatial or temporal trends in Pb concentration in freshwater fish or invertebrates from locations
across the United States were identified in this ISA. Many individual studies report Pb concentrations in
aquatic ecosystems and biota from specific sites across the United States; compilation of those data is
outside the scope of this ISA. Pb concentrations in water, sediment and other environmental media are
available in Section 11.1.3 and summarized in Table 11-1.
Since the 2013 Pb ISA, a few regional-scale studies, including a study in Canada, have assessed
trends in Pb concentrations in vegetation (peat bogs) or the water column. Peat bogs deposit and preserve
stable layers of accumulated moss and other plant material that can be used to reconstruct a record of
spatial and temporal distribution patterns of air Pb concentrations. Six peat cores collected in 2013 and
2014 in northern Alberta, Canada Shotvk etal. (2016) record the rates of atmospheric Pb deposition dated
from 1910 to 2014 using 210Pb and 14C dating in models, linking sample depth to age. Peak accumulation
rates were observed between the years 1950 and 2000 in each sample, and overall decreasing rates of Pb
accumulation were observed from 1980. Although this study was not in the United States, decreased Pb
accumulation rates coincided with the introduction of unleaded gasoline in the United States and Canada
in the mid-1970s and nearby potential point sources of Pb air pollution (industrial development including
bitumen mines and upgraders) are not attributed to the increase in Pb accumulation. The uppermost, most
recent, peat layers show near-zero modern atmospheric Pb deposition in the Alberta peat bogs.
In a 2015-2017 water quality survey of four Tennessee headwater Appalachian streams Olson et
al. (2019). the maximum observed Pb concentration and sole detectable measurement of this metal was
less than 1 |ig Pb/L. Reported mean concentration values at each site were less than the minimum
detection limit of 0.28 |ig Pb/L. These observations from remote streams without upstream anthropogenic
Pb sources suggest that long-range atmospheric deposition is not a major source of Pb contamination to
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this region. Limited evidence from regional studies of temporal trends in freshwater aquatic ecosystems
published since the 2013 Pb ISA suggest that modern atmospheric deposition of Pb is not a major
contributor to Pb concentrations in freshwater aquatic biota and ecosystems in remote locations.
11.3.4. Effects of Pb in Freshwater Systems
This section focuses on studies of the biological effects of Pb on freshwater plants and algae,
microbes, invertebrates, and vertebrates published since the 2013 Pb ISA. The biological effects of Pb in
the 2013 Pb ISA and in this appendix are generally presented in increasing complex levels of biological
organization from suborganismal responses (i.e., enzyme activities, changes in blood parameters) to
endpoints relevant to the population level and higher (growth, reproduction, and survival) up to effects on
ecological communities and ecosystems. Exposure-response studies that report toxicological dose
descriptors (e.g., LC50, EC50, lowest observed adverse effect level [LOAEL]) for effects on growth,
reproduction or survival endpoints are reported in Section 11.3.5.
11.3.4.1. Effects on Freshwater Microbes
The effects of Pb on microbial communities in freshwater ecosystems were not reviewed in detail
in the 2013 Pb ISA (U.S. EPA. 2013). except for a report that Pb could alter bacterial infection in the fish
Channapunctatus (Pathak and Gopal. 2009). In the 2006 Pb AQCD (U.S. EPA. 2006a). it was reported
that Pb could adsorb to biofilms, depending on pH, water hardness, polarity of matter, and amount of Fe
or Mn in the water and that methylation by microbes may result in Pb remobilization in aquatic
ecosystems; however, few studies directly report effects on microbes from Pb exposure. Since the 2013
Pb ISA (U.S. EPA. 2013). several experimental and observational studies have examined the relationship
between Pb concentration in the sediment and effects on freshwater microbes, as reviewed below. Several
of these studies report negative relationships between sediment Pb concentration and microbial abundance
or community structure, while some report no relationship or positive associations.
In a study from the United States, porewater and sediment Pb concentrations were negatively
correlated with bacterial RNA abundance, but not diversity or richness in Lake DePue, Illinois (Gough
and Stahl. 2011). Lake DePue is a shallow lake on the Illinois River located near a U.S. EPA Superfund
Site (the DePue/New Jersey Zinc/Mobil Chemical Site). Although the Zn smelting facility and phosphate
fertilizer plant are no longer operational, Lake DePue has received metal-contaminated sediments from
this site for over 80 years. Sediment Pb concentration in the lake was on average 180 mg Pb/kg (range:
68.6 and 541 mg Pb/kg). Porewater and sediment Pb were correlated with a low abundance of archaeal,
bacterial, and eukaryotic terminal restriction fragments (TRF); however, other metals were also correlated
to most of the same TRF profiles (particularly, Zn, As, Cd, Cu, and Fe). Specifically, porewater Pb was
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significantly correlated with the abundance of putative Mesophilic Crenarchaeota (positively correlated
with TRF 191 and negatively correlated with TRF 162 and TRF 510-512) but was not correlated with
the diversity of archaeal TRFs. Porewater Pb was also negatively correlated with a putative
Desulfobacterium and a eukaryotic TRF 108 with the nearest relative of /Y't'.v/t'na/Sccncdcsmaccac.
Sediment Pb also correlated with putative Mesophilic Crenarchaeota (positive correlation with TRF 191
and negative correlation with TRF 162), and unlike porewater, sediment Pb had a significant negative
correlation with archaeal TRF diversity. Sediment Pb did not significantly correlate with any reported
bacterial or eukaryotic TRF abundances. Finally, porewater and sediment Pb were negatively correlated
with relative bacterial RNA abundance. Interestingly, the high positive correlations between TRF 191
(TRF for Crenarchaeota) and metals, including Pb, suggest that methanogens, which are an archaeal
population typically expected to be the dominant group in freshwater systems, may not be able to
outcompete other archaeal groups in metal-contaminated sites. Overall, there were some differences in
overall community structure with regard to metal contamination observed using terminal restriction
fragment length polymorphism analysis of 16S rRNA genes, although variation in bacterial diversity,
richness and composition across a metal gradient was not detected. In a follow-up study using the same
samples, Kang et al. (2013) further explored the bacterial communities using a different approach, a
function gene microarray (GeoChip). Overall, the diversity of functional gene variants was similar across
all five sites, suggesting that heavy-metal concentrations in the sediments did not significantly affect
bacterial community structure; however, some individual gene categories were correlated with certain
porewater metal concentration, including Pb. Using a CCA, Pb, Zn, and Cd were all found as important
predictors for sulfate-reducing bacteria communities. Although significant correlations with Pb existed,
functional gene variants had similar relationships with other porewater metal concentrations, including
As, Cd, Cr, and Zn.
In another U.S. study, observational evidence suggests that the exchangeable Pb fraction
decreases microbial community diversity, while oxyhydroxide Pb concentration was correlated with an
increase in diversity in the mining district of Lake Coeur d'Alene, Idaho (Moberlv et al.. 2016). The
Coeur d'Alene Mining district stretches from Coeur d'Alene, Idaho to Superior, Montana and has had 90
mines in operation. Sediment cores were collected from sites in the Lake Coeur d'Alene delta and from
reference sites in the neighboring St. Joe River delta to characterize metal concentrations (Pb, Fe, Mn, and
Zn) and phase in the sediment and microbial community composition. Pb concentrations in the sediment
were high in the lake, ranging from 1540 to 3422 mg Pb/kg, while the St. Joe River delta site sediment Pb
concentration was 29 mg Pb/kg. More than 70% of the Pb was associated with the
exchangeable/carbonate phase, which is thought to the be most bioavailable phase. Pb in the
exchangeable/carbonate fraction was negatively correlated with the abundance of Aquificae and
Synergistes and positively correlated with candidate phylum LD1PA abundance (a phylum without many
cultured representatives); furthermore, this pattern is similar for Fe and Mn oxyhydroxides, as Pb
exchangeable/carbonate concentrations are highly correlated. Bacteroidetes OTU abundance was
negatively correlated with Pb-exchangeable/carbonate and positively correlated with Pb-(oxy)hydroxide.
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These results suggest that the phase of Pb is integral in determining the relationship between Pb
concentration in the sediment and microbial communities, as seasonal changes in Pb speciation could
affect microbial diversity.
To understand how heterotrophic bacteria in river sediments are affected by Pb, sediments were
collected from sites along three tributaries of the Nagara River in Japan, varying in land use types
(agricultural, industrial, or forested) and contamination (Du et al.. 2018). Sediment samples were brought
into the lab and used in a sequencing batch incubation experiment, in which the sediment and water from
each site were placed in flasks with a C, N, and P source. The flasks were then spiked with a control or
three nominal concentrations of Pb (100 |ig/L. 1000 |ig/L. or 10000 (.ig/L). The pH was held constant at
7.0 due to the added P; therefore, the dissolved concentrations for Pb were likely lower than the initial
concentration added but were not measured in this study. Bacterial abundance and activity were sampled
every 3 days for 30 days, and the community structure was sampled on the first and last days of the
experiment (day 0 and day 30). Bacterial abundance (heterotrophic bacterial density) and heterotrophic
activity were not affected by Pb exposure. The dominant species at the end of the experiment differed
from that at the start of the experiment for all treatments; however, there was no difference in the most
abundant bacterial species at the end of the experiment, suggesting little effect of Pb on heterotrophic
bacterial communities. Overall, Pb did not have significant effects on heterotrophic bacteria density,
activity, and community structure after 30 days of an incubation experiment.
The Pb enrichment factor, along with other heavy metals, was found to influence bacterial
community structure in the Poyang Lake river system, China (Zhang etal.. 2018). Fifty-nine sediment
samples were collected from five rivers for heavy-metal analysis and characterization of microbial
communities. Mean Pb concentration in the sediments ranged from 29.52 to 40.06 mg Pb/kg. The Pb
enrichment factor, which takes into account Fe as the normalizer element, along with the As and Cd
enrichment factors, pH, OC, and degree of contamination were the main variables affecting bacterial
community structure (redundancy analysis). The Pb enrichment factor, as well as Cd enrichment factor
and the degree of contamination, was strongly associated with higher abundances of Acidobacteria,
suggesting tolerance of the phyla.
In another study in freshwater systems in China, Pb concentration in the sediment was found to
negatively correlate with the relative abundance of major bacterial groups, but not with bacterial diversity
(Li et al.. 2020). Pb concentration in the sediment was 17.3 ± 7.3 mg Pb/kg (mean ± S.D.) and ranged
from 1.9-25.4 mg Pb/kg across 12 sites in Huangjinxia Reservoir in Shaanxi Province, China. Sediment
Pb concentration was highly correlated with Cr, Zn, and Ni but not significantly correlated with microbial
diversity indices (ACE, Chaol, Shannon, and Simpson's index). However, Pb sediment concentration
was significantly negatively correlated with the relative abundance of Bacteroidota, Nitrospirota, and
Verrucomicrobia and positively correlated with the relative abundance of Chloroflexi. Finally, similar to
Zn and Cr, Pb sediment concentration was negatively correlated with nitrification and aerobic nitrate
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oxidation, as predicted through Functional Annotation of Prokaryotic Taxa, and with metabolism, as
predicted through phylogenetic investigation of communities by reconstructing unobserved states.
Variation in bacterial community composition along an elevation gradient in Yangtze River,
China, was driven by OM, elevation, urbanization, and Pb concentration (Zhang et al.. 2020) Sediments
were collected from 24 sites along the Yangtze River, and environmental parameters (soil pH, total N,
total P, and OM), trace metals (Pb, Cu, Pb, Cd, As) and bacterial diversity (OTU abundance, Shannon
index) were characterized and correlated using Nonmetric multidimensional scaling and Pearson
correlation analysis. Pb concentration in the sediment ranged from 14.40 ± 0.80 mg Pb/kg to
87.01 ± 8.00 mg Pb/kg. Elevation (meters above sea level) was negatively correlated with Pb
concentration in the sediment as were many other physicochemical parameters and metal concentrations.
The population density and urbanization rate were not significantly correlated with Pb. Redundancy
analysis found that the first axis of variation explained 47.9% of the variation in microbial communities
and the second axis explained 18.6% of the variation. OM was the most significant variable, followed by
elevation (10.4%), urbanization rate (9.0%) and Pb (9.5%,). Above 400 meters above sea level (masl),
elevation was the strongest factor correlated with bacterial community structure. Below 400 masl, OM,
urbanization rate and Pb exerted the strongest influence. Bacterial community structure between 50 and
400 masl was most correlated with Pb, and below 50 masl community structure was most correlated with
urbanization rate. Above 400 masl, Pb concentration and OTU abundance were significantly correlated,
while the correlations between Pb and the Shannon index and evenness were not significant. Below 400
masl, the opposite pattern emerged: the relationship between Pb and OTU abundance was negative and
the relationships between Pb and the Shannon index and evenness were nonsignificant. Finally, Pb
concentration was positively correlated with the abundance of certain bacterial genera, negatively
correlated with others, and not correlated with most dominant bacteria taxa.
In summary, since the 2013 Pb ISA (U.S. EPA. 2013). several observational and experimental
studies examining the effects of Pb concentrations in freshwater sediment and porewater found negative
associations with bacterial or archaeal abundance, but not diversity (Li et al.. 2020; Kang et al.. 2013;
Gough and Stahl. 2011). while others found mixed associations between Pb and microbial diversity
(Moberlv et al.. 2016) or no relationship (Du et al.. 2018).
11.3.4.2. Effects on Freshwater Plants and Algae
The toxicity of Pb to freshwater algae and plants has been recognized in earlier EPA reviews of
the metal and the findings are briefly summarized here. In the 1977 Pb AQCD, differences in sensitivity
to Pb among different species of algae were observed, and concentrations of Pb within the algae varied
among genera and within a genus (U.S. EPA. 1977). The 1986 Pb AQCD (U.S. EPA. 1986) reported that
some algal species (e.g., Scenedesmus sp.) were found to exhibit physiological changes when exposed to
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high Pb concentrations in situ. Effects of Pb on algae reported in the 2006 Pb AQCD included decreased
growth, deformation, and disintegration of algae cells, and blocking of the pathways that lead to pigment
synthesis, thus affecting photosynthesis. Most studies on effects of Pb in freshwater algal species
reviewed in the 2013 Pb ISA and the AQCDs were conducted with nominal media exposures and effect
concentrations greatly exceeded Pb reported in surface water. In studies in which Pb was quantified,
effect concentrations for growth (EC50) for freshwater algae and macrophytes were much higher than
currently reported environmental Pb. Growth endpoints in freshwater algae reviewed in the 2013 Pb ISA
included significant inhibition of chlorophyll a content at 210 (ig Pb/L and higher in Wolffia arrhiza
(Piotrowska et al.. 2010). An increase in biomass was reported in L. minor exposed to 100 or
200 |ig Pb/L, with inhibition observed at higher concentrations (Dirilgen. 2011). There were also
numerous studies conducted at nominal Pb concentration that reported effects on enzyme activities and
protein content in freshwater aquatic plant species. Exposure-response relationships in which increasing
concentrations of Pb lead to increasing effects were consistently observed for freshwater aquatic plants. In
the 2013 Pb ISA, the body of evidence was sufficient to conclude there were likely to be causal
relationships between Pb exposure and freshwater plant physiological stress and between Pb exposure and
reduced freshwater plant growth. The body of evidence was inadequate to conclude there are causal
relationships between Pb exposure and freshwater plant reproduction and between Pb exposure and
freshwater plant survival.
New information on freshwater algae since the 2013 Pb ISA addresses the deficit of analytically
verified chronic toxicity data for these organisms. De Schamphelaere et al. (2014) conducted 72-hour
bioassays in standard test media to assess growth rate in three commonly tested algal species; P.
subcapitata, C. kesslerii, and C. reinhardtii. P. subcapitata was the most sensitive, with
EC50 = 83.9 |ig Pb/L, EC20 = 45.7 |ig Pb/ and EC10 = 32.0 |ig Pb/L based on filtered Pb concentration.
Furthermore, in subsequent tests with P. subcapitata at varying pH, 72-hour EC50 decreased from 72.0 (ig
filtered Pb/L at pH 6.0 to 20.5 |ig filtered Pb/L at pH 7.6. The authors noted that this species exhibited
greater sensitivity to Pb than two of the most chronically Pb-sensitive aquatic invertebrates (the
crustacean C. dubia and the snail L. stagnalis) at pH > 7.4 based on model-predicted chronic EC50 values.
Additionally, new information on Pb effects on the emergent freshwater macrophyte, the common
reed (Phragmites australis), shows an alteration in growth form and propagation strategy under Pb
exposure. In a phytotron experiment, reed plants were exposed to five Pb levels in sediment (measured
5.9 ± 0.2, 304 ± 4.38, 508 ± 7.89, 1513 ± 37.28, 3020 ± 120.41 mg Pb/kg) (Zhang et aL 2015a). In
addition to decreases in total biomass, photosynthesis and rhizome growth, the addition of Pb caused a
significant alteration in growth form. The numbers of axillary shoot buds and daughter apical rhizome
shoots were increased by Pb addition at the highest concentrations, and the bulk (80%) of daughter shoots
were from daughter axillary shoots. This clonal propagation strategy of increased formation and output of
axillary shoot buds, called the phalanx pattern, is an adaptive response to maintain population stability at
the lowest energetic cost. This same growth pattern alteration was also found in an additional study on the
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effects of Pb and drought in P. australis by the authors (Zhang et al.. 2015b). but clonal modular growth
and reproductive ability were significantly inhibited by the interaction between drought and Pb. These
propagation effects would cause a decline in P. australis populations in a dry environment under Pb
pollution.
In summary, information published since the 2013 Pb ISA does not substantially change what
was previously known about Pb effects on freshwater plants and algae. A few new studies assessed the
sensitivity of freshwater algal growth to Pb exposure and found a significantly negative effect in certain
species. New information on Pb effects on common reed (P. australis) shows significant decreases in
total biomass, photosynthesis, and rhizome growth as well as alterations in growth form and propagation
strategy under Pb exposure. The growth and reproductive ability of common reed have also been shown
to be significantly inhibited by an interaction between Pb exposure and drought, which may have
implications for future drought events. There is still little information on the relationships between Pb
exposure and freshwater plant or algal survival, particularly at exposure levels below the thresholds used
in this ISA.
11.3.4.3. Effects on Freshwater Invertebrates
Freshwater aquatic invertebrates are generally more sensitive to Pb exposure than other taxa.
Controlled studies at concentrations near the upper range of representative concentrations of Pb available
from surveys of U.S. surface waters (median: 0.50 |ig Pb/L; range 0.04 to 30 |ig Pb/L, 95th percentile
1.1 jxg Pb/L) (U.S. EPA. 2006a) (Table 11-1) reviewed in the 1986 AQCD, the 2006 Pb AQCD and the
2013 Pb ISA provide evidence for the effects of Pb on reproduction, growth and survival in sensitive
freshwater invertebrates. Freshwater invertebrate taxa that exhibit sensitivity to Pb include some species
of gastropods, amphipods, cladocerans and rotifers, although the toxicity of Pb is highly dependent upon
water quality variables such as DOC, hardness, and pH. Key studies reported in the 1986 AQCD include
increased mortality as low as 19 jxg Pb/L for the snail Lymnaeapalustris (Borgmann et al.. 1978) and
reproductive impairment at 30 (ig Pb/L (nominal values) for Daphnia sp. (Bicsingcr and Christcnscn.
1972). In a 42-day chronic study reviewed in the 2006 Pb AQCD, the LOEC for reproduction was
3.5 jxg Pb/L in the amphipod H. azteca receiving both waterborne and dietary Pb (Besser et al.. 2005).
In the 2013 Pb ISA, additional studies provided evidence for Pb effects on freshwater
invertebrates at low |ig Pb/L concentration. The growth of juvenile freshwater snails (L. stagnalis) was
inhibited at an EC20 of <4 |ig Pb/L (Grosell and Brix. 2009; Grosell et al.. 2006b'). In fatmucket mussel, L.
siliquoidea juveniles, a chronic value (geometric mean of no-observed-effect concentration [NOEC] and
LOEC) of 10 |ig Pb/L was obtained following 28-day exposures (Wang et al.. 2010). In a 7-day exposure
of the cladoceran C. dubia to 50 to 500 |ig Pb/L, increased DOC led to an increase in mean EC50 for
reproduction ranging from approximately 25 |ig Pb/L to >500 |ig Pb/L (Mager et al.. 201 la). The 48-hour
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LC50 values for the cladoceran C. dubia tested in eight natural waters across the United States varied from
29 to 1,180 |ig Pb/L and were correlated with DOC (Esbaugh et al.. 2011). The freshwater rotifer E.
dilatata 48-hour LC50 was 35 |ig Pb/L using neonates hatched from asexual eggs (Arias-Almeida and
Rico-Martinez. 2011). The EC20 for reduced growth and emergence of the midge C. dilutus was reported
to be 28 |ig Pb/L, observed in a 5 5-day exposure study, while the same species had a 96-hour LC50 of
3,323 |ig Pb/L (Mcbanc et al.. 2008) The EC10 for molting in the mayfly B. tricaudatus was 37 |ig Pb/L
(Mebane et al.. 2008). These studies provided evidence in the 2013 Pb ISA supporting determinations of
causal relationships between Pb exposure and growth, reproductive effects, and survival in freshwater
invertebrates (Table 11-4).
11.3.4.3.1. Suborganism-Level Response
The key studies described above from the 2013 Pb ISA and earlier AQCDs report effects on
reproduction, growth, and survival in freshwater invertebrates. Additional endpoints for Pb toxicity in
aquatic invertebrates considered in the 2013 Pb ISA and previous AQCDs included suborganism-level
effects such as enzyme function and oxidative stress. These suborganism-level effects were considered
together in the 2013 Pb ISA as "physiological stress" and the body of evidence was sufficient to conclude
that there is a likely to be causal relationship between Pb exposure and altered response. Although stress
responses are correlated with Pb exposure, they are nonspecific and may be altered with exposure to any
number of environmental stressors. An additional suborganism-level endpoint in the 2013 Pb ISA was
"hematological effects," which included changes to ALAD expression or the hematopoietic system
associated with Pb exposure. For this endpoint, the body of evidence was sufficient to conclude that there
is a likely to be causal relationship between Pb exposure and hematological effects in freshwater
invertebrates in the 2013 Pb ISA. These suborganism-level responses may serve as biomarkers for effects
at the organism level and higher; however, only a subset of studies that quantified response at the
suborganismal level concurrently assessed effects on growth, reproduction, development, or survival.
Only a few of the many studies identified in the literature search on suborganism-level response to Pb
exposure in freshwater invertebrates were conducted in the low |ig Pb/L range and hence met the criteria
for inclusion in the ISA.
Recent literature strengthens the evidence for Pb effects on enzymes and antioxidant activity in
freshwater invertebrates. New studies on physiological stress endpoints include changes in the activities
of antioxidant defense enzymes such as SOD, CAT and GPx with aqueous exposure to Pb. Juvenile D.
magna exposed nominally to 16 jxg Pb/L exhibited statistically significant decreased intracellular ROS
and increases in total GSH level and SOD activity in 48-hour exposure (Kim etal.. 2018). In the same
study, the expression patterns of several molecular biomarker gene transcripts were observed. Daphnid
neonates tested with the same concentration as juveniles showed a greater response to Pb exposure,
suggesting that the neonate lifestage is more susceptible to Pb. SOD and GPx activities were significantly
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reduced, and MDA levels were significantly increased in juvenile Oriental river prawn (Macrobrachium
nipponense) exposed to 25 |ig Pb/L for 60 days. CAT activity in the hepatopancreas increased at
12 |ig Pb/L and decreased in the 25 |ig Pb/L treatment (Ding et al.. 2019). In the same study, reductions in
weight gain and specific growth rate were observed in prawns exposed to 25 |ig Pb/L in chronic 60-day
exposure tests. No growth effects were observed in prawns at 12 |ig Pb/L (see Section 11.3.5).
Physiological stress in freshwater invertebrates was also assessed during sediment exposure to
Pb. Exposure of larval midge Chironomus riparius to Pb-spiked sediment (132 mg Pb/kg dry weight and
505.5 mg Pb/kg dry weight) for 16 days resulted in an antioxidant response (increase in metallothionein)
and cellular damage (increase in MDA) (Arambourou et al.. 2013). There was no significant change to
protein concentration, lipid was depleted while glycogen increased with increasing Pb in the sediment. In
the same organisms, Pb exposure via sediment did not result in statistically significant effects on growth,
survival, or number of mentum (mouthpart) deformities. In a separate study in C. riparius in Pb-spiked
sediment ranging from 18.1 to 456.9 mg Pb/kg dry weight, no significant differences were observed in the
frequency of mouthpart deformities (Arambourou et al.. 2012). In freshwater snail Bellamya aeruginosa
exposed for 28 days to Pb-spiked sediment, CAT activity and metallothionein were significantly induced
at the lowest concentration tested (29.7 mg Pb/kg dry wcightHLiu et al.. 2019b). In the bivalve Hyridella
australis also exposed 28-days to Pb-spiked sediments (205 ± 9 and 419 ± 16 mg Pb/kg dry mass), the
body burden of accumulated Pb was low (2.2 ± 0.2 mg Pb/kg dry mass and 4.2 ± 0.1 mg Pb/kg dry mass,
respectively); however, total antioxidant capacity significantly decreased while ROS and MDA increased
with Pb exposure compared with controls (Marasinghe Wadige et al.. 2014).
As reported in the 2013 Pb ISA, inhibition of ALAD enzyme activity, an important rate-limiting
enzyme needed for heme production, is a recognized biomarker of Pb exposure in some freshwater
invertebrate species that have hemoglobin. Previous studies have indicated considerable species
differences in ALAD activity in response to Pb. For example, the concentration at which 50% ALAD
inhibition was measured in the freshwater gastropod Biomphalaria glabrata (23 to 29 |ig Pb/L) was much
lower than that in the freshwater oligochaete L. variegatus (703 |ig Pb/L), based on nominal exposure
data (Aisemberg et al.. 2005). No recent studies quantifying ALAD activity in freshwater invertebrates at
environmentally relevant concentrations of Pb were identified for inclusion in this ISA. Furthermore, no
significant ALAD activity was detected at baseline metabolic conditions in hemolymph or tissue of the
freshwater unionoid mussel E. complanata, suggesting this is not a viable biomarker for the species
(Mosher et al.. 2012a).
11.3.4.3.2. Organism-Level Response
Organism-level endpoints include effects on behavior linked to Pb neurotoxicity. In the 2013 Pb
ISA, the body of evidence was sufficient to conclude there is a likely to be causal relationship between Pb
exposure and neurobehavioral effects in freshwater invertebrates (U.S. EPA. 2013) (see Table 11-4 of this
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appendix). In limited studies available on worms and snails, there is evidence that Pb may affect the
ability to escape or avoid predation. For example, in the tubificid worm T. tubifex, the 96-hour EC50 for
immobilization was 42 |ig Pb/L (Khangarot. 1991). Some organisms exhibit behavioral avoidance while
others do not seem to detect the presence of Pb (U.S. EPA. 2006b). Additional behavioral endpoints
reported in the Great Lakes Environmental Center draft Ambient Aquatic Life Water Quality Criteria for
Lead document GLEC (2008) include an EC50 of 140 |ig Pb/L for feeding inhibition in the freshwater
cladoceran C. dubia. In a study published since the 2013 Pb ISA, adult amphipods, G. fossarum exposed
to Pb for 5 days at a concentration at which survival was unaffected (2.7 |ig Pb/L) exhibited sublethal
behavioral and physiological responses. Locomotion was significantly decreased overtime (assessed 24,
48 and 120 hours) and respiration rate was significantly lower at 120 hours compared with unexposed
amphipods (Lebrun et al.. 2017). In a separate study with G. fossarum, both locomotion and respiration
were significantly decreased following exposure to 2.1 |ig Pb/L for 24-hour (Lebrun and Gismondi.
2020).
As described in the 2013 Pb ISA and previous AQCDs, Pb is neurotoxic to many organisms.
Alterations in neurotransmitter regulation and release may be an underlying mechanism for the behavioral
effects of Pb. Few studies in freshwater invertebrates have reported effects on neurotransmitters at lower
Pb concentrations. In prereproductive freshwater bivalve Lamellidens jenkinsianus obesa exposed for
21 days to either 68 or 763 |ig Pb/L, AChE activity (assessed on days 1, 7, 15 and 21 of the experiment)
was significantly inhibited at each timepoint compared with control (Brahma and Gupta. 2020). Several
locomotor behaviors (movement in the form of gliding, foot-siphon extension) were significantly reduced
or ceased completely in the Pb-exposed individuals compared with the control during a separate 5-day
exposure to either 69 or 776 |ig Pb/L. In the same study, reproductive-age individuals of another bivalve
species, Parreysia corrugata, were exposed to either 26 or 302 |ig Pb/L for 21 days. AChE activity was
significantly induced at 26 |ig Pb/L and significantly inhibited compared with control at 302 |ig Pb/L at
all timepoints. Behavioral response in the form of impaired movement with Pb exposure (25 and
304 |ig Pb/L) was also observed in this species. In 28-day chronic exposure of freshwater snail B.
aeruginosa to Pb-spiked sediment, the activity of the neurotransmitter AChE was significantly induced
starting at day 7 in the lowest concentration (29.7 mg Pb/kg dry weight) (Liu et al.. 2019b).
In the 2013 Pb ISA, the body of evidence was sufficient to conclude there is a causal relationship
between Pb exposure and growth in freshwater invertebrates (U.S. EPA. 2013) (see Table 11-4 of this
appendix). The growth of freshwater snail L. stagnalis was identified as one of the most sensitive
organisms and endpoints for Pb toxicity. At the time of the 2013 Pb ISA, the hypersensitivity of this
species to Pb was hypothesized to be from Pb inhibition of Ca2+ uptake. Subsequent experiments by Brix
et al. (2012) observed that effects on growth occur prior to effects on net Ca2+ flux, inhibition of carbonic
anhydrase activity in the snail mantle also showed no effect with Pb; therefore, the mechanism of Pb in
these highly sensitive organisms remains elusive. Additional studies reported in Section 11.3.5, Exposure
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and Response of Freshwater Species, support Pb effects on the growth of L. stagnalis in the low |ig Pb/L
range (Crcmazv et al.. 2018; Brix et al.. 2012).
Exposure-response studies discussed in Section 11.3.5. also add to the existing body of evidence
in the 2013 Pb ISA for a causal relationship between Pb exposure and reproductive effects as well as
survival in freshwater invertebrates. In summary, studies in freshwater invertebrates for suborganism-
level and organism-level endpoints are confirmatory with findings in the 2013 Pb ISA, with evidence in
additional species for some effects.
11.3.4.4. Effects on Freshwater Vertebrates
The 1977 Pb AQCD reported Pb effects in both fish and waterfowl. The available Pb studies on
waterfowl investigated exposure to Pb via accidental poisoning or ingestion of Pb shot (U.S. EPA. 1977).
Studies on aquatic vertebrates reviewed in the 1986 Pb AQCD were limited to hematological,
neurological, and developmental responses in fish (U.S. EPA. 1986). In the 2006 Pb AQCD, effects on
freshwater vertebrates included consideration of the role of water quality parameters on toxicity to fish, as
well as limited information on the sensitivity of turtles and aquatic stages of frogs to Pb (U.S. EPA.
2006a). In the 2013 Pb ISA, the body of evidence was sufficient to conclude there is a causal relationship
between Pb exposure and hematological effects, reproduction and survival in freshwater vertebrates
(based primarily on evidence from fish) (U.S. EPA. 2013) (see Table 11-4 of this appendix). There were
also likely to be causal relationships concluded between Pb exposure and physiological stress and
neurobehavioral effects. Newly available studies on the effects of Pb in fish and other freshwater
vertebrates are summarized below.
11.3.4.4.1. Fish
11.3.4.4.1.1. Suborganism-level Response
A large body of evidence supports sublethal biomarker perturbations with Pb exposure in
freshwater vertebrates; however, few studies were identified for this ISA that reported physiological
response at more environmentally relevant concentrations ofPb (< 10 |ig Pb/L; Section 11.1.1) or
concurrently assessed response at organism-level endpoints (i.e., from the cellular and subcellular level to
effects on growth, reproduction or survival). Various biomarkers of oxidative stress assessed in carp
(Carassius auratus gibelio) after 96 hours and 21 days were significantly altered at analytically verified
concentration of 10 and 30 |ig Pb/L (Khan et al.. 2015). For the acute exposure, CAT activities (liver and
kidney) were significantly reduced, and SOD was significantly upregulated in brain, kidney, and muscle
tissue. GPx activity in the liver and gill increased significantly, while activity in the muscle and kidney
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was significantly reduced. Biomarker response in chronic exposure showed significant reduction in CAT
(liver, gill, muscle) at 10 and 30 |ig Pb/L, whereas CAT was upregulated in the kidney. There was a
decline in GPx (liver and gill) as well as in SOD (liver, kidney, muscle), while the brain showed an
increase. Acetylcholine, a biomarker of neurotoxic stress, was significantly inhibited following chronic
exposure to 30 |ig Pb/L. Clemow and Wilkie (2015) observed no significant effect on respiratory stress,
mean cell hemoglobin concentration, plasma Ca2+ or Na2+ ion concentration or plasma protein in juvenile
rainbow trout (O. mykiss) over a 5-day exposure to 5.4 |ig Pb/L (26.1 nmol/L). In fingerling rainbow
trout, used in the same study for unidirectional Na+ flux measurement, there was an initial Na+ loss after
48 hours of exposure that recovered by 72 hours with exposure to 8.3 |ig Pb/L (40.2 nmol/L).
Hematological effects of Pb on fish reported in the 2013 Pb ISA and AQCDs include a decrease
in red blood cells and inhibition of ALAD with elevated Pb exposure under various test conditions.
Inhibition of ALAD is also reported in environmental assessments of metal-impacted habitats. For
example, as reported in the 2013 Pb ISA, lower ALAD activity has been significantly correlated with
elevated blood Pb concentrations in wild-caught fish from Pb-Zn mining areas, although there are
differences in species sensitivity (Schmitt et al.. 2007; Schmitt et al.. 2005). Few studies were identified
since the 2013 Pb ISA that quantify ALAD response in freshwater fish in laboratory exposure at
concentrations considered for this ISA (Section 11.1.1). Olson et al. (2018) reported gene transcription of
ALAD was significantly induced in zebrafish at 100 |ig Pb/L and higher nominal exposure. In a field
study of brown trout (Salmo trutta) collected from a lake in Norway contaminated with Pb (14 (ig Pb/L)
from an abandoned shooting range, ALAD activity in the trout population was approximately 20% of that
of a relatively uncontaminated reference lake (0.76 |ig Pb/L) (Mariusscn et al.. 2017).
11.3.4.4.1.2. Organism-Level Response
In the 2013 Pb ISA, studies supporting a likely to be causal relationship between neurobehavioral
endpoints in freshwater vertebrates and Pb exposure included research from early EPA reviews of the
metal. In the 1977 Pb AQCD, behavioral impairment of a conditioned response (avoidance of a mild
electric shock) in goldfish was observed at concentrations as low as 70 (ig Pb/L (Weir and Hine. 1970). In
the 2006 Pb AQCD, several studies were reviewed in which Pb was shown to affect predator-prey
interactions, including alteration in prey size choice and delayed prey selection in juvenile fathead
minnows following 2-week pre-exposure to 500 |ig Pb/L (Weber. 1996). Prey capture ability was
decreased in 10-day old fathead minnow larvae born from adult fish exposed to 120 |ig Pb/L for 300 days,
then subsequently tested in a 21-day breeding assay (Mageretal.. 2010).
Since the 2013 Pb ISA, there have been additional studies on neurobehavioral response in
freshwater vertebrates, particularly in zebrafish D. rerio. As a widely used model organism in
environmental toxicology, the zebrafish genome shares a high degree of homology with the human
genome (Dai et al.. 2014; Howe et al.. 2013). Zebrafish are used as an animal model for human health
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outcomes associated with Pb exposure such as neurogenerative disease (reviewed in Lee and Freeman
(2014)) and developmental and neurobehavioral alterations (Li et al.. 2019). Endpoints assessed in these
zebrafish assays, such as decreased locomotor activity and altered social interactions used as surrogates
for autistic behaviors in humans, can affect organism fitness in natural environments. Furthermore, many
of these studies link changes in gene expression, neurotransmitter levels or other molecular and cellular
responses to the observed behavioral outcomes. Experiments conducted in the low |ig/L range are
particularly representative of environmental concentrations (Table 11-1); therefore, zebrafish behavioral
assay studies conducted at low concentrations of Pb are reviewed below.
In zebrafish embryos exposed to 5.0, 9.7 or 19.2 |ig Pb/L there were no significant effects on
dorsal axon length up to 144 hours postfertilization (hpf); however, there was a significant reduction in
swimming speed at the highest Pb concentration tested (Zhu et al.. 2016). Alterations in the
neurotransmitter gamma-aminobutyric acid (GABA) were observed during development of zebrafish
embryos exposed to Pb (nominally to 10, 50 and 100 |ig Pb/L up to 72 hpf, Pb uptake was quantified in
embryos) (Wirbiskv et al.. 2014). The levels of this neurotransmitter varied with the dose of Pb and
developmental stage, with all three treatments resulting in a significant decrease in GABA by the end of
embryogenesis (72 hpf). Newly-hatched larval zebrafish exposed to Pb since 2.5 hpf exhibited
neuromuscular responses (increased muscular twitching) at concentrations of 49.6 and 100.7 |ig Pb/L at
72 hpf. No twitches were observed at lower concentrations or in the control group (Kataba et al.. 2022). In
another study, locomotor and social behavior responses were assessed in zebrafish larvae exposed to 4.5,
9.6 or 18.6 |ig Pb/L at 6 days postfertilization (dpf) during a dark and light photoperiod (Zhao et al..
2020). During the dark period, swimming activity was significantly decreased at 18.6 |ig/L. and at both
9.6 and 18.6 |ig/L. there was a decrease in clockwise turning; social contact time was significantly higher
in the light period at the highest Pb concentration. Downregulation of genes involved in brain neutrophic
factor signaling was observed in the Pb-exposed larvae, suggesting an underlying mechanism for the
observed responses. Hyperactivity (increased distance covered and speed) during the light period was
observed in larval zebrafish exposed to Pb (3.2, 93 or 252.6 |ig Pb/L) for 30 minutes in alternating light
and dark intervals of 10 minutes (Kataba et al.. 2020).
Several studies in zebrafish have considered the neurobehavioral effects of Pb at multiple
lifestages. Wang et al. (2022) exposed zebrafish embryos from 2 hpf to 120 hpf to nominal concentration
of 20, 50, 100 or 250 |ig Pb/L (0, 0.1, 0.25, 0.5 |iM) then examined whether the effects of the early-life
exposures persisted in juveniles and adults. Spinal curvature and hyper swimming activity were observed
in embryos exposed to the lowest concentration. Next, the fish were held in Pb-free conditions and 1-
month old juveniles and 4-month-old adult zebrafish exposed at the two lowest concentrations as embryos
(20 and 50 |ig Pb/L) were evaluated in various behavioral assays. Juvenile fish exposed to Pb in the
embryo stage exhibited significantly elevated hyperactivity and over-response to stimuli compared with
the control fish. Adult fish raised from Pb-exposed embryos were hyperactive and displayed anxiety-like
behaviors consistent with other studies in fish. Wang et al. (2018b) assessed swimming behavior in larval
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(15 dpf)) and juvenile (30 dpf) zebrafish that were continually exposed to Pb (analytically verified
concentration of 10 (ig Pb/L or 100 |ig Pb/L) from maternal exposure through egg fertilization and
subsequent larval development. Larval responses to Pb exposure included decreases in measures of
locomotion such as angular velocity, turn angle and inter-fish distance, a measure of social behavior.
Juvenile zebrafish exhibited similar behavioral responses to Pb; however, the inter-fish distance
increased, and there were increases in the percentage of fish moving up to the top of the tank. The
expression of key genes linked to behaviors, Ca channels and the metabolism of environmental
contaminants were altered with Pb exposure.
Reproductive outcomes in fish may also be affected by Pb-associated neurobehavioral alterations.
Courtship behaviors of adult male zebrafish exposed for 2 weeks to Pb (nominal concentration of 1, 10
and 100 |ig Pb/L) exhibited a biphasic response to Pb, with hyperactivity observed at low concentrations
and inhibitory effects at higher concentrations (Li et al.. 2019). The study used a video system optimized
for tracking zebrafish behavior to record the locomotion profiles of male fish interacting with female fish.
Movements including total velocity, vertical velocity, turning, and total distance were quantified to
evaluate changes in swimming trajectory patterns with Pb exposure. A U-shaped dose-response was
reported for total velocity and total distance while turning angle and turning speed were not significantly
affected by Pb treatment. Concurrent with the behavioral study, the transcription patterns of key genes
involved in testicular steroidogenesis and apoptosis were evaluated in tissue of testes of exposed males.
Most genes exhibited upregulation after low-level Pb exposure and downregulation after high-level Pb
exposure, consistent with the behavioral assays.
There is some evidence for parental transfer and transgenerational effects on fish learning and
avoidance behavior following Pb exposure. Zebrafish larvae (15 dpf) hatched from adult females
previously exposed to 19.5 (ig Pb/L were used as a model to test autism-like behaviors (Wang et al..
2016). Behaviors assessed included measures of locomotion, and repetitive, social and anxiety behaviors.
Analysis of larval swimming activity recorded on video indicated significant increases in distance moved
and swimming velocity compared with control larvae. No significant differences were observed in inter-
fish distance, angular velocity or turn angle. Additionally, changes in the expression of several genes
associated with autism-like behaviors were detected in the larvae hatched from the Pb-exposed fish. Adult
zebrafish exposed nominally to Pb (0.1 |iM [20 |ig Pb/L], 1.0 (j,M [200 |ig Pb/L] or 10.0 |iM
[2000 |ig Pb/L]) as embryos (to 24 hpf) and then raised in Pb-free medium were tested for avoidance
response and conditioning (Xu et al.. 2015). At the lowest concentration, adult zebrafish learned
avoidance responses during training and testing, while fish exposed to the higher concentrations of Pb
displayed no significant changes in avoidance response. In F3 offspring of the Pb-exposed embryos, these
learning deficits persisted at the two higher Pb concentrations.
In the 2013 Pb ISA, evidence was inadequate to establish a causal relationship between Pb
exposure and growth effects in freshwater vertebrates. Since the 2013 Pb ISA, a few additional studies in
fish have assessed the effects on growth following dietary or aqueous exposure to Pb. In chronic dietary
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exposure (24 months) to 8-49 mg Pb/kg in food pellets, there were no significant differences in fish body
weights or the survival of Prussian carp C. gibelio females (Luszczek-Trojnar et al.. 2013). In another
study with adult female carp C. carpio exposed to Pb via diet (68.4 mg Pb/kg dry weight in food pellets),
there were no significant differences in mean body weights at the end of the study (three exposure
seasons), although Pb-exposed fish weighed significantly less than control fish after the first exposure
season (Luszczek-Trojnar et al.. 2016). This is consistent with dietary studies reviewed in the 2013 Pb
ISA (Alves and Wood. 2006). In aqueous exposure studies, zebrafish embryos exposed to Pb
(19.3 |ig Pb/L) to 6 dpf (144 hpf) showed no significant differences in hatching success, body length or
body weight compared with the control (Chen et al.. 2016b). Similarly, exposure of zebrafish embryos to
Pb (5.0, 9.7, 19.2 |ig Pb/L) up to 144 hpf did not affect growth rate or survival (Zhu et al.. 2016). No
differences in head length, head width or total body length were observed in 72 hpf embryos exposed
nominally to 10, 50 or 100 |ig Pb/L (Wirbiskv et al.. 2014).
For the effects of Pb on reproduction and development in freshwater vertebrates, the weight of
evidence for the causal relationship in the 2013 Pb ISA was primarily from studies with fish. Pb AQCDs
have reported developmental effects in fish, specifically spinal deformities in brook trout (Salvelinus
fontinalis) exposed to 1 19 |ig Pb/L for three generations (U.S. EPA. 1977). as well as in rainbow trout
exposed to concentrations as low as 120 |ig Pb/L (U.S. EPA. 1986). In the 2006 Pb AQCD (U.S. EPA.
2006a'). decreased spermatocyte development in rainbow trout was reported at 10 (ig Pb/L, and testicular
damage occurred in fathead minnow at 500 |ig Pb/L. In the 2013 Pb ISA, a 300-day chronic toxicity study
was conducted by Mager et al. (2010) in fathead minnows treated with both 31 and 112 |ig Pb/L with
HCO3 and with 130 (ig Pb/L with DOC. The total reproductive output was decreased, and average egg
mass production increased as compared with egg mass size in controls and in low HCO3 and DOC
treatments with Pb. Other supporting evidence for the causal determination in the 2013 Pb ISA for
reproductive effects in aquatic vertebrates included alteration of steroid profiles and additional
reproductive parameters, although most of the available studies were conducted using nominal
concentrations of Pb. Additionally, a study in frogs in the 2006 AQCD showed Pb delayed
metamorphosis, decreased larval size and caused skeletal malformations at nominal concentration of
100 |ig Pb/L; however, tissue concentrations quantified in frogs following exposure fell within the range
of tissue concentrations in wild-caught tadpoles (Chen et al.. 2006).
Several new early lifestage fish studies add to the existing evidence for Pb effects on endocrine
and developmental endpoints. In a study that quantified Pb in the exposure water, hatching success rates
in zebrafish embryos were reduced at 4.5, 9.6 and 18.6 |ig Pb/L. At 72 hpf, the hatching success rates in
all three concentrations were significantly decreased compared with the control, indicating that Pb caused
a hatching delay, which was also observed at the end of the experiment at 96 hpf (Zhao et al.. 2020).
Curcio et al. (2021) also reported a hatching delay in zebrafish embryos at 102 hpf with nominal exposure
to 5 |ig Pb/L. In this study, various embryo developmental effects were noted at 5 |ig Pb/L and at the
lower concentration of 2.5 |ig Pb/L. All individuals showed spinal and tail deformities after 144 hours of
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exposure. In contrast, in adult zebrafish exposed nominally to 10 (ig Pb/L for 3 months, there were no
significant effects on mortality, malformation, egg production and subsequent growth of larval offspring
(Chen et al.. 2017). In another zebrafish study, endocrine disruption in larvae was assessed by quantifying
changes in thyroid hormone following exposure to Pb (analytically verified concentration of 2, 5, 10, 15,
20, 30 |ig Pb/L) in embryos from 2 hpf to 144 hpf (Zhu et al.. 2014). Triiodothyronine (T3) and thyroxine
(T4) levels were significantly reduced at 30 |ig Pb/L. Pb did not significantly affect the percentage of
hatched larvae; however, Pb exposure significantly increased malformations and reduced survival at
30 |ig Pb/L compared with the control. In comparison to these studies showing reproductive and
endocrine responses in fish early lifestages, no endocrine disruption was observed in adult male common
carp (C. carpio) at 7, 14 or 21 days of Pb exposure, even at the lowest analytically verified concentration
(120 (ig Pb/L) (Korkmaz et al.. 2022).
Reproductive and endocrine effects of exposure to Pb via diet were assessed in dietary exposure
with female Prussian carp C. gibelio. At 12 months, there was a significant increase in luteinizing
hormone (LH) secretion after hormonal stimulation at the two highest analytically verified concentrations
(24 and 49 mg Pb/kg), whereas (8 mg Pb/kg) spontaneous LH secretion significantly decreased at the
lowest dose tested (Luszczck-Troinar et al.. 2014). At 24 months, differences in LH secretion between
treatment groups were not significant. There were also differences in oocyte size and maturation. At
12 months, oocytes in the 8 mg Pb/kg treatment group were significantly larger than those in the control
and other treatment groups. After 24 months, oocyte maturity and oocyte diameter were not significantly
different between the control and Pb-treated fish.
An emerging area of ecotoxicology involves the assessment of pollutant effects on the
microbiome and subsequent fitness of the host organism (Evariste et al.. 2019). Since the 2013 Pb ISA,
gut microbiota as a target for Pb toxicity have been assessed in zebrafish. In adult male zebrafish exposed
for 7 days to a nominal concentration of 10 or 30 (ig Pb/L, gut mucus production increased. The relative
abundance of a-Proteobacteria decreased significantly and the relative abundance of Firmicutes
significantly increased at 30 (ig Pb/L relative to the control (Xia et al.. 2018). Approximately 30 kinds of
microorganisms responded to Pb, and concurrent with altered gut microbiota composition, a total of 41
metabolites associated with metabolic pathways and liver function were significantly changed.
11.3.4.4.2. Birds
A new study in mallards (A. platyrhynchos) expands existing information on Pb effects in birds
frequenting aquatic habitats contaminated with Pb and other metals. Prior AQCDs and the 2013 Pb ISA
include evidence for changes in ALAD activity and other oxidative stress biomarkers. Adding to this
evidence, there was a positive relationship between the lipid peroxidation index and blood Pb in female
mallards sampled in northeastern Spain. Lysozyme levels were negatively correlated with blood Pb
concentrations (Vallverdu-Coll et al.. 2016). Additionally, in male mallards, there were significant
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relationships between blood Pb and beak and leg hue. In mallards, male leg and beak color typically
ranges from orange-red to yellow-orange and from yellow-orange to green, with redder beaks and
yellower legs typically being more attractive to females. In this study, the leg redness of males had a
significant negative relationship with blood Pb levels, as did beak yellowness. This indicates that male
mallards with higher blood Pb levels are likely to be less attractive to females, and therefore could
potentially have lower reproductive success. Another study from the same author investigated how blood
Pb levels in mallard chicks can affect multiple suborganismal and organismal-level effects (Vallverdu-
Coll et aL 2015). This study on the same population of mallards in northeastern Spain found that
ducklings with blood Pb levels above 180 ng/mL showed reduced body mass and died during the first
week posthatching. Additionally, cellular immune function at day 15 in ducklings was negatively
correlated with Pb levels in blood on the same day.
11.3.4.4.3. Amphibians
Since the 2013 Pb ISA, new laboratory studies on the effects of Pb exposure on freshwater
amphibians have focused on tadpole growth, development, and survival. Two different studies evaluated
the effects of Pb-contaminated water on Asiatic toad (Bufo gargarizans) tadpole growth and development.
Chai et al. (2017) reared Asiatic toad embryos and tadpoles in different nominal concentration of Pb-
contaminated water (0, 10, 50, 100, 500, 1000, and 2000 |ig Pb/L). At 5 days of exposure, the total length
of embryos was significantly lower in 1000 and 2000 |ig Pb/L treatments than in controls; however, the
total length was significantly higher at 50 (ig Pb/L than the length in controls. Similar results were seen in
the mean weight of embryos on day 5, with embryos from the two highest exposures being significantly
lighter than controls, while embryos from the three lowest treatments were significantly heavier than
controls. Malformations (edema in the tail, wavy fin, abdominal edema, stunted growth, hyperplasia, and
axial flexures) were observed starting at the 500 |ig Pb/L treatment, with the incidence of malformation
increasing with Pb concentration. Yang et al. (2019) performed a similar experiment and obtained similar
results. Asiatic toads were reared in water with different concentrations of Pb (0, 10, 50, 100, 500, and
1000 (ig Pb/L, nominal values; 0, 9.85, 48.73, 97.69, 497.34, and 998.27 (ig Pb/L, measured values). On
day 10, there was a significant increase in total length and body mass at 50 (ig Pb/L and a significant
decrease in snout-to-vent length at 1000 |ig Pb/L compared with controls. However, farther along in
development at day 20, there was a significant decrease in snout-to-vent length at 100 and 500 |ig Pb/L
compared with controls.
Huang et al. (2014) examined the effect of Pb on these endpoints in dark-spotted frogs
(Pelophylax nigromaculata). Tadpoles were reared in different concentrations of Pb (40, 80, 160, 320,
640, 1280 (ig Pb/L nominal values; 38.2, 79.3, 158.4, 318.7, 638.1, 1278.9 (ig Pb/L analytically verified
concentration) from heartbeat to complete tail reabsorption. The threshold concentrations for effects on
body mass, snout-vent length, forelimb length, and hindlimb length were 160, 160, 160, and 320 |ig Pb/L,
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with total malformation rate increasing linearly with Pb concentration. Metamorphosis time was
significantly affected by Pb concentration and exhibited a linear increase with increasing Pb concentration
(0 ng Pb/L = 76.4 ± 0.5 days, 160 pg Pb/L = 90.8 ± 0.5 days, 1280 pg Pb/L = 118.4 ± 0.5 days). Pb
concentration also significantly affected the survival rate, which decreased with increasing Pb
concentration (0 |ig Pb/L = 98.3 ± 1.7%, 160 |ig Pb/L = 93.3 ± 1.7%, 1280 |ig Pb/L = 80.0 ± 0.3%).
Other than the studies in fish described above and in the following section on exposure-response,
there is limited new information regarding Pb toxicity in freshwater vertebrates. For fish, studies are
largely confirmatory with studies in the 2013 Pb ISA. Additional research with zebrafish augment
existing understanding of Pb effects on neurobehavior and reproductive endpoints.
11.3.5. Exposure and Response of Freshwater Species
Evidence regarding exposure-response relationships and potential thresholds for Pb effects on
aquatic populations can provide tools for quantitative analyses of risks in freshwater ecosystems (Section
11.1.7.3). Exposure-response data for the reproduction, growth, and survival of freshwater biota
(including microalgae, invertebrate, amphibian, and fish species) were summarized in Table 6-5 of the
2013 Pb ISA (U.S. EPA. 2013). Additionally, the Annex of the 2006 Pb AQCD (U.S. EPA. 2006b)
summarized data on exposure-response functions for invertebrates (Table AX7 2.4.1) and fish (Table
AX7 2.4.2) available at the time. For Pb exposure-response, there is significant new research reporting
results from bioassays of freshwater algae, invertebrates and fish based on measured rather than nominal
concentration of Pb. In some cases, effects were observed in sensitive species at concentrations
comparable to or lower than those reported in the 2013 Pb ISA (Table 11-5) or earlier EPA reviews of Pb.
Some of the studies report LCio and LC20 toxicity values and/or calculate the free-ion concentration.
In the 2006 AQCD and 2013 Pb ISA, available exposure-response data for freshwater plants and
algae did not indicate any effects on growth or survival at environmentally relevant concentrations. In the
2006 AQCD, EC50 values for growth inhibition in various freshwater algal and aquatic plant species were
between approximately 1000 and >100,000 |ig/L and were mostly based on nominal concentration data
(U.S. EPA. 2006b). An important advancement since the 2013 Pb ISA is the availability of bioassay data
for algal growth rate in several freshwater species based on measured Pb concentration instead of nominal
concentration, which strengthens confidence in the findings for the concentrations assessed (De
Schamphelaere et al.. 2014). In chronic 72-hour bioassays in standard test media to assess the growth rate
in three commonly tested algal species (P. subcapitata, C. kesslerii, C. reinhardtii), P. subcapitata was
the most sensitive, with EC50 = 83.9 |ig Pb/L, EC20 = 45.7 |ig Pb/L and EC10 = 32.0 |ig Pb/L based on
filtered Pb concentrations (De Schamphelaere et al.. 2014). Furthermore, in subsequent tests with P.
subcapitata at varying pH, the 72h EC50 decreased from 72.0 |ig filtered Pb/L at pH 6.0 to 20.5 |ig filtered
Pb/L at pH 7.6. Inhibitory concentration (IC) values calculated using a specific growth rate at 72 hours
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with a linear interpolation method for Raphidocelis subcapitata (formerly P. subcapitata) were
ICio = 0.15 (j,M, (31 ng Pb/L), IC25 = 0.39 (81 pg Pb/L) and IC50 = 0.78 (161 Pb/L) (Mho et
al.. 2019).
In addition to freshwater algae, there is new toxicity information based on measured Pb
concentration for freshwater plants. The toxicity of Pb to duckweed Lemna minor expressed as percent
net root elongation was assessed in chronic bioassays of seven U.S. surface waters with different water
chemistries (Antunes and Krcagcr. 2014). The 20% IC in 7-day static renewal tests with the waters ranged
from 306 nM to >6920 nM (63 |ig Pb/L to >1,433 |ig Pb/L) expressed as total dissolved Pb indicating that
Pb speciation, solubility, subsequent bioavailability, and toxicity varied under the range of water
hardness, pH, and DOC in the tested waters.
For freshwater invertebrates, effects in sensitive species of amphipods, gastropods, cladocerans
and mussels were reported at low |ig Pb/L concentrations in exposure-response studies reviewed in the
1986 AQCD, the 2006 AQCD and the 2013 Pb ISA. Additional toxicity data for these taxonomic groups
discussed below support and expand upon what was known in the previous Pb assessment in terms of the
relative sensitivity of these freshwater biota to Pb.
Toxicity testing with amphipods reported in the 2006 AQCD and 2013 Pb ISA indicate a
response to Pb at <10 |ig Pb/L under some water conditions. At higher pH and water hardness, these
organisms are less sensitive to Pb (U.S. EPA. 2006b). For example, a 7-day LC50 of 1 |ig Pb/L was
observed in soft water with the amphipod H. azteca (Borgmann et al.. 2005). In this same species, the 96-
hour LC50 for Pb at pH 5 was 10 fxg Pb/L (Mackie. 1989). In 42-day chronic exposures of H. azteca
exposed to Pb via water and diet, the LC50 was 16 jxg Pb/L (Besser et al.. 2005). In a chronic 42-day
bioassay w ith H. azteca, published after the 2013 Pb ISA, survival was similar to that observed by Besser
et al. (2005) under two different experimental diets conducted concurrently (LC20 =15 jxg Pb/L and
LC20 =13 jxg Pb/L) and support the findings of effects in amphipods in the low |ig/L range (Besser et al..
2016).
Some species of freshwater gastropods have exhibited sensitivity to Pb at <20 |ig Pb/L. In the
1986 AQCD, Borgmann et al. (1978) found increased mortality at Pb concentration as low as 19 |ig Pb/L
in the freshwater snail Lymnaea palutris exposed from hatching to reproductive maturity (approximately
120 days). To follow-up on the set of studies reviewed in the 2013 Pb ISA (Grosell and Brix. 2009;
Grosell et al.. 2006b) that identified the freshwater snail L. stagnalis as highly sensitive to Pb
(EC20 = <4 |ig Pb/L in 30-day exposure experiments) several additional chronic studies have since been
undertaken with this species. In growth bioassays conducted in a variety of natural waters across the
United States with different water chemistries 14-day EC20 and EC50 values ranging from 1.5 to 49.5 and
from 3.6 to 244.6 |ig Pb/L, respectively, were reported for L. stagnalis (Esbaugh et al.. 2012). Munlev et
al. (2013) conducted full life cycle bioassays with a duration of 56 days to assess the effects on survival,
growth, reproduction, and development in L. stagnalis and determine if there was any recovery from
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growth inhibition effects reported in the 30-day exposures. Survival was significantly decreased at the
highest concentration of Pb tested (8.4 |ig Pb/L) after 21-days of exposure until the end of the experiment,
for a final NOEC = 2.7 |ig Pb/L and LOEC = 8.4 |ig Pb/L. Consistent with the earlier 30-day exposures,
growth was significantly decreased at day 28, even at the lowest tested concentration (1.0 (ig Pb/L), for
NOEC < 1.0 |ig Pb/L and LOEC =1.0 jxg Pb/L. By day 56, growth remained significantly lower than that
of the controls in the 2.7 and 8.4 |ig Pb/L concentration; however, snails exposed to 1.0 |ig Pb/L
surpassed the growth rates of the unexposed snails. Inhibition of the specific growth rate at the
2.7 (ig Pb/L exposure was observed during the last week of the experiment. Conducting a 56-day life
cycle bioassay with L. stagnalis enabled assessment of reproductive and developmental endpoints
(Munlev et al.. 2013). The reproductive phase started at day 32 and continued till the end of the study. For
the number of egg masses and time until first egg mass, the NOEC <1.0 jxg Pb/L and
LOEC =1.0 jxg Pb/L. No effects of Pb on the number of embryos per egg mass were observed at any
concentration tested. Individuals exposed to the highest concentration (8.4 |ig Pb/L) did not reproduce
during the life cycle test. Egg capsule and embryo diameters after 7 days of development were
significantly reduced at 2.7 |ig Pb/L (the highest concentration in which snails reproduced in the study).
Although growth exhibited some recovery in L. stagnalis in the longer 56-day life cycle tests, growth
effects observed at 28 days were predictive of the reproductive effects observed in the longer exposure
(Munlev et al.. 2013). Additional growth studies conducted by Brix et al. (2012) reported an EC20
(biomass) at 8 days of exposure of 3.2 |ig 1 1 Pb and 3.5 |ig 1 1 Pb after 16 days of exposure. Under similar
experimental conditions. Cremazv et al. (2018) reported a 14-day EC10 of 4 |ig Pb/L, an EC20 of
7.67 |ig Pb/L and an EC50 of 23.4 |ig Pb/L for juvenile growth from compiled results of multiple toxicity
tests. The corresponding chronic growth effect concentrations based on free-ion activity were
EC10 = 0.157 ng Pb/L, EC20 = 0.320 ^g Pb/L and EC50 = 1.08 ^g Pb/L.
New acute data for cladocerans include a 48-hour EC50 = 280 |ig Pb/L for immobilization in D.
magna (Okamoto et al.. 2015). Among the studies reviewed in the 2013 Pb ISA was a series of 48-hour
acute toxicity tests using a variety of natural waters across North America. The cladoceran C. dubia. LC50
values in that study ranged from 29 to 180 |ig Pb/L, and DOC was well correlated with protection against
the toxicity of Pb(Esbaugh et al.. 2011). In this same species, increasing DOC led to an increase in the
mean EC50 for reproduction, ranging from approximately 25 |ig Pb/L to >500 |ig Pb/L in 7-day chronic
toxicity bioassays (Mager et al.. 201 la). In a study published after the 2013 Pb ISA in this same species, a
series of 7-day reproductive toxicity tests to assess the effects of metal mixtures reported an EC50 range of
1 1 1 to 302 |ig Pb/L in the Pb-only treatments (Nvs et al.. 2016a). In another study with C. dubia, the EC50
for reproduction ranged from 99.8 |ig Pb/L at pH 6.4 to 320 |ig Pb/L, at pH 8.2, and 81.2 (ig Pb/L at
0.25 mM Cato 130 (ig Pb/L at 1.75 mM Ca (Nvs et al.. 2014). In comparison, in a series of chronic Pb
toxicity tests conducted in a variety of natural waters across the United States with different water
chemistries which expanded upon the findings of Esbaugh et al. (2011). 7-day EC20 for reproduction in C.
dubia ranged from 12.1 to 223.3 |ig Pb/L, and 7-day-ECso ranged from 20.1 to 573.4 |ig Pb/L (Esbaugh et
al.. 2012).
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Using the same set of waters from across the United States, reproduction (as population growth)
was also assessed in rotifer/', rapida over a 4-day exposure period (Esbaugh et al.. 2012). Chronic EC20
and EC50 in this species based on dissolved Pb concentration ranged from 3.2 to 103.3 and 10.6 to
154.9 |ig Pb/L, respectively. The variability in toxic response to Pb was linked to water chemistry; DOC
had a protective effect for C. dubia and snail L. stagnalis, while rotifer response was most closely
associated with Ca and pH, not DOC. In comparison, another species of rotifer, B. calyciflorus, was less
sensitive to Pb; 4-day chronic reproductive toxicity EC20 ranged from 75 jxg Pb/L to 336 (ig Pb/L and
EC50 ranged from 138 to 634 |ig Pb/L in natural waters of varying chemistry (Nvs et al.. 2016b).
In response to a lack of chronic toxicity data in freshwater isopods based on measured
concentrations Van Ginneken et al. (2017) conducted a series of exposure-response studies with trace
metals including Pb in adult A aquaticus. The authors determined LC10, LC20 and LC50 effect values for
this species (14-day LCio=49.7 |ig Pb/L, LC2o=130 |ig Pb/L, LCso=677 |ig Pb/L) and also calculated lethal
concentrations based on free-ion activity using the Windermere Humic Aqueous Model
(LC10 = 0.04 |ig/L. LC20 = 0.31 |ig/L and LC50 = 9.13 (.ig/L). In a separate study w ith A. aquaticus, the 10-
day LC50 was 443 |ig Pb/L (Van Ginneken et al.. 2015). In another crustacean, juvenile prawns (M
nipponense), no statistically significant effects on mortality were reported at 12 or 25 |ig Pb/L
concentration in chronic 60-day exposure trials; however, reductions in weight gain and specific growth
rate were observed in the prawns exposed to 25 |ig Pb/L (Ding et al.. 2019).
In freshwater mussels, sensitivity to Pb has been demonstrated to vary with lifestage. In a study
from the 2013 Pb ISA, newly transformed juvenile freshwater mussels (Lampsilis siliquoidea) were more
sensitive than older juveniles in acute exposures. A chronic value (geometric mean of NOEC and the
LOEC) of 10 |ig Pb/L was reported in 28-day exposures of 2-month-old iuvenilesCWang et al.. 2010). The
lowest median effect concentration for glochidia (larvae) ofZ. siliquoidea at 24 and 48 hours was
>299 |ig/L. A more recent study in glochidia of six different freshwater mussel species found in
southeastern Australia (Hyridella australis, Hyridella depressa, Velesunio ambiguus, Alathyria profuga,
Cucumerunio novaehollandiae, Hyridella drapeta) indicated these species were more sensitive in acute
tests than glochidia of L. siliquoidea (native to the United States). The 24-hour EC50 values for valve
closure ranged from 176 to 274 |ig Pb/L (Markich. 2017). Following 72-hour Pb exposure in the same
species, the EC50 values ranged from 65 to 110 (ig Pb/L. Calculated no-effect concentrations (NECs) at
72 hours ranged from 11 to 21 |ig Pb/L.
Other recent tests with freshwater invertebrates have illustrated the range in the sensitivity of
North American species to Pb. In a battery of acute toxicity tests using resident invertebrates collected
from the South Fork Coeur d'Alene River watershed, Idaho, U.S. and tested in the river water, the lowest
EC50 concentration for Pb (96-hour EC50 = 253 |ig Pb/L) was obtained with the stonefly Sweltsa sp.,
however, in other tests with Sweltsa sp., mortalities occurred at Pb concentrations up to three times
greater, indicating a high degree of variability in repeated tests with the same species (Mebane et al..
2012). Additional invertebrates were tested in waters from the South Fork Coeur d'Alene River
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watershed, Idaho, U.S., and their lowest corresponding 96-hour EC50 values (some invertebrate species
were tested multiple times) were: four mayfly species (Baetis tricaudatus [96-hour LC50 = 322 to
<1,250 |ig Pb/L tested at varying water hardness], Rhithrogena sp. [96-hour LC50 = >166 |ig Pb/L],
Drunella sp. [96-hour LC50 = >267 |ig Pb/L], Epeorus sp. [96-hour LC50 = >346 |ig Pb/L] and
Leptophlebiidae [96-hour LC50 = >346 |ig Pb/L]), a caddisfly (Arctopsyche sp. 96-hour
LC50 = >1,255 |ig Pb/L), a Simuliidae black fly (96-hour LC50 = 415 |ig Pb/L), Chironomidae midge (96-
hour LC50 = 1,955 |ig Pb/L), a Tipula sp. Crane fly (96-hour LC50 = >1,035 |ig Pb/L), a Dytiscidae beetle
(96-hour LC50 = >1,035 |ig Pb/L) and two snail species (Physa sp. [96-hour LC50O = 1,159 |ig Pb/L] and
Gyraulus sp [96-hour LC50 = 380->l,035 |ig Pb/L] tested at varying water hardness).
Since the 2013 Pb ISA, additional exposure-response information has been obtained from
sediment bioassays for freshwater invertebrates. In 21-day whole sediment chronic toxicity bioassays, no
negative effect was noted for larvae of the North American mayfly species, Hexagenia limbata, exposed
up to 2,903 mg Pb/kg sediment (highest concentration tested); for survival, the porewater
LOEC = >130 |ig/L and overlying water LOEC = >53.6 |ig/L (Nguyen et al.. 2012). In the same study, for
a European species Ephoron virgo, 21-day EC50 and LOEC of 2,201 and 2,071 mg Pb/kg were found,
respectively, with a porewater LOEC = 105 fxg Pb/L and overlying water LOEC =19 fxg Pb/L. In long-
term whole-sediment toxicity tests with three benthic organisms exposed to various concentrations of Pb;
L. variegatus (16 to 5,746 mg Pb/kg), G. pulex (21 to 2,734 mg Pb/kg) and mayfly Ephoron virgo (15 to
2,972 mgPb/kg), in which Pb-spiked sediments were allowed to fully equilibrate 35 or 40 days prior to
testing and metal concentrations were monitored throughout, the survival of E. virgo (21-day
EC10 = 1,455 mg Pb/kg dry weight) and the biomass of L. variegatus (28-day EC10 = 1,870 mg Pb/kg dry
weight) were more sensitive endpoints compared with the growth of G. pulex (3 5-day
EC10 = 2,541 mg Pb/kg dry weight) (Vandcgchuchtc et al.. 2013).
For freshwater vertebrates, the majority of available exposure-response data are for fish. In the
studies reviewed for the 2006 Pb AQCD, freshwater fish demonstrated negative effects at concentrations
ranging from 10 to >5,400 |ig Pb/L, generally depending on exposure duration and water quality
parameters (e.g., pH, hardness, salinity) as summarized in Table AX7 2.4.2 of the 2006 AQCD (U.S.
EPA. 2006b'). In the 2013 Pb ISA, several acute and chronic bioassay studies with fish further elucidated
the role of water chemistry in toxicity (Esbaugh et al.. 2011; Grosell et al.. 2006b; Grosell et al.. 2006a).
In a series of 96-hour acute toxicity tests with fathead minnow (P. promelas) conducted in a variety of
natural waters across North America, LC50 values ranged from 41 to 3,598 |ig Pb/L in this species
(Esbaugh et al.. 201IV Chronic assays with rainbow trout reported in the 2013 Pb ISA provided
additional exposure-response data for this species. In a 69-day test with rainbow trout, the following
chronic values were observed for survival: NOEC = 24 |ig Pb/L, maximum acceptable toxicant
concentration (MATC) = 36 |ig Pb/L, EC10 = 26 |ig Pb/L, EC20 = 34 |ig Pb/L and LC50 = 55 |ig Pb/L
(Mebane et al.. 2008). Results from a 62-day test, with fish length as the endpoint, were
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NOEC = 8 ng Pb/L, MATC = 12 |ig Pb/L, ECio = 7 jig Pb/L, EC20 = 102 |ig Pb/L and LC50 = 120 |ig Pb/L
(Mebane et al.. 2008).
New evidence since the 2013 Pb ISA includes additional studies on fish species native to North
America. In 96-h acute toxicity tests with white sturgeon (A. transmontanus), which is experiencing
population declines in the U.S. and Canada, two early lifestages (8 and 40 dph) were tested in lab water
and in water from the Columbia River upstream of the Teck Trail smelter facility, British Columbia,
Canada (Vardv et al.. 2014). For 8 dph larvae, 96-hour LC50 = 177 |ig/L (lab water) and 96-hour
LC50 >410 |ig/L (river water); for 40 dph, 96-hour LC50 = 528 |ig/L (lab water) and 96-hour
LC50 = 1,556 |ig/L (river water) (Vardv et al.. 2014). In 27 dph juvenile white sturgeon exposed to Pb
concentrations in water ranging from 0.03 to 60 |ig Pb/L for 28 days, there was an EC20 > 60 |ig Pb/L for
survival, length, and biomass (Wang et al.. 2014a). Considering that the early lifestages of white sturgeon
are in close contact with sediment and porewater Balistrieri et al. (2018) reported an EC20 = 0.9 nM Pb2+
(0.18 |ig Pb2+/L) developed from predictive response modeling using in situ measurements of Pb in
Columbia River sediment and porewater, free-ion concentrations from equilibrium speciation calculations
and the laboratory toxicity testing results of Wang et al. (2014a) of Pb to the early lifestages of sturgeon.
Similar dose-response curves based on free metal ion concentration were observed for effective mortality
and for reduction in biomass at Pb2+ concentrations higher than quantified in sediment porewater,
indicating young sturgeon at the sediment-water interface are unlikely to be affected by toxic
concentrations of Pb in the upper reaches of the Columbia River. Mebane et al. (2012) tested westslope
cutthroat trout (Oncorhynchus clarkii lewisi) a native subspecies of conservation concern, in a series of
bioassays using water from various locations within the South Fork Coeur d'Alene River watershed,
Idaho. EC50 values for the effective mortality for this species ranged from 47 to 487 |ig Pb/L.
In native rainbow trout (O. mykiss), 7-week waterborne-only exposure (4, 11,21, 82, 251 and 907
ug Pb/L) conducted as part of a larger study to assess the toxicity of different dietary pathways in juvenile
rainbow trout, survival was assessed daily, and fish were weighed weekly (Alsop et al.. 2016). At 96-h,
toxicity values were LC10 = 304.3 |ig Pb/L, LC20 = 357.7 |ig Pb/L and LC50 = 487.3 |ig Pb/L. At 7 weeks,
LC10 = 55.6 |ig Pb/L, LC20 = 96.9 |ig Pb/L and LC50 = 280.2 |ig Pb/L. All fish exposed at the highest
concentration did not survive, and no significant effects on growth were reported for any concentration
for the duration of the experiment. In 27 dph juvenile rainbow trout, EC20 > 128 |ig Pb/L for survival,
length and biomass following 28 days of Pb exposure (Wang et al.. 2014a). In tests with larval trout, EC20
values were the same as observed in the juveniles. In addition to studies on native fish species, other
studies in fish support previous understanding of the role of water chemistry in Pb toxicity. For larval
zebrafish (D. rerio) acute toxicity, 96-hhour LC50 values varied with water hardness; in soft water
LC5o= 52.9 |ig Pb/L and in hard water LC50 = >590 |ig Pb/L (Alsop and Wood. 2011). |ig Pb/L (Alsop and
Wood. 2011).
As discussed in Section 11.1.7.3, the existing U.S. EPA AWQC for Pb for the protection of
aquatic life are CMC of 65 |ig Pb/L (for acute exposure) and CCC of 2.5 |ig Pb/L (for chronic exposure)
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at a hardness of 100 mg/L (U.S. EPA. 1985a). Since these criteria were developed in 1984, there have
been additional acute and chronic toxicity data and improved characterization of modifying factors that
affect Pb bioavailability and toxicity. Taking these advances into consideration Deforest etal. (2017)
proposed updated acute BLM-based aquatic life criteria, ranging from 18.9 to 998 |ig Pb/L and chronic
BLM-based Pb freshwater criteria ranging from 0.37 to 41 |ig Pb/L (Table 11-5). The lowest criteria were
for water with low DOC (1.2 mg/L), pH (6.7) and hardness (4.3 mg/L as CaCCh). and the highest criteria
were for water with high DOC (9.8 mg/L), pH (8.2) and hardness (288 mg/L as CaCOs). which
encompasses varying water quality conditions of North American surface waters and the importance of
DOC and pH as modifying factors compared with hardness. The updated data sets in Deforest et al.
(2017) incorporated toxicity information for L. stagnalis, C. dubia, H. azteca and P. rapida, freshwater
invertebrates that are relatively sensitive to Pb exposure. The number of genera with acute toxicity data
for Pb increased from 10 to 32, and for chronic toxicity, from 4 to 13, which enabled the proposed chronic
criteria to be based on bioassay data rather than an acute-to-chronic ratio that was used in 1984 for
derivation of the CCC.
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Table 11-5 Studies in freshwater biota with analytically verified Pb concentrations and that report an effect on
growth, reproduction or survival comparable to, or lower than, the lowest effect concentrations
reported in previous Pb AQCDs or the 2013 Pb ISA.
Species
Concentration Exposure Method
Modifying
Factors
Effects on Endpoint
Reference
Effect Concentration (published
since the
2013 Pb ISA)
Algae/Plants
Green algae
(Pseudokirchneriella
subcapitata),
Green algae
(Chlorella kessleri)
Green algae
(Chlamydomonas
reinhardtii)
P. subcapitata
Total Pb:
<1,19, 42, 85,
228.5,
412 Pb |jg/L
Filtered Pb:
<1, 16, 37, 77,
201, 418 Pb |jg/L
C. kesslerii
Filtered Pb:
<1, 7, 18, 39, 80,
164, 417 |jg Pb/L
C. reinhardtii
Filtered Pb: <0.8,
9.5, 19.8, 43.3,
89.4, 194, 452,
783,1613 |jg Pb/
L
Standard 3-d toxicity
tests conducted in
OECD standard test
medium with nominal
addition of 4 mg/L of
Suwannee River
Fulvic Acid. Cell
densities were
measured after 24,
48 and 72 h of
exposure using a
particle counter. The
growth rates of C.
vulgaris and C.
reinhardtii were not
considered
exponential during
the third day of
exposure, so the 2-d
ECx values were
calculated for these
two species.
Additional tests were
conducted with P.
subcapitata with
varying pH and fulvic
acid
Temperature:
24°C
pH = 6
Growth:
Interspecies comparison of algal
growth rate indicated that P.
subcapitata is the most sensitive
and C. kesslerii the least
sensitive. In P. subcapitata, as
pH increased from 6.0 to 7.6, the
72-h ECso decreased from 72.0
to 20.5 |jg filtered Pb/L
P. subcapitata
2-d ECso = 89.9 pg Pb/L
2-d EC20 = 44.7 |jg Pb/L
2-d EC10 = 29.7 |jg Pb/L
3-d ECso = 83.9 pg Pb/L
3-d EC20 = 45.7 pg Pb/L
3-d EC10 = 32.0 pg Pb/L
C. kesslerii
2-d ECso = 388 pg Pb/L
2-d EC20 = 185 pg Pb/L
2-d EC10 = 120 pg Pb/L
C. reinhardtii
2-d ECso = 172 pg Pb/L
2-d EC20 = 108 pg Pb/L
2-d EC10 = 82.3 pg Pb/L
De
Schamphelae
re et al.
(2014)
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Reference
Species Concentration Exposure Method Effects on Endpoint Effect Concentration (published
r r Factors r since the
2013 Pb ISA)
Green algae
(Raphidocelis
subcapitata formerly
known as
Pseudokirchneriella
subcapitata)
(1.20; 2.41; 4.82
and 12.06 pM)
Nominal, stock
solution
analytically
verified
72-h toxicity test with
triplicates,
maintained in a
temperature-
controlled room. Cell
density assessed
every 24 h
Temperature: Growth:
25 ± 2°C Pb significantly inhibited algal
growth. All treatments differed
significantly (p < 0.05) from the
control group at 72 h of exposure.
Pb completely inhibited algal
growth at 12.06 pM
72-h IC10 = 0.15 pM,
(31 pg Pb/L)
72-h IC25 = 0.39 pM
(81 pg Pb/L)
72-h IC50 = 0.78 pM
(161 pg Pb/L)
Alho et al.
(2019)
Duckweed
(Lemna minor)
A range of
concentrations
as low as
10 pg Pb/L to as
high as
9,740 pg Pb/L.
Total Pb added
to each water
was varied
because waters
differed in
hardness, DOC,
and pH. All
waters were
equilibrated for
24 h prior to
bioassays
A series of 7-d static
renewal tests with L.
minor were
conducted with seven
different surface
waters collected from
across the United
States with varied
chemistries and
spiked with a
concentration series
of Pb(N03)2. Plants
were held in a growth
chamber and growth
was assessed as %
net root elongation
Temperature:
25 ± 2°C
pH:
5.4-8.3
depending on
surface water
DOC:
0.5-12.5 mg/L
depending on
surface water
Hardness:
8-266 mg/L
CaC03 depending
on surface water
Growth: The inhibition of net root 20% inhibitory
elongation varied widely
depending upon the chemistry of
the assayed waters and its
effects on Pb speciation
concentration in 7-d static
renewal tests with the
waters ranged from
306 nM (63 pg Pb/L) to
>6920 nM to
(>1,433 pg Pb/L) total
dissolved Pb
Antunes and
Kreaaer
(2014)
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Species
Concentration Exposure Method
Modifying
Factors
Effects on Endpoint
Reference
Effect Concentration (published
since the
2013 Pb ISA)
Invertebrates
Amphipod
(Hyalella azteca)
Control 5, 10, 20,
40, 80 |jg Pb/L.
Pb aqueous
concentrations
varied among
diet treatments
and overtime,
suggesting that
food inputs
modified Pb
concentration
and
bioavailability
7-d-old amphipods in
flow-through water-
only exposure to Pb
as Pb-nitrate in 42-d
chronic bioassays.
Amphipods were fed
one of two
experimental diets: a
suspension of yeast,
cereal leaves, and
trout pellets (YCT) or
a diatom + Tetramin
(DT) fish food diet.
Assays conducted
concurrently in test
water from the same
diluter system
Besser et al.
Hardness
100 mg/L as
CaCC>3
pH about 8.2
Alkalinity 95 mg/L
Survival: Lowest reliable toxicity
Survival was similar with aqueous va',^eJ°r e®crt?1 enc'P0int in (2016)
Pb exposure in amphipods fed
two different diets
Growth:
Biomass significantly reduced in
amphipods fed YCT, not
significantly reduced in
amphipods fed DT up to
63 |jg Pb/L
Reproduction:
Fecundity significantly reduced in
amphipods fed YCT, not
significantly reduced in
amphipods fed DT up to
63 |jg Pb/L. (Note: fecundity and
total young endpoints did not
meet test acceptability criteria for
YCT diet).
|jg/L filtered Pb:
DT diet:
42-d EC2o= 13 |jg Pb/L
42-d NOEC = 5.9 pg Pb/L
42-d LOEC = 13 |jg Pb/L
YCT diet:
42-d EC2o= 15 pg Pb/L
42-d NOEC = 6.1 pg Pb/L
42-d LOEC = 14 pg Pb/L
Lowest biotic ligand
model-normalized effect
concentrations:
EC20 = 8.2 pg Pb/L (total
young for the DT test)
ECso = 6.6 pg Pb/L
(biomass for the YCT test)
Isopod
(Asellus aquaticus)
15.1, 31.1, 74.7,
203, 443 pg Pb/L
Various metal
mixtures and single
metals were tested in
a 10-d exposure with
individuals of equal
length (9.43 ± 0.17
mm) in a climate
chamber. The Pb-
only treatment was
Pb as PbCb
Temperature:
20 ± 1°C
Hardness:
117 mg L"1
CaC03
Survival: Focus of study was on
mixture toxicity. Only LC50 was
calculated for Pb-only treatment
10-d LCso = 443 pg Pb/L Van Ginneken
et al. (2015)
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Reference
Species Concentration Exposure Method Effects on Endpoint Effect Concentration (published
r r Factors r since the
2013 Pb ISA)
Isopod
(Asellus aquaticus)
0.71 (control),
25.6, 110, 358
and
37,616 |jg Pb/L
(measured
values for
effective
concentration)
<0.1, <0.1, 0.36,
4.67 and
18,982 |jg Pb/L
(free-ion
activities of the
measured
effective
concentrations
calculated using
the Windermere
Humic Aqueous
Model with 100%
of DOC as fulvic
acids)
Chronic 14-day
exposure to Pb(NC>3)2
with adult A.
aquaticus. Assay
water sampled on
days 0,1,4, 7 and 14,
isopods were
removed from
exposure containers
for 4 h on day 7 for
feeding
Temperature:
15 ± 1°C
pH:
7.72 ± 0.03
DOC:
5.94 ±0.13 mg/L
Dissolved oxygen:
8.68 ± 0.03 mg/L
Survival: Severe mortality was
only observed at the highest
concentration tested after 14-d
exposure. Low mortality was
observed in the other
concentrations. During the
exposure period, LC values
declined until day 4, then
continued to slowly decrease.
The free-ion activities produced
the lowest LC values
14-d survival
LC10 = 49.7 |jg Pb/L
LC10 for
FIA = 0.04 |jg Pb/L
LC20 = 130 |jg Pb/L
LC20 for
FIA = 0.31 |jg Pb/L
LC50 = 677 |jg Pb/L
LC20 for
FIA = 9.13 |jg Pb/L
7-d survival
LC10 = 97.4 |jg Pb/L
LC20 = 602 |jg Pb/L
LCso = 13,562 |jg Pb/L
Van Ginneken
etal. (2017)
(LC10, 20 and 50 values
were also calculated for
day 1 and day 4).
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Reference
Species Concentration Exposure Method Effects on Endpoint Effect Concentration (published
r r Factors r since the
2013 Pb ISA)
Cladoceran
(Ceriodaphnia dubia)
pH 6.4, 7, 7.6
series: (nominal
concentration 80,
110, 140, 170,
220,
320 |jg Pb/L)
pH 8.2 test:
(nominal
concentration
100, 160 220
280, 340,
400 |jg Pb/L)
Ca test series:
(nominal
concentration 50,
100, 150, 220,
320,
400 |jg Pb/L)
Total and filtered
Pb in each series
quantified but not
reported for
individual assays
Reproductive effects
of Pb (PbCh)
assessed in 7-d
chronic assays.
Juveniles (<24 h old)
exposed to Pb and
varying Ca or pH in
static renewal
assays. Mortality and
number of juveniles
noted daily
PH
4 series:
6.4; 7; 7.6; 8.2
Hardness
4 series:
Ca = 0.25 mM;
1.0 mM; 1.75 mM;
2.5 mM
DOC
3.2-3.3 mg/L in
pH series
3.8-4.0 in
hardness series
Reproduction
Total reproduction (number of
juveniles per female) relative to
the mean control reproduction
varied with Ca or pH over 7-d
chronic exposure to Pb. High pH
was protective of Pb toxicity and
water hardness had less effect on
chronic toxicity than pH
7-d ECso for reproduction
ranged from 99.8 |jg Pb/L
at pH 6.4 to 320 ug Pb/L
at pH 8.2
7-d EC50 for reproduction
ranged from 81.2 |jg Pb/L
at 10 mg/L (0.25 mM) Ca
to 130 |jg Pb/L at 70 mg/L
(1.75 mM) Ca
Nvs et al.
(2014)
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Cladoceran
(Ceriodaphnia dubia)
Rotifer
(Philodina rapida)
Snail (Lymnaea
stagnalis)
Each species
was tested in a
range of
concentrations
starting at low
|jg Pb/L. Actual
concentrations
were measured
but not reported
for the individual
assays
All three species
exposed to Pb as
Pb(NC>3)2, in a range
of representative
surface waters
across North America
C. dubia: (<24-h-old
neonates) 7-d
chronic reproductive
bioassays conducted
in a temperature-
controlled chamber
with a combination of
dietary and aqueous
exposure and
monitored daily for
survival and
reproduction
P. rapida'. 4-d chronic
Pb toxicity with adults
was assessed using
a population growth
rate endpoint which
conformed to
classical
concentration-
dependent
responses.
L. stagnalis'. 14-d
chronic toxicity test
for growth starting
with 7 to 10 dph
snails. Water
changes and food
replacement every
48 h
Representative
surface waters for
the bioassays had
varying pH, DOC,
and water
hardness
C dubia:
pH: 6.51-8.47
DOC: 114-1443
Temperature:
26°C
P. rapida:
pH: 7.23-8.44
DOC: 79-1405
Temperature:
26°C
L. stagnalis:
pH: 5.79-8.61
DOC: 36-1314
Temperature:
26°C
Reproduction:
Highest reproductive toxicity in C.
dubia was observed in soft water,
most protective water had high
DOC. For P. rapida population
growth, DOC was not predictive
of chronic toxicity
Growth:
Effects on growth occurred at low
|jg/L concentration in L. stagnalis
in some of the tested waters. For
the snails, the greatest effects on
growth occurred with low-DOC
waters
C. dubia:
7-d-EC5os for reproduction
ranged from 20.1 to
573.4 |jg/L in
representative surface
waters of varying
chemistries. EC20S ranged
from 12.1 to 223.3 pg/L.
P. rapida:
EC20 and EC50 ranged
from 3.2-103.3 and 10.6-
154.9 |jg/L dissolved Pb,
respectively
L. stagnalis:
EC20S and ECsos for
growth ranged from 1.5 to
49.5 and 3.6 to 244.6 pg/L
dissolved Pb,
respectively, in the natural
waters
Esbauqh et al.
(2012)
Rotifer
For the Ca and
pH series:
(nominal
Reproductive effects
of Pb (PbCh)
assessed in recently
pH:
Reproduction:
For population size in Nvs et al.
natural waters: (2016b)
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Species
Concentration Exposure Method
Modifying
Factors
Effects on Endpoint
Effect Concentration
Reference
(published
since the
2013 Pb ISA)
(Brachionus
calyciflorus)
concentration
range 46-
2,200 |jg Pb/L)
For DOC test
series: (nominal
concentration
range 100-
10,000 |jg Pb/L).
Total and filtered
Pb in each series
was quantified
hatched rotifers
exposed to Pb for 48-
h (three generations).
Tests were
performed in four
series (varying Ca,
varying pH, varying
DOC, and natural
waters collected from
five unpolluted
waterbodies in
different locations in
Europe)
ranged from 6.8 to
8.2 in natural
waters
DOC:
ranged from 3.2 to
31.5 in natural
waters
Temperature:
25°C
The ECso (based on filtered Pb)
for population size differed by up
to 4.6-fold in the natural waters.
The highest toxicity was
observed in the synthetic
reference water. For the
modifying factor bioassays, both
population growth rate and
population size generally
decreased with increasing pH.
For DOC, toxicity expressed as
filtered Pb decreased significantly
with increasing DOC. Ca was not
protective
EC10 ranged from 52
(synthetic reference
water) to 231 |jg Pb/L
EC20 ranged from 75
(synthetic reference
water) to 336 |jg Pb/L
ECso ranged from 138
(synthetic reference
water) to 634 |jg Pb/L
(based on filtered Pb
concentration)
Snail
(Lymnaea stagnalis)
6, 12.5, 25,
100 |jg Pb/L
(analytically
verified)
Juvenile snail growth
was assessed in a
static renewal assay
over a 16-d period.
Primary focus of the
study was to
investigate possible
mechanisms of Pb
toxicity
Temperature: Growth:
23-25°C After 4 d, a moderate effect of
pH = 7.8 Pb on juvenile snail growth was
observed, severity of growth
inhibition increased after 8 d,
effects on growth occurred prior
to net Ca2+ flux in the snails,
inhibition of carbonic anhydrase
activity in the snail mantle also
showed no effect with Pb
EC20 (biomass) at 8 d of
exposure was 3.2 |jg L~1
Pb
EC20 (biomass) was
3.5 |jg L~1 Pb after 16 d of
exposure
Brix et al.
(2012)
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Snail
(Lymnaea stagnalis)
0.18 (control),
2.7 and
8.4 |jg Pb/L
(measured)
Newly hatched
juvenile snails were
exposed to Pb (as
Pb(N03)2 in Milli-Q
water) for 56-d in a
full life cycle
assessment toxicity
test in a flow-through
system to assess
effects on survival,
growth and
reproduction (number
of egg masses, time
until first egg mass,
number of embryos
per egg mass). The
reproductive phase
started at day 32
(egg masses
appeared in the
control) and
continued till the end
of the study
Temperature:
24.8 ± 0.2°C
pH:
6.89 ± 0.06
DOC:
330 ± 7.02 |jM C
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Survival:
Survival was significantly
decreased at the highest
concentration (8.4 |jg Pb/L) after
21-d exposure to the end of the
experiment
Growth:
Growth was significantly
decreased, even at the lowest
tested concentration (1 |jg Pb/L)
at day 28. By day 56, growth
remained significantly lower than
the controls in the 2.7 and
8.4 |jg Pb/L concentration;
however, snails exposed to
1.0 |jg Pb/L surpassed the
growth rates of the unexposed
snails. Inhibition of specific
growth rate at the 2.7 |jg Pb/L
exposure was observed during
the last week of the experiment.
Survival:
56-d chronic toxicity
NOEC = 2.7 |jg Pb/L
LOEC = 8.4 |jg Pb/L
Growth:
28-d
NOEC<1.0 |jg Pb/L
LOEC = 1.0 |jg Pb/L
Reproduction:
NOEC<1.0 |jg Pb/L
LOEC = 1.0 |jg Pb/L
Munlev et al.
(2013)
Reproduction:
For the number of egg masses
and time until first egg mass, the
NOEC<1.0 |jg Pb/L and
LOEC = 1.0 |jg Pb/L. No effects
on the number of embryos per
egg mass were observed at any
concentration tested. Individuals
exposed to the highest
concentration (8.4 |jg Pb/L) did
not reproduce during the life
cycle test. Egg capsule and
embryo diameter after 7 d of
development were significantly
reduced at 2.7 |jg Pb/L (the
highest concentration in which
snails reproduced in the study)
DRAFT: Do not cite or quote
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Reference
Species Concentration Exposure Method Effects on Endpoint Effect Concentration (published
r r Factors r since the
2013 Pb ISA)
Snail
(Lymnaea stagnalis)
Low |jg Pb/L
concentrations
(Pb was
measured in
each assay) EC
values are from
combined results
of Pb data from
multiple toxicity
tests
Series of 14-d
chronic toxicity
assays with single
metals (Pb as
Pb(NC>3)2) and binary
metal mixtures with
juvenile L. stagnalis
to assess effects on
relative growth rate.
Concentration-
response curves
were obtained by
compiling all the
single-metal toxicity
tests performed at
different times over a
2-yr period
Temperature:
25 ± 1°C
pH = 7.81 ± 0.20
DOC = 0.76 ± 0.0
8 mg L"1
Alkalinity = 0.80 ±
0.05 mEq-L-1
Growth:
Inhibition of relative growth rate
was observed at low |jg Pb/L
concentrations, consistent with
other bioassays with L. stagnalis
14-d chronic toxicity:
EC10 = 4.0 |jg Pb/L
EC20 = 7.67 |jg Pb/L
ECso = 23.4 |jg Pb/L
Corresponding chronic
effect concentrations
based on free-ion activity:
EC10 = 0.157 |jg Pb/L
EC20 = 0.320 |jg Pb/L
ECso = 1.08 |jg Pb/L
C re mazy et
al. (2018)
Mussel
(Hyridella australis)
(Hyridella depressa)
(Velesunio
ambiguus)
(Alathyria profuga)
(Cucumerunio
novaehollandiae)
(Hyridella drapeta)
Each acute
toxicity test
consisted of a
control and 10
concentrations,
which were
based on
preliminary
range-finding
tests. Individual
test
concentrations
were not
reported.
Concentrations
were measured
Glochidia (larvae)
from gravid females
collected from two
different river
catchments in
southeastern
Australia were used
in the bioassays.
Four static tests were
conducted for each
mussel species and
exposure time (24,
48 or 72 h) with PbCI
in reconstituted
freshwater. Viability
(as assessed by
valve closure) was
determined at the
end of the exposure
period
Temperature:
22 ± 1°C
pH 7.0 ± 0.2
Hardness
42 ± 4 mg CaCC>3
L"1
Alkalinity
22 ± 2 mg CaCC>3
L"1
Survival:
Pb sensitivity significantly
increased with each exposure
time and varied by species, with
greatest toxicity observed in C.
novaehollandiae
24-h ECso (for valve
closure as a proxy for
viability) ranged from 176
to 274 |jg Pb
48-h ECso ranged from
102-165 |jg Pb/L
72-h EC50 ranged from 65
to 110 |jg Pb/L
72-h calculated NEC
ranged from 11 to
21 |jg Pb/L
Markich
(2017)
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Reference
Species Concentration Exposure Method Effects on Endpoint Effect Concentration (published
r r Factors r since the
2013 Pb ISA)
Prawn
(Macrobrachium
nipponense)
0, 5, 10, 20, 40,
80, 160, 320 and
640 |jg Pb/L
(nominal values)
Acute toxicity
bioassay
12 |jg Pb/L,
25 |jg Pb/L
(measured)
Chronic growth
bioassay
For the 96-h acute Temperature:
toxicity assay, 26 ± 1°C
juveniles were
exposed to Pb as Pb
acetate in semistatic pH 7.0-7.3
renewal (every 24 h)
conditions, survival
was assessed every
24 h. For the chronic
growth assay,
prawns were
exposed for 60 days
under the conditions
described for the
acute bioassay.
Prawns fed a
commercial diet twice
daily
dissolved oxygen
>6.5 mg/L
DOC: 190 pmol/L
Survival: LCso values decreased
overtime in the acute bioassay
from 24 to 96 h. Mortality was not
significantly affected by Pb
(12 |jg Pb/L or 25 pg Pb/L) in the
60-day chronic bioassay.
Growth: reductions in weight
gain and specific growth rate in
prawns exposed to 25 pg Pb/L,
but not in prawns exposed to
12 pg Pb/L
Acute toxicity test: Ding et al.
24-h LCso = 646 pg Pb/L (2019)
48-h LCso = 250.6 pg Pb/L
72-h
LCso = 175.6 pg Pb/L
96-h LCso= 131.3 pg Pb/L
60-d chronic bioassay:
Reduction in weight gain
observed at 25 pg Pb/L
(approx. 20% of the 96-h
LCso)
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Reference
Species Concentration Exposure Method Effects on Endpoint Effect Concentration (published
r r Factors r since the
2013 Pb ISA)
Vertebrates
Zebrafish
(Danio rerio)
0-
33,300 |jg Pb/L
(nominal values).
There was low
solubility of Pb in
the hard water;
the highest
concentration of
dissolved Pb
measured in
hard water was
590 |jg Pb/L with
total Pb
concentration of
630 |jg Pb/L
(1,000 |jg Pb/L
nominal values).
Highest
concentration
tested was
33,300 |jg Pb/L
(nominal values),
which was
3,830 |jg Pb/L
(measured) in
the hard water
Newly hatched larvae
were tested in either
soft water or hard
water with Pb as Pb-
nitrate for 96-h.
Experiments were
conducted in six-well
culture plates with 10
mL water and 10
larvae per well.
Water was changed
every 24 h
Temperature:
28°C
Soft water
Hardness:
11.7 mg CaCOs/L
pH: 7.48
Na+ = 220 M,
K+ = 14 M
Ca2+ = 75 M
Mg2+ = 42 M
DOC = 0.9 mg/L.
Hard water:
hardness = 141 m
g CaCC>3/L
pH = 7.8
Na+ = 700 M
K+ = 38 M
Ca2+ = 1,350 M
Mg2+ = 336 M,
DOC = 3.5 mg/L
Survival: Pb was more toxic to
larvae in soft water than hard
water. No mortalities were
observed in the bioassays with
hard water even at the highest
tested concentration
Soft water:
96-h LCso = 52.9 pg Pb/L
Hard water:
96-h LCso = >590 pg Pb/L
Alsop and
Wood (2011)
External Review Draft
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Reference
Species Concentration Exposure Method Effects on Endpoint Effect Concentration (published
r r Factors r since the
2013 Pb ISA)
Zebrafish
(Danio rerio)
2, 5, 10, 15
30 |jg Pb/L;
analytically
verified
concentration
20,
Embryos/larvae were
exposed to Pb
acetate tri hydrate
from 2 h
postfertilization (hpf)
embryos to 144 hpf
50% of the exposure
solution was renewed
daily
Temperature: Reproduction
28±0.5°C No significant effect on
percentage of hatched larvae at
any of the tested concentrations
Growth
Significant increase in prevalence
of malformations at 30 |jg Pb/L
compared with the control
Zhu et al.
(2014)
Survival
Significant decrease in survival at
30 |jg Pb/L compared with the
control
Zebrafish
(Danio rerio)
5, 9.7,
19.2 |jg Pb/L;
measured
6-hpf embryos
exposed to Pb
acetate tri hydrate
until 144-hpf. 50% of
exposure solution
was renewed daily
Temperature: Reproduction
28 ± 0.5 °C No significant difference on
hatching success rate at any of
the tested concentrations
Growth
No significant differences were
found for body length or body
weight at tested concentrations
compared with control
Zhu et al.
(2016)
Survival
No significant effect on survival
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Species
Concentration Exposure Method
Modifying
Factors
Effects on Endpoint
Effect Concentration
Reference
(published
since the
2013 Pb ISA)
Zebrafish
(Danio rerio)
19.3 |jg Pb/L
6-hpf embryos
exposed to Pb
acetate trihydrate
until 144-hpf. 50% of
exposure solution
was renewed daily.
Mortality rate,
malformation rate
(e.g., pericardial
edema and axial
spinal curvature) and
hatching success
recorded each day.
After exposure, body
length and body
weight of each
zebrafish larva was
measured
Temperature:
28 ± 0.5°C
Reproduction
No significant difference on
hatching success rate at
19.3 |jg Pb/L compared with
control.
Growth
No significant differences were
found for body length or body
weight at 19.3 |jg Pb/L compared
with control
Survival
No significant effect on survival at
19.3 |jg Pb/L
Chen et al.
(2016b)
Zebrafish
(Danio rerio)
4.5, 9.6,
18.6 |jg Pb/L
analytically
verified
concentration
6-hpf embryos
exposed to Pb
acetate tri hydrate
until 144-hpf. 50% of
exposure solution
was renewed daily.
For each treatment,
malformation,
survival rate and
hatching rate were
recorded at 24, 48,
72 and 96 hpf.
Additional behavioral
assays were
conducted at 144 hpf
Temperature: Reproduction:
28.5°C Hatching success rate
significantly decreased in all
concentrations at 72 hpf
compared with control; this delay
in hatching rate also observed at
96 hpf.
Survival
Survival rate of Pb-exposed
embryos at all tested
concentrations significantly lower
than controls at 96 hpf.
Zhao et al.
(2020)
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Reference
Species Concentration Exposure Method Effects on Endpoint Effect Concentration (published
r r Factors r since the
2013 Pb ISA)
rainbow trout
(Oncorhynchus
my kiss)
Waterborne-only
study:
4, 10, 20, 80,
240 and
800 |jg Pb/L
(nominal
concentration
reported,
concentrations
analytically
verified)
Waterborne, diet
and combined
exposure study:
0, 8.5, 20, 60
and 110 |jg Pb/L
(measured)
In waterborne
exposure to establish
LC/EC values,
juveniles (average
size = 2-4 g) were
exposed for 7 wk to
Pb as Pb-nitrate;
growth (weighed
weekly) and survival
were assessed at
various timepoints
including 96-h. In the
second study,
juvenile fish were
exposed for 7 wk via
waterborne Pb only,
dietary Pb only in the
form of live prey
(worms Lumbriculus
variegatus pre-
exposed for 28-d to
the same
concentration of Pb
as the fish) or
simultaneously to
waterborne and
dietary Pb
Temperature:
13°C
pH:
7.8-8.0
Hardness:
140 mg/L as
CaCC>3
DOC:
2.5 mg/L
Survival:
In the waterborne-only study to
establish LC/EC values, all fish in
the highest concentration tested
(800 |jg Pb/L) did not survive. In
the second study, survival in all
treatments (waterborne only,
dietborne only or combination)
and tested concentrations were
comparable to the control
(>90%).
Growth:
Waterborne Pb exposure had no
significant effects on specific
growth rate or biomass in either
experiment. In the dietary
combination experiment,
marginal (nonsignificant)
reductions were observed in the
dietborne and combined
exposures only at 110 |jg Pb/L
96-h:
LC10 = 304.3
H9
Pb/L
LC20 = 357.7
H9
Pb/L
LCso = 487.3
H9
Pb/L
Alsop et al.
(2016)
7-w:
LC10 = 55.6 |jg Pb/L
LC20 = 96.9 |jg Pb/L
LCso = 280.2 |jg Pb/L
External Review Draft
11-160
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Reference
Species Concentration Exposure Method Effects on Endpoint Effect Concentration (published
r r Factors r since the
2013 Pb ISA)
rainbow trout
(Oncorhynchus
my kiss)
white sturgeon
(Acipenser
transmontanus)
Trout:
0, 10, 20, 40, 80,
160 |jg Pb/L
(nominal values)
Sturgeon:
0, 5.0, 10, 20,
40, 80 |jg Pb/L
(nominal values)
Measured
concentrations of
metals (not
provided) were
used for
calculation of
effect
concentration
A series of chronic
tests with two
lifestages (newly
hatched larvae and
approximately 1-mo-
old juveniles) of trout
and sturgeon were
conducted in
aqueous-only
exposure with Pb as
Pb-nitrate.
For trout: C1: 1-dph
larval trout in a 21-d
exposure; C2: 26-dph
juvenile trout in a 28-
d exposure; CC: 1-
dph larval trout in a
52-d exposure.
For sturgeon: C1: 2-
dph larval sturgeon in
a 25-d exposure C2:
27-dph juvenile
sturgeon in a 28-d
exposure; CC: 2-dph
larval sturgeon in a
53-d exposure. An
additional (C1-R) test
was conducted with
1-dph larval sturgeon
in a 24-d exposure
Trout:
Temperature:
12 ± 1°C
Hardness:
Approximately
100 mg/L as
CaC03,
Alkalinity:
approximately
90 mg/L as
CaCC>3
pH:
approximately
8.0
Sturgeon:
Temperature:
15 ± 1°C
Hardness:
Approximately
100 mg/L as
CaCC>3
Alkalinity:
approximately
90 mg/L as
CaCC>3
pH:
approximately
8.0
Growth/Survival
Note: Effect concentrations
reported in this study are based
on the most sensitive endpoint
(mortality, immobility, fish length
or biomass).
Trout: No acute effects observed
in larval or juvenile fish after 4-d.
Generally, trout were tolerant to
Pb concentration used in the
study
Sturgeon:
No mortality or immobilization of
newly hatched sturgeon was
observed by 4-d. The 53-d
exposures did not meet the test
acceptability criteria (due to
control mortalities); therefore,
there are no 53-d EC20S for the
survival. However, the EC20S
based on the length and weight
of surviving fish throughout the
53-d exposures were reported
Trout:
Acute 4-d EC50
C1 (larvae): >136 |jg Pb/L
C2 (juvenile):
>143 |jg Pb/L
CC (larvae) >136 |jg Pb/L
Chronic EC20
C1 (larvae 21-d)
>128 |jg Pb/L
C2 (juvenile 28-d)
>128 |jg Pb/L
CC (larvae 52-d)
>126 |jg Pb/L
Sturgeon:
Acute 4-d EC50
C1 (larvae): >55 |jg Pb/L
C2 (juvenile): >61 |jg Pb/L
CC (larvae) >55 |jg Pb/L
Chronic EC20
C1 (larvae 14-d)
>56 |jg Pb/L
C2 (juvenile 28-d)
>60 |jg Pb/L
CC (larvae 53-d)
>27 |jg Pb/L (note: low
control survival in this
experiment)
Wang et al.
(2014a)
External Review Draft
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Reference
Species Concentration Exposure Method Effects on Endpoint Effect Concentration (published
r r Factors r since the
2013 Pb ISA)
white sturgeon
(Acipenser
transmontanus)
8 dph
Lab:
0.1, 0.8, 2.3, 6.4,
19, 65, 210,
414 |jg Pb/L
Columbia River:
0.2, 0.4, 1.4, 6.1,
17, 60, 191,
410 |jg Pb/L
40 dph
Lab:
0.1, 21, 46, 97,
208, 396, 809,
1610 |jg Pb/L
Columbia River:
0.3, 20, 37, 95,
192, 325, 799,
1685 |jg Pb/L
96-h acute toxicity
assays conducted
with two lifestages (8
and 40 dph)under
static renewal
conditions with Pb as
Pb-nitrate in
laboratory water and
field-based tests with
Columbia River
water. The
laboratory- and field-
based tests were
conducted in parallel,
under the same
exposure conditions
and following the
same experimental
protocols. Water from
the Columbia River
was pumped into a
trailer retrofitted for
toxicity testing
Laboratory water Survival
Temperature:
16 ± 0.9°C
PH
7.5 ± 0.2
Ca2+ to Mg2+
Ratio: -1.3:1
Columbia River
Water
Temperature:
16 ± 0.7°C
PH
7.7 ± 0.1
Ca2+ to Mg2+
Ratio: ~4:1
Fish exposed at 8 dph were more
sensitive than fish exposed at 40
dph. Fish exposed in lab water
were more sensitive than fish
exposed to Columbia River
water. There was a lack of
mortality observed in 8 dph fish
exposed to river water even at
the highest concentration tested.
8 dph
96-h LCso= 177 pg Pb/L
(lab water)
96-h LCso = >410 pg Pb/L
(Columbia River water)
40 dph
96-h LCso = 528 pg Pb/L
(lab water)
96-h LCso= 1,556 pg Pb/L
(Columbia River water)
Vardv et al.
(2014)
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Species
Concentration Exposure Method
Modifying
Factors
Effects on Endpoint
Effect Concentration
Reference
(published
since the
2013 Pb ISA)
Asiatic toad
(Bufo gargarizans)
0, 10, 50, 100,
500 and
1000 |jg Pb/L,
nominal; 0, 9.85,
48.73, 97.69,
497.34 and
998.27 |jg Pb/L,
measured
First larval stage
(Gosner stage 26)
tadpoles exposed to
Pb acetate in static
renewal (every 48 h)
solutions up to
Gosner stage 42
(forelimb emergence
starting at 31 to 35 d
depending on Pb
treatment group).
Tadpole growth and
developmental stage
assessed at day 10
and day 20.
Exposure continued
until day 60 to
determine mean
percent
metamorphosis
Temperature: Growth
~20°C On days 10 and 20, significant
increase reported in total tadpole
length and body mass at
50 |jg Pb/L. At Gosner
developmental stage 42
(metamorphic climax), snout-vent
length was significantly longer
than control in the 10 |jg Pb/L
treatment group. Snout-vent
length and total length were
significantly longer in tadpoles
exposed to 50 |jg Pb/L compared
with control. No statistically
significant difference in body
mass or tail length in any
treatment.
Survival
No mortality observed in control,
10, 50 or 100 |jg Pb/L during 60-
d exposure.
Yang et al.
(2019)
Dark-spotted frog
(Pelophylax
nigromaculata)
40, 80, 160, 320,
640,
1280 |jg Pb/L
nominal; 38.2,
79.3, 158.4,
318.7, 638.1,
1278.9 |jg Pb/L
analytically
verified
concentration
Embryos exposed to
Pb-nitrate in static
renewal assays from
heartbeat (Gosner
stage 19) to full
metamorphosis
(Gosner stage 46).
Chronic exposure
duration was up to
70 d
Temperature
19-25°C (room
temperature)
PH
7.04-7.69,
DO
6.8-7.3mg/L
Hardness
249-258 mg
CaCOs/L
Growth
Growth was inhibited at higher Pb
concentrations; total
malformation rate increased
linearly with Pb concentration.
Survival
No significant effect on survival at
40, 80, 160 or 320 pg Pb/L
Lowest threshold Huang et al.
concentration = 160 pg Pb (2014)
/L for effects on
metamorphosis time, body
mass, snout-vent length,
and forelimb length
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Reference
Species Concentration Exposure Method Effects on Endpoint Effect Concentration (published
r r Factors r since the
2013 Pb ISA)
Multiple
37 species and 32
genera of
invertebrates and
fish
(acute toxicity data
included in
derivation of
proposed updated
acute freshwater
quality criterion for
Pb)
15 species and 13
genera of
invertebrates and
fish (chronic toxicity
data included in
derivation of
proposed updated
chronic freshwater
quality criterion for
Pb)
Pb was
analytically
verified in all
studies
U.S. EPA guidelines
(U.S. EPA. 1985b)
were followed to
identify acceptable
studies. Water
chemistries over a
wide range of
conditions were
predicted from the
biotic ligand model.
Acute: All included
assays were
waterborne Pb
exposures reporting
48 to 96-h EC50S.
The four lowest
genus mean acute
values (Hyalella,
Ceriodaphnia,
Gammarus and
Daphnia) and a total
of 32 genus mean
values were used to
determine a 50th
percentile critical
accumulation
concentration to
derive the proposed
acute criterion based
on U.S. EPA
methods
Acute toxicity end points
included survival,
immobilization, and loss of
equilibrium
The proposed updated acute
criterion is based on expanded
toxicity data sets and BLM
predictions that demonstrate the
influence of water hardness, used
in the calculation of the current
water quality criteria, is less
important as a modifying factor
relative to DOC.
Chronic toxicity endpoints
included survival, growth, and
reproduction
There is sufficient new chronic
toxicity data for Pb since the 1984
water quality criteria to allow for
direct determination of criteria
from toxicity data, rather than the
use of an acute-to-chronic ratio.
Proposed Freshwater
Acute Water Quality
Criterion based on BLM of
North American surface
water chemistry
conditions ranged from
18.9 to 998 |jg Pb/L.
Proposed Freshwater
Chronic Water Quality
Criterion based on BLM of
North American surface
water chemistry
conditions ranged from
0.37 to 41 |jg Pb/L
Deforest et al.
(2017)
Chronic: Based on
EC20 values from life
cycle tests in
External Review Draft
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Reference
Species Concentration Exposure Method Effects on Endpoint Effect Concentration (published
r r Factors r since the
2013 Pb ISA)
freshwater
invertebrates as well
as partial life cycle or
early lifestage tests in
fish. The four lowest
genus mean chronic
values (Lymnaea,
Philodina, Hyalella,
Ceriodaphnia) and a
total of 13 genus
mean values were
used to identify a
chronic 5th percentile
waterborne Pb
concentration
following EPA
guidelines
Ca2+ = calcium ion; CaC03 = calcium carbonate; d = day; DOC = dissolved organic carbon; dph = days posthatch; DT = diatom + Tetramin; ECX = X% effect concentration;
hpf = hours postfertilization; K+ = potassium ion; LCX = X% lethal concentration; Mg2+ = magnesium ion; mo = months; Na+ = sodium ion; Pb = lead; Pb(N03)2 = lead nitrate;
wk = weeks; YCT = yeast, cereal leaves and trout; yr = year.
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11.3.6. Freshwater-Community and Ecosystem Effects
Field studies in the 2006 Pb AQCD (U.S. EPA. 2006b) and the 2013 Pb ISA (U.S. EPA. 2013)
report reductions of species abundance, richness, or diversity, particularly in benthic macroinvertebrate
communities coexisting with multiple metals where the sources of Pb were from mining or urban
effluents. Changes to aquatic plant community composition have been observed in the presence of
elevated surface water Pb concentrations. Additionally, field studies have linked Pb contamination to
reduced primary productivity and respiration, and to altered energy flow and nutrient cycling. In the 2013
Pb IS A (U.S. EPA. 2013) the body of evidence was sufficient to conclude there is a likely to be causal
relationship between Pb exposure and freshw ater-community and ecosystem effects. Studies reviewed in
that document noted ecological effects on invertebrate communities can occur at environmental Pb
concentrations lower than those required to affect plant communities. High sediment Pb concentrations
were linked to shifts in amphipod communities inhabiting plant structures, and potentially to alterations in
ecosystem nutrient processing. Although the presence of Pb is associated with shifts in biological
communities, this metal rarely occurs as a sole contaminant in natural systems, making the contribution of
Pb to the observed effects difficult to ascertain. New information on the effects of Pb at the population,
community, and ecosystem levels is reviewed below.
Several studies reviewed here reported negative associations between sediment Pb concentration
and invertebrate community composition. A series of studies conducted in Caddo Lake, Texas has further
elucidated the effects of Pb on benthic macroinvertebrate communities and Pb as a modifying factor in
leaf-litter decomposition. Caddo Lake is a shallow, eutrophic lake which neighbors a superfund site
(Longhorn Army Ammunition Plant, Texas). Qguma and Klerks (2015) found evidence that Pb
contamination may affect leaf-litter decomposition in the lake. Litter decomposition (relative change in
dry weight of American lotus [Nelumbo lutea] leaves deployed in litter bags) was determined after
30 days at sites spanning a gradient of sediment Pb concentration. Sediment Pb concentration in Caddo
Lake ranged from 4.3 to 148.9 mg Pb/kg, with some sites exceeding the Probable Effects Concentration
for sediment (128 mg Pb/kg). In a principal component analysis, total sediment Pb and sediment
porewater Pb were positively correlated, and benthic macroinvertebrate abundance was negatively
correlated with sediment Pb concentration and porewater Pb concentration. The authors suggested that the
combination of sediment Pb content and decreased macroinvertebrate abundance, among other untested
factors, may lead to reduced leaf-litter decomposition in Caddo Lake. Macroinvertebrate assemblage from
sediments collected from a contaminated region of the lake and a control area were evaluated to assess
community tolerance to Pb in 48-hour aqueous exposure to a range of nominal Pb concentrations (0, 20,
200, 2,000, 200,000, or 2,000,000 (ig Pb/L) (Qguma and Klerks. 2017). Mortality for benthos under
increasing [Pb2+] concentration was lower than for those macroinvertebrates collected from the control
site, suggesting community tolerance. The interaction between the collection site (control versus
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contaminated site) and [Pb2+] on survival was nonsignificant. Macroinvertebrate density was similar
between the two sediments. When benthic macroinvertebrate species composition was compared between
sites, the community at the control site included more metal-sensitive taxa (gastropods and amphipods)
compared with sediments from the contaminated site.
In another study on sediment macroinvertebrates in Caddo Lake, sediment Pb concentration was
negatively correlated with the diversity and abundance of benthic macroinvertebrates although amphipod
sensitivity to Pb and Cu was unrelated to sediment Pb and Cu concentrations (Oguma and Klerks. 2020).
Using a univariate approach between benthic community metrics and heavy-metal concentrations, the
benthic macroinvertebrate abundance, family richness, and Shannon H' Index were negatively correlated
with sediment Pb concentrations. Although this study provides correlational evidence that Pb sediment
concentration affects benthic macroinvertebrate community structure, % sand/clay content, % OM, and
Cu sediment concentration among other principal components are correlated with benthic
macroinvertebrate community metrics. A sensitive amphipod (H. azteca) was exposed to sediment, and
reproduction, survival and growth were assessed at 28, 35, and 42 days. The survival (28, 35, and
42 days), reproduction (35 and 42 days) and growth (42 days) of H. azteca were not affected by Pb
sediment concentration.
Crayfish density was negatively correlated with sediment Pb concentration in the Old Lead Belt
mining district in Missouri where Pb-Zn mining occurred from the 1700s to the 1970s (Allert et al..
2013). Parts of the district were designated as U.S. EPA Superfund sites. To test whether benthic
macroinvertebrate, fish, and crayfish communities differed along Pb and other heavy-metal gradients in
the Big River, benthic fish, crayfish, macroinvertebrates, sediment, and surface waters were sampled from
riffles from eight sites (two reference sites where no mining activities occurred, two mining sites with
high contamination, and four sites downstream of the mining sites with slightly lower contamination).
The density of fish including sculpins (Cottus spp.), darters (Etheostoma spp. And Percina spp.), and
madtoms (Noturus), and crayfish (Orconectes spp.) was estimated in situ. Individuals of the Missouri
saddled darter {Etheostoma tetrazonum) and golden crayfish {(). luteus) were collected and used for metal
analyses. Additionally, an in situ toxicity test on juvenile O. luteus and O. hylas was conducted at the two
reference sites and two mining sites over 56 days, and the growth and survival of crayfish were assessed
at the end of the test. Surface water Pb concentrations were lowest at the reference sites
(0.06 ± 0.01 |ig Pb/L, mean ± S.D.) and highest at the mining sites (7.85 ± 1.63 |ig Pb/L). Sediment Pb
concentrations followed the same pattern, with the lowest concentrations at the reference site
(12.5 ±2.1 mg Pb/kg dry weight), followed by the downstream sites (710 ± 530 mg Pb/kg dry weight)
and the highest concentrations at the mining sites (1170 ± 467 mg Pb/kg dry weight). Pb in the sediment
at the mining and downstream sites was significantly higher than the Probable Effects Concentration for
sediment derived by (Macdonald et al.. 2000) (128 mg Pb/kg dry weight). Pb concentration in detritus
was significantly lower in reference sites compared with mining sites. Moving up the food web, Pb
concentration in macroinvertebrates was lower in reference sites than in mining sites
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(12.7 ± 4.4 mg Pb/kg dry weight for reference sites and 720 ± 276 mg Pb/kg dry weight for mining sites,
respectively). Similarly, in two different larval species of caged crayfish ((). luteus and O. hylas), Pb
concentration was lower in reference sites compared with the mining site. Field-collected adult O. luteus
Pb concentration followed the same pattern, reference Pb
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(Qguma and Klerks. 2015). Macroinvertebrate community composition was found to be sensitive to mild
Pb contamination in a freshwater lake (Qguma and Klerks. 2020. 2017). Crayfish and fish density was
negatively correlated to surface water Pb concentrations and sediment concentrations for crayfish in a
river system (Allcrt et al.. 2013). Pb accumulated in reeds were found to be negatively, positively, or not
correlated with abundance of some periphyton families (Obolewski et al.. 2011) Finally, larval and adult
insect community structures were affected by natural gradients of Pb in a lake system (Lidman et al..
2020).
11.4 Saltwater Ecosystems
11.4.1. Summary of New Information on Effects of Pb in Saltwater Ecosystems
and Causality Determination Update Since the 2013 Pb ISA
Historically, the effects of Pb were less well characterized in saltwater biota compared with
freshwater biota. Few studies on Pb toxicity have been conducted on saltwater plant and algal species, and
the observed effects generally occurred at concentrations that greatly exceeded reported concentrations of
Pb from coastal waters (Table 11-1). Evidence in the 2013 ISA was inadequate to infer causality
relationships between Pb exposure and effects on physiological stress, growth, survival, and reproduction
in saltwater plants and algae (U.S. EPA. 2013). In the 1977 Pb AQCD and the 1986 Pb AQCD, there
were no studies that reported the effects of Pb in saltwater invertebrates. In the 2006 AQCD, few effects
were noted in saltwater invertebrates including gender differences in sensitivity to Pb in copepods,
increasing toxicity of Pb with decreasing salinity in mysids and effects on embryogenesis in bivalves
(U.S. EPA. 2006a). In the 2013 Pb ISA, available evidence was sufficient to be suggestive of a causal
relationship between Pb exposure and the endpoints of physiological stress, hematological effects, and
reproduction for saltwater invertebrates (U.S. EPA. 2013). Evidence for effects on neurobehavior, growth
and survival in saltwater invertebrates and vertebrates, as well as effects on ecological populations and
communities, was concluded to be inadequate to infer a causality relationship.
For many of the endpoints for saltwater biota (Table 11-7), evidence remains inadequate to assess
causality. For other endpoints, new evidence continues to support, or expands somewhat, the basis for the
causality determination in the 2013 Pb ISA. For suborganism-level endpoints, evidence was suggestive of
a causal relationship between Pb exposure and physiological stress in saltwater invertebrates in the 2013
Pb ISA, and this remains the case. There is very little new evidence for hematological effects of Pb in
saltwater invertebrates, which, at the time of the 2013 Pb ISA, was suggestive of, but not sufficient to
infer, a causal relationship (U.S. EPA. 2013). Evidence for hematological effects in previous AQCDs and
the 2013 Pb ISA were primarily from field monitoring studies of marine bivalves using ALAD as a
biomarker for Pb exposure and correlated ALAD inhibition to increased Pb tissue content. For the
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organism-level endpoints of neurobehavior and growth effects associated with Pb exposure, there is
inadequate experimental evidence to assess causality for saltwater species.
Since the 2013 Pb ISA, there is additional research for saltwater organisms that supports a change
in causality determinations for some endpoints. Several newer studies quantify Pb in exposure media and
report effects on endpoints at lower concentration than previously observed for saltwater biota. The
increased availability of studies that report analytically verified concentrations have enabled updated
estimates of effects criteria. For example, an increase in toxicological data for saltwater organisms over
the last several years and the availability of studies that analytically verified Pb exposure concentration
has led to a study proposing updates to the acute and chronic AWQC for Pb (Church et al.. 2017). For the
acute criterion, the proposed update of 100 |ig Pb/L is less than the current acute criterion of 210 (ig Pb/L
due to more recent toxicity data from relatively sensitive early lifestages of Echinodermata and Mollusca.
In the 2013 Pb ISA, the evidence at that time for Pb effects on the survival of saltwater
vertebrates was inadequate to infer a causal relationship with Pb exposure (U.S. EPA. 2013). New
evidence (Section 11.4.5) is limited to laboratory-based bioassays in a few fish species. Toxicity data for
other saltwater vertebrates remains lacking. Several recent chronic bioassays conducted with early
lifestages of three saltwater fish species reported NOEC in the range of 11-14 |ig Pb/L (Table 11-7).
Furthermore, Pb in these bioassays was analytically verified. In the larval fish topsmelt (Atherinops
affinis), LC50 = 15.1 jxg Pb/L and NOEC<13.8 |ig Pb/L were obtained at a salinity of 14 ppt (Reynolds et
al.. 2018). Calculated chronic values for additional saltwater fish species that are consistent with the range
reported above include grey mullet (Mugil cephalus) fingerling survival and Tiger perch (Terapon
jarbua) fingerling survival (Hariharan et al.. 2016). Based on these new chronic studies in saltwater fish,
the evidence is suggestive of, but not sufficient to infer, a causal relationship between Pb exposure
and saltwater vertebrate survival.
In the 2013 Pb ISA the evidence was concluded to be suggestive of, but not sufficient to infer, a
causal relationship between Pb exposure and reproduction and developmental effects in saltwater
invertebrates (U.S. EPA. 2013). Endpoints reported in the previously available studies included a delay in
the onset to reproduction (amphipod Elasmopus laevis) (Ringenarv et al.. 2007). impaired larval
development (Wang et al.. 2009)and embryogenesis inhibition (Wang et al.. 2009; Beiras and Albentosa.
2004) in bivalves and a decrease in the fertilization rate of eggs (marine polycheate annelid Hydroides
elegans) (Gopalakrishnan et al.. 2008). Since the 2013 Pb ISA, the evidence base for Pb effects on
reproductive and developmental endpoints in saltwater invertebrates has expanded, primarily due to
multiple new embryo-larval developmental assays in Mollusca and Echinodermata (Section 11.4.5 and
Table 11-7). Several of these acute exposure bioassays analytically verified the concentration of Pb at
which effects were observed (Markich. 2021; Romero-Murillo et al.. 2018; Nadella et al.. 2013) and
reported effects at lower concentrations than those reported in the 2013 Pb ISA. The 48-hour EC10 larval
development in the mussels Mytilus trossulus and Mytilus galloprovincialis, was 9 and 10 jxg Pb/L
respectively, and 72-hour EC10 was 19 jxg Pb/L in the sea urchin Strongylocentrotus purpuratus (Nadella
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ct al.. 2013). In the scallop Argopectenpurpuratus, there was a 48-hour EC50 = 44 |ig Pb/L for abnormal
larval development (Romcro-Murillo et al.. 2018). These effects concentrations are comparable to those
reported for larval developmental assays from two species of oysters Magallana gigas (48-hour
EC50 = 49.5 |ig Pb/L, 48-hour NEC = 9.9 |ig Pb/L) and Saccostrea glomerata (48-hour
EC50 = 52.1 |ig Pb/L, 48-hour NEC = 10.1 |ig Pb/L) (Markich. 2021). Considering the coherence of
reproductive and developmental effects of Pb across species, observations in saltwater invertebrates are
consistent with terrestrial and freshwater invertebrates (both "causal" in the 2013 Pb ISA) As a result of
the newly available evidence since the 2013 Pb ISA, the evidence is sufficient to conclude there is
likely to be a causal relationship between Pb exposure and reproductive and developmental effects
in saltwater invertebrates.
For community and ecosystem effects, evidence was inadequate in the 2013 Pb ISA to assess
causality between Pb exposures and the alteration of species richness, species composition and
biodiversity in saltwater ecosystems. Reduced species abundance and the biodiversity of protozoan and
meiofauna communities were observed in laboratory microcosm studies with marine water and marine
sediments reviewed in the 2006 Pb AQCD, as summarized in Table AX7 2.5.2 (U.S. EPA. 2006b). In the
2013 Pb ISA, there were a few additional studies including effects on community structure and nematode
diversity that were altered in a microcosm study with marine sediments (Mahmoudi et al.. 2007). Since
then, new experimental and observational studies have examined the relationship between Pb in sediment
and microbial abundance and/or diversity (Section 11.4.4.1), as well as Pb associations with saltwater
foraminiferal communities (Section 11.4.6). Several of the benthic foraminifera studies reported effects
on community richness, diversity, and abundance. In other studies with foraminifera, there were changes
in the abundance of certain taxa associated with Pb, but not diversity metrics. Considering the new
evidence, Pb quantified in sediment is a factor that explains variations in microbial diversity and
foraminiferal species distributions and abundance in a variety of distinct geographic locations. In these
studies, Pb was often correlated with other heavy metals.
These effects observed in saltwater biota are coherent with the observed community and
ecosystem-level effects of Pb in terrestrial and freshwater environments, which were reported as "likely
causal" in the 2013 Pb ISA (U.S. EPA. 2013). In addition to the available studies assessing Pb effects on
saltwater communities, primarily foraminifera, the effects of Pb on reproduction in sensitive saltwater
invertebrates and possible effects on survival in early lifestages of some saltwater vertebrates, especially
when considered cumulatively, could affect populations as well as community and ecosystem structure
and function. Population, community, or ecosystem-level studies are typically conducted at sites that have
been affected by multiple stressors (several chemicals alone or combined with physical or biological
stressors), which increase the uncertainty of attributing the observed effects to Pb. Therefore, for saltwater
the evidence is suggestive of, but not sufficient to infer, a causal relationship between Pb exposure
and community and ecosystem effects.
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Table 11-6 Updated causality determinations for Pb in saltwater organisms
and ecosystems.
Level
Effect
Saltwater
2013 Pb ISA3
2023 Pb ISAb
Community and
Ecosystem
Community and Ecosystem Effects
Inadequate
Suggestive
Reproductive and Developmental Effects
- Plants
Inadequate
Inadequate
Reproductive and Developmental Effects
- Invertebrates
Suggestive
Likely Causal
Population-
level
Reproductive and Developmental Effects
- Vertebrates
Inadequate
Inadequate
Growth - Plants
Inadequate
Inadequate
Endpoints
Organism-level
Responses
Growth - Invertebrates
Inadequate
Inadequate
Growth - Vertebrates
Inadequate
Inadequate
Survival - Plants
Inadequate
Inadequate
Survival - Invertebrates
Inadequate
Inadequate
Survival - Vertebrates
Inadequate
Suggestive
Neurobehavioral Effects - Invertebrates
Inadequate
Inadequate
Neurobehavioral Effects - Vertebrates
Inadequate
Inadequate
Hematological Effects - Invertebrates
Suggestive
Suggestive
Hematological Effects - Vertebrates
Inadequate
Inadequate
Suborganismal
Responses
Physiological Stress - Plants
Inadequate
Inadequate
Physiological Stress - Invertebrates
Suggestive
Suggestive
Physiological Stress - Vertebrates
Inadequate
Inadequate
Conclusions were based on the weight of evidence framework for causal determination in Table II of the 2013 Pb ISA (U.S. EPA.
2013). Ecological effects observed at or near Pb concentrations measured in sediment and water in Table 6-2 of the 2013 Pb ISA
were emphasized, and studies generally within one to two orders of magnitude above the reported range of these values were
considered in the body of evidence for saltwater (Section 6.4.21) (U.S. EPA. 2013). bChanges from the 2013 Pb ISA are indicated
as bolded text.
1
2 The 2013 Pb ISA concluded that the body of evidence was suggestive of a causal relationship
3 between Pb exposure and physiological stress, hematological effects, and reproductive and developmental
4 effects in saltwater invertebrates (Table 11-6). Evidence was inadequate at the time to assess causality for
5 additional effects in saltwater invertebrates and for marine algae and vertebrates. Key uncertainties from
6 the last review for saltwater ecosystems included the uncertainties associated with generalization of
7 effects observed in controlled laboratory studies to conditions in coastal environments where many
8 modifying factors affect Pb bioavailability and toxicity. In general, Pb toxicity to marine or estuarine
9 plants, invertebrates and vertebrates was less well characterized than toxicity to Pb in freshwater systems
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in the 2013 Pb ISA due to an insufficient quantity of studies on saltwater organisms. Specifically, there
was a lack of chronic toxicity data, and relatively few studies reported analytically verified Pb
concentration in the experimental media. Information regarding the contribution of atmospheric Pb to
total Pb in coastal environments was sparse. This was attributed to multiple sources of Pb, confounding
effects of transport from terrestrial and freshwater systems and the lack of studies connecting the air
concentration of Pb and saltwater ecosystem exposure.
Studies published since the 2013 Pb ISA (literature cutoff for inclusion in the 2013 Pb ISA was
September 2011) that characterized bioavailability, uptake, bioaccumulation, and effects of Pb in
saltwater ecosystems or that decreased uncertainties identified in the prior NAAQS review of this criteria
air pollutant are presented throughout the following sections. Saltwater ecosystems considered encompass
a range of salinities from just above that of freshwater (<1 ppt) to that of seawater (generally described as
35 ppt). Coastal ecosystems may receive Pb from multiple sources such as contributions from
atmospheric deposition and via inputs from terrestrial systems including runoff and riverine transport
(Appendix 1: https://cfpub.epa.gov/ncea/isa/recordisplay.cfm?deid=357282).
Habitats associated with coastal areas include salt marshes, estuaries, shallow open waters, sandy
beaches, mud and sand flats, rocky shores, oyster beds, coral reefs, mangrove forests, river deltas, tidal
pools, and seagrass beds (U.S. EPA. 2016). Estuaries, where freshwater inflows gradually mix with salt
water, are dynamic, heterogeneous environments characterized by gradients of salinity. Salinity is one of
the modifying factors affecting Pb speciation in coastal systems, and changes in salinity affect the ionic
strength of the water (Section 11.4.2). The Pb2+ ion, which is the most bioavailable form of Pb, is not
common in seawater; rather, Pb primarily exists as a carbonate complex and to a lesser extent as a
chloride complex (Church et al.. 2017; Millero et al.. 2009).
Brief summaries of conclusions from the 1977 Pb AQCD (U.S. EPA. 1977). the 1986 Pb AQCD
(U.S. EPA. 1986). the 2006 Pb AQCD (U.S. EPA. 2006a) and the 2013 Pb ISA (U.S. EPA. 2013) are
included where appropriate. Recent research on the bioavailability and uptake of Pb into saltwater
organisms including plants, invertebrates and vertebrates is presented in Section 11.4.2. Section 11.4.3
covers environmental concentrations of Pb in saltwater biota and ecosystems in the United States at
different locations and over time. The toxicity of Pb to marine flora and fauna including growth,
reproductive and developmental effects (Section 11.4.4) is followed with data on exposure and the
response of saltwater organisms (Section 11.4.5). Responses at the community and ecosystem levels of
biological organization are reviewed in Section 11.4.6.
11.4.2. Factors Affecting Bioavailability, Uptake and Bioaccumulation, and
Toxicity in Saltwater Biota
Factors affecting bioavailability of Pb to saltwater organisms are many of the same factors
affecting bioavailability to freshwater biota (Section 11.3.2), notably OM and pH. Other factors, such as
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salinity, play a greater role in Pb fate, transport, and bioavailability in saltwater systems, especially in
dynamic estuarine environments characterized by gradients of salinity. Since the 2013 Pb ISA, there is
additional information (summarized below) on these chemical factors which can be quantified and
directly related to toxicity. Studies have further explored the effects of varying DOM composition and
changing pH on Pb uptake and bioaccumulation in saltwater biota. Other factors that affect the uptake and
toxicity of Pb, such as biological adaptations by organisms, are more difficult to link quantitatively to
toxicity. As discussed in previous EPA reviews of Pb, species differences in metabolism, sequestration,
and elimination rates have been shown to control the relative sensitivity and vulnerability of exposed
organisms and influence the potential for effects on survival, reproduction, growth, metabolism, and
development. Diet and lifestage at the time of exposure also contribute significantly to sensitivity and
vulnerability in populations and communities. The 2006 Pb AQCD (U.S. EPA. 2006b') reviewed the
effects of genetics, age, and body size on Pb toxicity. While genetics appears to be a significant
determinant of Pb sensitivity, the effects of age and body size are complicated by environmental factors
that alter the metabolic rates of saltwater organisms. Literature reviewed in the 2013 Pb ISA corroborated
these findings and discussed seasonal physiological changes and lifestage as important determinants of
differential sensitivity to Pb.
11.4.2.1. Dissolved Organic Matter
In seawater, DOM is a major factor controlling bioavailability of Pb (U.S. EPA. 2013). Studies
reviewed in the 2013 Pb ISA showed that different components of DOM have different effects on Pb
bioavailability in marine systems. Increasing humic acid concentrations increased Pb uptake by mussel
gills and increased toxicity to sea urchin (Paracentrotus lividus) larvae (Sanchez-Marin et al.. 2007).
while in contrast, fulvic acid reduced Pb bioavailability (Sanchez-Marin et al.. 2011). Continuing their
research in a study published after the 2013 Pb ISA Sanchez-Marin and Beiras (2012) observed that more
soluble DOM (fulvic acids and DOM extracted from the Suwannee River) also increased the
bioavailability and toxicity of Pb to sea urchin embryos, although not to the same extent as humic acid.
Furthermore, the experimental evidence suggests that the mechanisms by which DOM enhances Pb
uptake and toxicity implies direct contact of the organic compounds with the plasma membrane. In
another study examining the effects of different forms of DOM, Tang et al. (2020) observed that the
bioaccumulation of Pb in saltwater shrimp was likely affected by the quality of OM; with more
autochthonous OM present, there was less bioaccumulation compared with the levels in winter months
when more allochthonous OM is present. Additionally, because the ingestion of DOM bound to metals is
the major route of entry for metals, this suggests that the allochthonous OM may have a greater
percentage of functional groups that bind Pb (e.g., fluorophores).
Several studies published since the 2013 Pb ISA have explored the protective effects of different
types of OM by quantifying enzymatic activity and oxidative response in saltwater invertebrates.
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Nogueira et al. (2018) examined the toxicity of Pb alone and in combination with natural OM (NOM)
from different sources (allochthonous, autochthonous, and mixed) on larvae of the Canadian native bay
mussel (Mytilus trossulus). With 48-hour exposure to Pb alone (20 |ig Pb/L, nominal value) there was an
increase in carbonic anhydrase activity and lipid peroxidation. Various NOMs did not protect against Pb
toxicity, and lipid peroxidation increased significantly with some types of NOM. A parallel study
conducted on the invasive Mediterranean mussel (Mytilus galloprovincialis) (Nogueira et al.. 2017) also
showed that various sources of NOM differentially induced increases of enzyme activities and oxidative
stress to a greater extent than Pb alone; however, M. galloprovincialis was less sensitive than native M
trossulus overall. In these studies, no protective effects of NOM were observed. The interaction of NOM
with metals is influenced by the source and composition of NOM, and some forms of NOM may exert a
sublethal response independently. In a series of bioassays, Nadella et al. (2013) assessed the influence of
DOM on the embryo development of two mussels, M. galloprovincialis and M. trossolus, and the pacific
purple sea urchin (S. purpuratus). Addition of DOM from a freshwater source and a seawater source
decreased the toxicity of Pb to embryos of the mussels compared with toxicity tests in 100% seawater.
However, there was no concentration-dependent relationship with increasing addition of DOM.
Unexpectedly, DOM exacerbated Pb toxicity in 48-hour embryo toxicity tests with S. purpuratus. In the
absence of Pb, one of the DOM sources resulted in 100% mortality of S. purpuratus embryos. The
authors speculated that this is a species-dependent response, attributable to DOM interaction with the
epithelial interface.
11.4.2.2. pH
The importance of pH in the speciation of Pb in saltwater environments and as a modifying factor
of Pb toxicity was previously reported (U.S. EPA. 2013. 2006a'). Several additional studies published
since the 2013 Pb ISA further describe pH effects on Pb uptake and toxicity in saltwater organisms. A
decrease in pH under the scenario of increasing ocean acidification may lead to additional bioavailable Pb
(Pb2+) in marine environments (Figure 11-5) and associated toxic effects on biota as reviewed in Ivanina
and Sokolova (2015). Belivermis et al. (2020) demonstrated that a decrease in pH (from 7.94 to 7.16)
resulted in a significant increase in 210Pb in the soft tissues, but not the shells, of blue mussels (M edulis)
after a 9-day exposure. Pb uptake in mussels was highly variable, likely due to the variability of the
physiological status of individual mussels. The lower Ca2+ in acidified seawater can make Pb2+ more
available to mussels due to decreased competition, and the lower pH means a higher partial pressure of
CO2, which can result in decreased biomineralization that may facilitate the uptake of Pb.
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In a study reviewed in the 2006 AQCD, Verslvcke et al. (2003) exposed the estuarine mysid
Neomysis integer to individual metals, including Pb, and metal mixtures under changing salinity. At a
salinity of 5%, the reported LC50 for Pb was 1140 |ig/L (95% CL = 840, 1440 (.ig/L). At an increased
salinity of 25%, the toxicity of Pb was substantially reduced (LC50 = 4,274 |ig/L [95% CL = 3,540,
5710 |ig/L|). The reduction in toxicity was attributed to increased complexation of Pb2+ with Cl~ ions.
Studies published since the 2013 Pb ISA have further considered salinity as a modifier of Pb uptake and
toxicity in saltwater invertebrates. The relationships between tissue concentration of Pb and inorganic
cations (Na+, Mg2+, K+ and Ca2+) were assessed in the Hong Kong oyster (Crassostrea hongkongensis) at
four different salinities at a nominal concentration of 3 |ig Pb/L under laboratory conditions (Yin and
Wang. 2017). All four cations were negatively correlated with trace metal uptake by oysters; the tissue
concentration of Pb was lower at higher salinities during the 6-week exposure (due to decreasing free-ion
concentration of Pb at higher salinity). For the rotifer Proales similis, exposed nominally to Pb (13, 25,
50, 100 |ig Pb/L) in 5-day chronic reproductive toxicity tests conducted at four salinity conditions (5, 15,
25 and 35 ppt), population density was highest at the lowest salinity, and toxicity increased with
increasing Pb concentration (Rebolledo et al.. 2021). As salinity increased, population density decreased
in all treatments and the control; however, across all salinities, the population growth rate was lowest at
100 |ig Pb/L (the highest tested concentration). In contrast, embryo development assays in larval mussels
(bay and Mediterranean) and pacific purple sea urchins conducted at two salinities (33 ppt and 21 ppt)
reported no effect of salinity on Pb toxicity (Nadella et al.. 2013).
Recent studies in saltwater fish have examined the modifying effect of salinity. In chronic
exposure with larval topsmelt fish (A. affinis), Pb was consistently more toxic at lower salinity (14 ppt)
than at higher values (28 ppt) (Reynolds et al.. 2018). Free Pb2+ ion concentrations, the most bioavailable
form of Pb, were higher in the lower-salinity water, determined based on Pb speciation calculations in the
study. Lower-salinity water contains fewer cations, leading to decreased competition of free ionic Pb with
binding sites. Differential responses to salinity have also been reported in other studies in fish including
juvenile yellowfin seabream (Acanthopagrus latus); the LC50 was significantly higher in fish acclimated
to 17 ppt salinity compared with fish acclimated to 0 ppt, 9 ppt, 25 ppt and 34 ppt salinity (Tsui et al..
2016).
11.4.2.4. Association with Sediments
Habitat type is a factor in the bioaccumulation of trace metals, as invertebrates closely associated
with benthic environments have greater contact with porewater and sediments, where metal
concentrations are higher than those in seawater. Several new studies published since the 2013 Pb ISA
reported differences in the biouptake of Pb associated with sediment characteristics. Belzunce-Segarra et
al. (2015) compared bioaccumulation in the benthic bivalve Tellina deltoidalis with two sediment types
(silty, sandy) in the lab and deployed in the field. During the 31-day exposure period, Pb bioaccumulation
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from sediments generally increased in a linear fashion with increasing sediment Pb concentration and was
greater in sandy sediments. For the silty sediments, there was more bioaccumulation in field-deployed
bivalves compared with bivalves in a parallel laboratory exposure, whereas the opposite was observed
with sandy sediments. Bioaccumulation in bivalves was attributed primarily to dietary exposure via
ingestion of particles due to the poor relationship between dissolved Pb in overlying waters (1 to
2.2 |ig Pb/L) and bioaccumulation. The authors noted that under laboratory exposure conditions, the
absence of processes occurring in the natural environment such as sediment resuspension, dilution of
surface sediments by deposition, and avoidance behaviors by organisms, likely lead to overestimation of
bioavailability. Battuello et al. (2018) quantified trace metals in two predaceous marine invertebrates
native to coastal waters of Italy: Eurydice spinigera (Isopoda), which burrows in sediments during the day
and rises to feed in the pelagic zone at night, and Flaccisagitta enflata (Chaetognatha), a zooplanktonic
species. Although the invertebrates have a similar feeding behavior and occupy the highest invertebrate
trophic level, Pb was an order of magnitude higher in E spinigera (3.1 mg Pb/kg wet weight) compared
with F. enflata.
Fan et al. (2014) observed that the accumulation of Pb in polychaetes (marine annelid worms)
was significantly related to the total metal concentrations in sediment; however, metal concentrations in
polychaetes were less strongly correlated with metal concentrations in sediments if normalized for OC
concentration. The correlation improved when the metal concentrations in sediments were normalized for
Mn content, whereas normalization for Fe did not affect the correlation between Pb in sediment and Pb
accumulation in polychaetes. This suggested that Mn content in the sediment may be the driving factor
affecting bioaccumulation, while OM content in the sediment played little role in controlling the
bioaccumulation of Pb in polychaetes. Additionally, Pb accumulation in polychaetes was highly
positively correlated with its concentrations in FeMn oxides and organic fractions, and Pb
bioaccumulation in polychaetes was not related to its partitioning in different geochemical fractions.
11.4.2.5. Seasonality
Seasonal differences in Pb uptake and concentration in bivalves were noted in several European
field monitoring studies included in the 2013 Pb ISA (Carvalho et al.. 2011; Couture et al.. 2010; Pearce
and Mann. 2006). These differences could be due to seasonal changes in anthropogenic inputs or to
altered organism physiological condition in warmer versus colder months. Newer studies also reported
seasonal fluctuations in Pb uptake in saltwater invertebrates. Seasonal and spatial variation of trace metal
accumulation was observed in M. galloprovincialis mussels collected from sites around Port Phillip Bay,
Australia in the summer and winter (Shen et al.. 2020). In mussels collected from locations identified as
high risk for contamination, Pb body burden was higher in summer than in winter. In mussels collected
from less affected sites, there was no significant difference in Pb burden with season. This suggests that
the increase in trace metals detected in mussels at more affected sites was due to greater anthropogenic
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influence in summer. Metal bioaccumulation in red cherry shrimp (Neocaridina denticulata, now N.
davidi) sampled from a brackish wetland in Taiwan showed a seasonal variation in body residues, with
the highest accumulation of Pb in winter (Tang et al.. 2020). The saltwater shrimp could accumulate more
metal when wetlands shifted to a more heterotrophic system, as observed by the negative correlation
between net ecosystem production and Pb accumulation in shrimp. The highest ratios of Pb in shrimp to
waterborne Pb levels were found in winter (February), during the wetland's highest season of
heterotrophy. Hernandez-Almaraz et al. (2016) measured heavy-metal content including Pb of white sea
urchins (Tripneustes depressus) and slate pencil sea urchins (Eucidaris thouarsii) collected in the
southwestern Gulf of California, Baja Sur California, Mexico in summer and winter and reported that Pb
concentrations were higher in E. thouarsii in the summer compared with the winter, likely due to diet.
11.4.2.6. Diet Composition
Few studies in saltwater biota have examined the role of diet composition on Pb uptake and
toxicity. Several studies in the 2013 Pb ISA reported tissue distribution patterns of Pb or assessed toxicity
to biota following dietary exposure (U.S. EPA. 2013). A study published since the 2013 Pb ISA
comparing the gut contents and Pb concentration of field-collected white sea urchins (T. depressus) and
slate pencil urchins (E. thouarsii) suggested different diets may influence Pb concentrations in these
organisms (Hernandez-Almaraz et al.. 2016). Specifically, Pb concentrations in the gonads of T.
depressus were below the detectable limit at all sites (<0.07 mg Pb/kg dry weight), while Pb
concentrations in the gonads of E. thouarsii ranged from 12.8 ±1.7 mg Pb/kg dry weight (mean ± SE) to
38.6 ± 4.2 mg Pb/kg dry weight. The diet for T. depressus varied with season and site and included both
brown and red macroalgae (mainly Sargassum, Gracilaria and Laurencia). The main food source for E.
thouarsii was red macroalgae, although they are considered a generalist omnivore that also fed on some
invertebrates, which was confirmed by higher 615N than T. depressus. Given Pb was only detected in E.
thouarsii, the authors suggested that these urchins might be exposed to Pb via macroalgae, specifically,
crustose macroalgae (Lithophyllum) or articulated coralline macroalgae (Amphiroaj, as well as
invertebrates including mollusks, and/or barnacles.
In another dietary study Guo et al. (2013) examined whether the burned nassa sea snail
(Nassarius siquijorensis) showed differences in bioaccumulation patterns after being fed either Japanese
carpet shell clams (Ruditapes philippinarum), Asian green mussels (Perna viridis), Fistulobalanus
albicostatus (barnacles) or Portuguese oysters (Crassostrea angulata) for 8 weeks. The prey items were
collected from an intertidal zone in Xiamen, southeastern China. N. siquijorensis were sampled every
2 weeks and muscle and viscera metal concentrations, including Pb, were determined. In addition to the
body burden of metals in the snails, metal concentrations were also determined for the subcellular
fractions of the snails (heat-sensitive protein fraction, metallothionein-like protein fraction, MRG, cellular
debris and organelles). Pb concentrations differed between the four prey items (P. viridis:
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0.66 ±0.19 mg Pb/kg dry weight, mean + S.D., n = 8;R. philippinarum: 1.1 ±0.3 mg Pb/kg dry weight;
C. angulata: 2.4 ± 0.3 mg Pb/kg dry weight; F. albicostatus: 5.9 ±1.1 mg Pb/kg dry weight). Subcellular
metal distribution in N. siquijorensis viscera and muscle at the beginning of dietary exposure was
concentrated in the cellular debris (44.3%). After exposure to four prey items over 8 weeks, the dominant
pool for Pb in the muscle was the cellular debris, while MRG became the dominant storage pool for
viscera across most prey items. Throughout feeding, MRG became a more important storage pool for Pb
relative to cellular debris. Pb was largely accumulated in the cellular debris and MRG for all prey items.
11.4.2.7. Lifestage
Additional studies on Pb effects in saltwater biota published since the 2013 Pb ISA provide
further evidence for variance in response to Pb at different lifestages. Embryo and juvenile lifestages are
commonly tested in bioassays due to their increased sensitivity to pollutant exposure. Many studies in
saltwater invertebrates discussed in the following sections continue to support findings in prior AQCDs
and the 2013 Pb ISA of differential toxicity with organism lifestage and increased sensitivity of larval or
other early lifestages compared with adults. In saltwater vertebrates, chronic toxicity bioassays with
topsmelt (A. afftnis) at two lifestages (larvae and 2.5-month-old juveniles) lend further support to greater
sensitivity of earlier lifestages to Pb in saltwater fish (Reynolds et al.. 2018).
11.4.2.8. Historical Exposure
In the 2013 Pb ISA, the few studies that reported the development of tolerance to prolonged Pb
exposure were limited to freshwater invertebrates and fish: information was lacking for saltwater. A
recent study with the mangrove crab (Ucides cordatus) collected from two locations in Brazil suggests
that a crab population inhabiting an historically polluted area may have developed mechanisms to cope
with elevated metals, resulting in differences in Pb accumulation compared with individuals from a
relatively pristine mangrove (Duarte et al.. 2020). After 28 days of laboratory exposure to low
concentration of Pb (10.6 |ig Pb/L), crabs collected from the protected site accumulated statistically
significantly more Pb in four of the six quantified tissues (gills, carapace, gonads, and muscle) and almost
double the total concentration of Pb compared with the crabs from the historically contaminated location.
The population from the protected site also took up more Pb in the biologically active form and exhibited
greater genotoxic effects (assessed by frequency of micronucleated cells and DNA strand breaks).
Furthermore, metallothionein induction in crabs from the historically contaminated location was more
than twice as high as that from the clean site.
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11.4.2.9. Species Sensitivity
As is the case for terrestrial and freshwater organisms, there are considerable differences in
response to Pb among saltwater biota. This information serves as the basis for the species sensitivity
distributions (Section 11.4.5) for saltwater invertebrates and fish reported by (Church ct al.. 2017). Both
inter and intraspecific differences in Pb uptake and bioaccumulation may occur in macroinvertebrates of
the same functional feeding group (U.S. EPA. 2013). For example, in the 2013 Pb ISA, data from
20 years of monitoring of contaminant levels in filter-feeding mussels of the Mytilus genus and eastern
oysters (C. virginica) sampled along the U.S. coast, as part of the NOAA Mussel Watch program, indicate
that Pb is on average three times higher in mussels than in oysters (Kimbrough et al.. 2008). Wang et al.
(2014b) compared acute toxicity data (hazard toxicity ratios based on LC50 values; EC50 values for algal
responses) for temperate and tropical saltwater species sensitivity distributions across five broad
taxonomic groups (algae, crustaceans, fish, mollusks, worms). Based on the hazardous concentration for
10% of the species (HC10) ratios, temperate saltwater species are more sensitive to Pb than tropical
saltwater biota. In the meta-analysis, algae were the most sensitive taxa to Pb (HC10 = 29 |ig Pb/L, [95%
CI 9.5, 86], n = 8) followed by fish (HC10 = 166 (ig Pb/L [95% CI 49], n = 10), crustaceans
(HCio = 428 ng Pb/L [95% CI, 263, 696], n = 22), mollusks (HC10 = 1230 ^g Pb/L [95% CI 412, 3,660],
n = 7), and worms (HC10 = 2,430 ^g Pb/L [95% CI 1,200, 4,610], n = 9).
11.4.2.10. Uptake and Bioaccumulation in Saltwater Plants and Algae
In the 1977 Pb AQCD, the cordgrass Spartina alterniflora was found to reduce the quantity of Pb
in sediments by a small amount (U.S. EPA. 1977). Limited data on marine algae and saltwater plants
reviewed in the 1986 Pb AQCD, 2006 Pb AQCD, the 2013 Pb ISA and this appendix provide evidence
for species differences in Pb uptake and bioaccumulation rates.
One study examined element concentrations in pelagic Sargassum that washed up along the coast
of the Yucatan peninsula in Mexico from the Caribbean (Rodriguez et al.. 2020). Of 63 different samples
collected across eight sites from August 2018 to June 2019, only five samples had Pb levels at 2-
3 mg Pb/kg dry weight (as measured by X-ray fluorescence, which has a detection limit of 2 ppm). Other
metals such as As were detected in much higher amounts. Though Pb is not present in high amounts in
Sargassum, the study showed that pelagic seaweed may be an avenue of transport across large distances
and contribute to Pb levels in coastal environments where it washes ashore.
An additional area of new research is the uptake of Pb by mangroves and the mechanisms that
may limit or confer tolerance. Mangrove swamps are coastal wetlands found in tropical and subtropical
regions. They are characterized by halophytic woody plants growing in brackish to saline tidal waters.
One greenhouse experiment aimed to investigate the possible function of root lignification/suberization
on Pb uptake and tolerance in two pacific mangrove species with different degrees of root lignification
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and suberization: holly mangrove (Acanthus ilicifolius) and red mangrove (Rhizophora stylosa) (Cheng et
al.. 2015). Plants were grown in pots with three nominal Pb treatments applied to the sediment—low
(250 mg Pb/kg), medium (500 mg Pb/kg) and high (1000 mg Pb/kg)—and one control with no Pb; Pb
exposure was a period of 3 months. In the species with little lignification and suberization, A. ilicifolius,
biomass yield decreased significantly as plants were exposed to increasing concentrations of Pb; about 20,
35 and 50% reductions were observed in low, medium, and high Pb treatments when compared with the
respective controls. R. stylosa, however, was not affected by low and medium Pb exposure. A significant
decrease in relative Pb was observed within the outer cortex cell layers, indicating that lignified/suberized
exodermis acts as a barrier to the movement of Pb. A further study with six pacific mangrove species
subjected to different levels of a metal mixture (Pb with Zn and Cu) corroborates these findings and
suggests that mangrove species, which possess more extensive lignification and suberization within their
root exodermis, exhibit higher tolerance for heavy metals (Cheng et al.. 2014).
The EPA Framework for Metals Risk Assessment states that the latest scientific data on
bioaccumulation do not currently support the use of BCFs and BAFs when applied as generic threshold
criteria for the hazard potential of metals (U.S. EPA. 2007); however, such metrics are useful to provide
information about the amount of uptake of metals into plants, compartmentalization into different plant
tissues, and differences between species. In a field study conducted in four marine and four inland
wetlands in Sicily with differing levels of anthropogenic impacts, Pb concentrations were quantified in
soil, water, and plant tissues of two Mediterranean seagrasses, Posidonia Oceania and Cymodocea
nodosa, and five freshwater species were quantified (Bonanno et al.. 2017). Sediment Pb levels ranged
from 2.56 ± 0.33 mg Pb/kg at the lowest impacted site to 11.5 ± 1.57 mg Pb/kg at the most impacted site
for the marine sites and 1.05 ± 0.21 to 17.2 ± 4.58 mg Pb/kg for the freshwater sites. BCFs (C root/ Csediment)
were higher for the two marine seagrasses than those for any of the freshwater species, more than twice as
high as the values for the highest freshwater species (0.71 and 0.84 for P. Oceania and C. nodosa,
respectively, compared with 0.03-0.30 for the freshwater species). For both marine species, Pb was
concentrated in root tissue, but translocation factors into different tissues differed between species. An
additional study Bonanno et al. (2020) examining the seagrass C. nodosa and marine green algae Ulva
lactuca showed that both species are comparable in their ability to sequester high levels of trace elements
including Pb.
11.4.2.11. Uptake and Bioaccumulation in Saltwater Invertebrates
At the time of the 1977 AQCD, it was understood that shellfish concentrate Pb in their tissues and
shells (U.S. EPA. 1977). Uptake and subsequent bioaccumulation of Pb in marine invertebrates varies
greatly between species and across taxa as previously characterized in the 2006 AQCD (U.S. EPA.
2006a) and the 2013 Pb ISA (U.S. EPA. 2013). In the case of invertebrates, Pb can be bioaccumulated
from multiple sources, including the water column, sediment, porewater and dietary exposures, and
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factors such as the proportion of bioavailable Pb, lifestage, age and metabolism can alter the accumulation
rate. Since the 2013 Pb ISA additional information on uptake rates, Pb sequestration patterns and Pb
accumulation from aqueous and dietary exposures has been published for saltwater invertebrates.
As reported in studies in previous reviews, major sites for Pb accumulation following aqueous
exposure include the gill and digestive gland or hepatopancreas; current studies continue to support these
findings. In pacific oyster (C. gigas) exposed nominally to 5 |ig Pb/L for 9 days, Pb concentration in the
gill and digestive glands were 19- and 24-fold higher, respectively, than Pb measured at the beginning of
the experiment (Mens et al.. 2018). Following 28-day exposure to a low concentration of Pb
(10.6 |ig Pb/L), the highest concentration of Pb was accumulated in the gill, followed by the carapace in
the mangrove crab (U. cordatus) (Duartc et al.. 2020). The crabs sequestered Pb in detoxified forms, with
differences in Pb accumulation and storage observed in two distinct populations (crabs collected from a
protected mangrove area and those collected from a historically contaminated site).
Adult female Atlantic Horseshoe crabs (Limulus polyphemus) collected from several different
beaches in Long Island, NY, had higher Pb concentration in gills than legs or eggs; Pb in leg tissue was
significantly and positively correlated with egg Pb burden, suggesting maternal transfer of the internalized
metal to eggs (Bakker et al.. 2017b). Pb quantified in field-collected horseshoe crab embryos (range 0.05-
0.43 mg Pb/kg dry weight) and developing larvae (range 0.07-0.59 mg Pb/kg dry weight) was compared
with Pb concentration in eggs, sediment, porewater and overlaying water (Bakker et al.. 2017a). Although
Pb measured in environmental media varied between sites, the concentration of Pb significantly increased
from egg to embryo at four out of five sampling locations, indicating uptake of Pb from the surrounding
substrate following hatching since the embryonic lifestage develops in the sediments. There was no
significant change in Pb concentration when comparing embryos to larvae; however, the authors noted
that it is possible some trace metals are lost at the larval stage during molting.
Embryos of the sea urchin S. purpuratus exposed to an analytically verified Pb concentration of
55 fxg Pb/L during 96-hour embryo toxicity assays showed significant Pb accumulation after 12 hours
through 96 hours of development, with a peak at 84 hours (Tellis et al.. 2014). Pb disrupted Ca uptake
during initial development stages, especially during gastrulation, and there was a corresponding increase
in Ca2+ATPase activity in the embryos; however, Ca levels in Pb-exposed embryos returned to control
amounts by 72 hours.
A few dietary exposure studies in marine invertebrates have been conducted since the 2013 Pb
ISA. In sea hare (Aplysia californica) exposed to Pb solely through diet (green seaweed U. lactuca
previously exposed to an analytically verified concentration of either 10 jxg Pb/L or 100 |ig Pb/L for
48 hours), the Pb accumulation pattern in the mollusk was greatest in the hepatopancreas followed by the
gill and crop(Jarvis et al.. 2015). In sea cucumbers (Apostichopus japonicus) fed a Pb-supplemented diet
(100, 500 or 1000 mg Pb/kg dry weight) for 30 days, the profile of tissue Pb accumulation was body
wall>intestine>respiratory tree (Wang et al.. 2015a). The bioavailability of Pb from food and subsequent
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trophic transfer is affected by how Pb is stored in the prey. In a feeding study, the common prawn
Palaemon serratus was fed for 28 days with either tissues from the mussel (M. galloprovincialis) exposed
to 100 |ig Pb/L for 48 hours or tissues from the field-collected clam Dosinia exolete, wherein Pb is stored
primarily in nonbioavailable MRG in the kidney (Sanchez-Matin and Beiras. 2017). Although the Pb
concentration in both food items was similar (15 and 17 mg Pb/kg wet weight, respectively), Pb
accumulation in prawns was 10x higher when fed tissue from the mussels, in which Pb was in a more
soluble subcellular faction, compared with the prawns consuming D. exolete, in which Pb was in a less
bioavailable form.
Pb uptake is influenced by feeding strategy. In the filter-feeding bivalve Andara trapezia, uptake,
and bioaccumulation from Pb-spiked sediments (analytically verified concentration of 100 and
300 mg Pb/kg) to the gill and mantle, hemolymph and hepatopancreas were quantified on days 0, 14, 28,
42 and 56 of a 56-day exposure (Taylor and Maher. 2012). At the end of the experiment, total Pb
concentration in the mollusk was 1 mg Pb/kg at the low concentration and 12 mg Pb/kg at the high
concentration. In the highest Pb treatment, an increase in Pb in hemolymph was observed from day 42 to
day 56, resulting in a doubling of Pb tissue concentration. The authors speculated this could be related to
greater availability of dissolved Pb in porewater over time due to oxidation of the sediments. Generally,
the order of tissue accumulation was hemolymph>gill and mantle>hepatopancreas over the 56-day
exposure. In contrast, the deposit-feeding bivalve Tellina deltoidalis exhibited a distinct pattern of Pb
uptake under similar experimental conditions and exposure to spiked sediments (28-day exposure to
analytically verified concentrations of 100 and 300 mg Pb/kg) (Taylor and Maher. 2014). Individuals in
the 100 mg/kb Pb-spiked sediment rapidly accumulated Pb early in the exposure period (day 3) followed
by continued uptake over the remainder of the experiment, to reach a final tissue concentration
(96 mg Pb/kg) equal to that of the spiked sediment. In the 300 mg Pb/kg microcosm, the bivalves seemed
to exhibit a pattern of uptake and loss over the 28-day period, with the highest Pb concentration at day 21
and a final total Pb concentration of 430 mg Pb/kg.
Aquatic invertebrate strategies for detoxifying Pb reviewed in the 2006 Pb AQCD and 2013 Pb
ISA included sequestration of Pb in lysosomal-vacuolar systems, excretion of Pb by some organisms and
deposition of Pb to molted exoskeleton. Pb can be stored in two forms: biologically detoxified metal
(which includes MRG) and biologically available metal. Following the biouptake experiments described
above, subcellular partitioning of Pb was determined in the bivalves (Taylor and Maher. 2014. 2012). Of
the recovered Pb in A. trapezia tissues, Pb was associated to the greatest extent with the biologically
detoxified metal fraction (ranging from 66% to 69% in the gill and mantle to 56% in the hepatopancreas),
distributed fairly evenly between the metallothionein-like proteins and MRG fractions (Taylor and Maher.
2012). In T. deltoidalis, Pb was also primarily found in the biologically detoxified metal fraction
(approximately 70%), with 74% of the total detoxified Pb converted to MRG and the remainder in the
metallothionein-like protein fraction (Taylor and Maher. 2014).
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In a 96-hour exposure to analytically verified concentrations of Pb (0-1,800 |ig Pb/L),
intracellular partitioning data in adult clams Venerupis decussata showed that most Pb accumulated in the
insoluble fraction (>80%), a form not readily bioavailable to consumers at higher trophic levels (Frcitas et
al.. 2014). Total Pb in clams increased with increasing water concentration up to 230 |ig Pb/L then
decreased at higher concentrations. The clams bioconcentrated Pb in the soluble fraction more efficiently
at low water concentrations (BCF > 26) compared with higher concentrations (>450 |ig Pb/L; BCF<16).
Similar results were observed with the clam Venerupis corrugata following 96-hour nominal exposure to
Pb (100 to 800 |ig Pb/L). Most of the metal was found in the insoluble fraction and associated with MRG
(42-72%) (Frcitas ctal.. 2014).
11.4.2.12. Uptake and Bioaccumulation in Saltwater Vertebrates
Studies reviewed in prior AQCDs and ISAs report Pb accumulation in tissues sampled from
seabirds, saltwater fish, and marine mammals (U.S. EPA. 2013. 2006a. 1977); however, there are fewer
biouptake studies of Pb in saltwater than in freshwater. Because marine fish drink seawater to maintain
osmotic homeostasis, Pb can be taken up from the water column via both the gills and intestine (Lee et al..
2019; Wang and Rainbow. 2008). In the 2013 Pb ISA, storage of Pb in metal granules was reported as a
detoxifying mechanism in mummichogs (Fundulus heteroclitus). Fish at more polluted sites stored a
higher amount of Pb in MRG as compared with other detoxifying cellular components such as heat-stable
proteins, heat-denaturable proteins and organelles (Goto and Wallace. 2010). Since the 2013 Pb ISA,
additional studies have further elucidated the role of subcellular fractions in metal detoxification in
saltwater fish. Metal binding to subcellular fractions in the livers of wild-caught yelloweye rockfish
(.Sebastes ruberrimus) collected from the southeast coast of Alaska was assessed to gain a better
understanding of the degree to which this long-lived endangered fish species can detoxify nonessential
metals including Pb (Barst et al.. 2018). Combining data from the rockfish, Pb was detected to a greater
extent in the detoxified compartment (46%); however, detoxification was incomplete given that Pb was
also present in metal-sensitive fractions (a total of 35%, divided between heat denatured proteins [12.2%],
mitochondria [11.4%], microsomes and lysosomes [10.8%]). Metals associated with sensitive subcellular
fractions indicate a risk of disruption to cellular processes; however, the concentrations of Pb in rockfish
were low compared with other detected metals. These patterns were consistent with results from
subcellular partitioning in livers of yellow eels native to North America (Anguilla rostrata) and Europe
(Anguilla anguilla) (Rosabal et al.. 2015). In both eel species, the granule-like detoxification fraction
showed the strongest increase in Pb concentrations among all subcellular fractions, with the metal-
sensitive mitochondrial fraction representing a significant binding compartment for Pb.
A novel study exploring the use of fish eyes as an organ for monitoring Pb exposure compared Pb
concentration in mullet (Liza aurata) eyes, water column and sediment in a metal-contaminated location
and reference area within the same estuary (Pereiraetal.. 2013). Eyes from individuals collected from the
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contaminated site (0.81 |ig Pb/L water column, 417 mg Pb/kg sediment) had significantly higher Pb
accumulation (10x) than the less affected site (0.032 |ig Pb/L water column, 61 mg Pb/kg sediment),
suggesting the eye is a target organ for Pb. It is not known if the accumulation of metals in the eye is from
direct contact with water or redistribution of Pb taken up by the fish via other routes of exposure.
Studies that considered uptake of Pb in saltwater birds and mammals are limited to surveys of
field-collected individuals that reported Pb concentration in tissue or trace-element patterns of tissue
distribution.
11.4.2.13. Uptake and Bioaccumulation Through Marine Food Web
Trophic transfer of Pb in marine food webs was found to be negligible in the 2006 Pb AQCD
(U.S. EPA. 2006a') and the 2013 Pb ISA (U.S. EPA. 2013). In many studies reported in previous
assessments and those reviewed here, Pb was found to decrease with increasing trophic levels, although
some studies found evidence of bioaccumulation. Whether Pb is biodiluted or bioaccumulated in marine
food webs depends on the sediment and porewater Pb, the type of marine ecosystem, the organisms
examined, and other contaminants. In a review published in 2013, Cardwell et al. (2013) compiled
laboratory and field studies to examine the transfer of Pb and other heavy metals through marine food
webs. In most of the field studies reviewed, no evidence was found for biomagnification of Pb across
trophic levels. Specifically, nine studies examined trophic transfer of heavy metals through marine food
webs in the field. Eight of these studies found no evidence of biomagnification of Pb, and one did not
examine Pb or did not present data on Pb. More recent studies are presented below.
Biodilution of Pb in marine food webs was supported by an environmental gradient study on a
green sea turtle food web in San Diego Bay, California, U.S. (Komoroske et al.. 2012). Green sea turtles
(Chelonia mydas) largely forage on eelgrass (Zostera marina) and invertebrates, and exposure to heavy
metals occurs primarily through foraging, as these organisms breathe air and do not feed during
migration. At each of eight eelgrass sites, sediment samples, eelgrass, red algae (Gracilaria spp.), green
algae (Ulva spp.), soft-bodied invertebrates (i.e., Zoobotryon spp.), sponges, and green sea turtle carapace
tissues were collected and analyzed for trace metals. Mean Pb concentrations in sediments and organisms
varied across season and site in San Diego Bay. Pb did not bioaccumulate in eelgrass or algae: Pb in the
sediment was significantly higher than Pb in eelgrass and red algae, but not higher in green algae.
Biodilution of Pb was also reported across six intertidal sites in New England (four in the Gulf of
Maine and two in Narragansett Bay, Rhode Island) spanning a gradient of watershed land use and
urbanization (Chen et al.. 2016a). Sediments, invertebrates, and benthic and pelagic fish were sampled
and analyzed for heavy metals. Trophic position was characterized using stable-isotope analysis on biotic
tissues. Specifically, S13C is correlated with the relative proportion of pelagic diet sources, while S15N is
related to trophic position. Invertebrate and fish samples were categorized into five taxonomic groups, as
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the same species were not collected at all sites. Biota-sediment accumulation factors (BSAFs) were
calculated for each taxonomic group (amphipod, crab, Fundulus, mussel, and shrimp) as the metal
concentration in the organisms divided by the metal concentration in the sediment. Positive logio BSAF
values indicate bioaccumulation, while negative values indicate biodilution. Pb concentrations in the
sediment across six sites in the Gulf of Maine and Narragansett Bay ranged from 4.7 mg Pb/kg to
79.6 mg Pb/kg, and these concentrations increased linearly with the percent of total OC. All logio BSAF
values were negative for Pb, indicating that organisms in higher trophic levels contained less Pb than
organisms occupying lower trophic levels. Pb concentration across five taxonomic groups (mussel,
shrimp, crab, Fundulus, and amphipod) showed considerable variation across taxa and sites.
Simultaneously extracted metal-AVS in the sediment were marginally predictive of biota Pb content,
while trophic level and pelagic feeding were not predictive of biota Pb.
Trophic level positions of a marine invertebrate community and body Pb concentrations of a
marine invertebrate community were not correlated in the Bay of Fundy, Nova Scotia, Canada suggesting
Pb does not bioaccumulate in this system (English et al.. 2015). The invertebrate community included
barnacles (Balanus balanus), worms (Cerebratulus lacteus, Clymenella torquata, Glycera dibranchiate,
Hediste diversicolor), amphipods (Corophium volutator, Gammarus oceanicus), clams (Ensis directus,
Mya arenaria, Macoma balthica), snails (Ilyanassa obsoleta, Littorina littorea), mussels (Mytilus edulis),
and crabs (Pagurus pubescens). Stable isotopes were used to characterize the relative trophic position as
organisms in higher trophic levels contain higher levels of S15N, while S13C is often associated with lower
trophic levels. Although the species sampled likely did not belong to the same food web, they occupy
similar trophic levels in different food webs and are all important prey items for species in higher trophic
levels. In this study, S15N was negatively correlated with S13C for most species. Pearson correlation
coefficients were calculated between stable-isotope and trace-element content for each species to test for
bioaccumulation or biodilution through the food web. Pb concentration varied among invertebrate species
in the community; however, no single species had higher Pb concentrations than the others. Pb ranged
from 0.07 ± 0.01 mg Pb/kg (mean ± S.D.) in Glycera dibranchiate (Polychaeta) to 1.25 ± 1.40 mg Pb/kg
in I. obsoleta (Gastropoda). There were no significant correlations between trophic level position (S15N or
S13C) and logio Pb concentration, suggesting Pb does not show considerable bioaccumulation in the food
web.
In another example, trophic level position determined using stable isotopes of white sea urchins
(T. depressus), slate pencil sea urchins (E. thouarsii), and nine types of macroalgae food sources in four
Sargassum beds in the southwestern Gulf of California in Baja California Sur, Mexico were not correlated
(Hernandez-Almaraz et al.. 2016). Out of the macroalgae and two sea urchins studied, E. thouarsii had
the highest Pb concentrations (ranging from 12.8 ± 1.7 mg Pb/kg dry weight [mean ± SE] to
38.6 ± 4.2 mg Pb/kg dry weight) and stable-isotope content.
Pb accumulation in a tropical estuarine lagoon in Mexico decreased with increasing trophic level
(Mendoza-Carranza et al.. 2016). Sediment Pb concentration was 20.86 ± 5.80 mg Pb/kg (mean ± S.D.),
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and the suspended load Pb concentration was 16.59 ± 2.79 mg Pb/kg in the San Pedrito Lagoon, which is
impacted by wastewater discharge and petroleum extraction. Pb concentration was only above the limit of
detection in two plant species, spider lily (Hymenocallis littoralis) and mangrove fern (Acrostichum
aureum), and fish samples (2.9 mg Pb/kg) from the lagoon. In general, BCFs of Pb were low, and BCFs
were higher in plants than in fish, suggesting trophic dilution.
Bioaccumulation, but not biomagnification, was found in a semiarid coastal lagoon in Sonora,
Mexico along the Gulf of California (Jara-Marini et al.. 2020). The community consists of primary
producers (phytoplankton, algae, mangrove), primary consumers (zooplankton, barnacles, oysters, clams,
snails, shrimp, crab, snapper, and juveniles of flathead mullet), secondary consumers (adults of flathead
mullets, crab, snapper, mojarra, and grunt), and tertiary consumers (night herons, great blue herons,
magnificent frigate, and cormorants). BMF was corrected for the trophic position, and the trophic BMF
(TBMF) was estimated from the antilogarithm of the slopes of the linear correlation between the trophic
level and the metal concentration. BMF and TBMF values above 1.0 indicate that a metal is being
transferred upward through the trophic levels, while values below 1.0 indicate biodilution along the food
web. Pb values in suspended particulate matter and sediment varied between seasons, ranging from
0.70 mg Pb/kg in autumn to 1.03 mg Pb/kg in winter. Pb concentrations among primary producers (range:
0.63 to 1.03 mg Pb/kg), secondary consumers (range: 0.80 to 1.53 mg Pb/kg), and most tertiary
consumers did not vary seasonally. Only two tertiary consumers, neotropic cormorant (Phalacrocorax
brasilanus) and magnificent frigatebird (Fregata magnificens), showed the highest Pb concentrations in
the summer. Pb only showed a positive relationship between log-transformed Pb concentrations and
trophic level in the summer. Pb concentrations generally decreased through the food web, depending on
the season. The BMF ranged from 0.50 to 3.57 for Pb across organisms, and TBMF ranged from 1.02 to
1.15. Although TBMF values were above 1.0, biomagnification was unlikely because the relationship
between trophic level and Pb concentration was only significantly positively correlated in the summer.
In addition to evidence from field studies, laboratory findings also suggest a decrease in the
concentration of Pb with trophic transfer. In an 8-week feeding study, trophic transfer factors were
calculated for sea snail (Nassarius siquijorensis) fed either venerid clams (Ruditapes philippinarum),
mussels (Perna viridis), barnacles (Fistulobalanus albicostatus), or oysters (Crassostrea. angulata)
collected from an intertidal zone in Xiamen, southeastern China (Guo et al.. 2013). The net trophic
transfer factor, which is the ratio of net accumulated metal concentrations over the experiment to metal
concentrations in the prey was well below 1 for barnacles and 0 for oysters, clams, and mussels,
suggesting biodilution in this system. Although not tested statistically, the variation in trophic transfer
factors across prey species demonstrated prey-specific bioavailability.
Although most observational studies suggest biodilution of Pb occurs through marine food webs,
Pb was found to bioaccumulate in mummichog (Fundulus. heteroclitus) in the Goose Pond estuary in
Brooksville, Maine (Broadlev et al.. 2013). The Goose Pond estuary was impacted by the former Callahan
Mine, which is one of the few documented open-pit hard-rock mining sites in an intertidal zone. The
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sediment concentration of Pb was above the probable effects level in some of the sample sites. BAFs
ranged from 3.3-3.65 across Goose Pond sites and 2.62-3.77 at the reference sites. The reference site
values bioaccumulation factors were conservative as they included water and tissue Pb concentrations
below the instrument detection limit. The sediment-to-F. heteroclitus and w ater-to-/'', heteroclitus ratios
were high for Pb. The ratio of metal enrichment to background levels (concentrations at the Goose Pond
site adjacent to the tailings pile / the mean concentrations at a reference site) were 34.2 for sediment, 32.3
for water, and 45.6 for /¦'. heteroclitus.
Environmental gradient field studies outside of North America provide additional evidence to
support the biodilution of Pb in marine food webs. For example, trophic transfer of Pb was low in a
seagrass food web in an estuarine lake in Australia, as the trophic level was negatively correlated with Pb
concentration (Schneider et al.. 2018). In another example, Pb concentrations decreased with increasing
trophic level in a Mediterranean coastal lagoon (Vizzini et al.. 2013) and in a small pelagic fish marine
food web along a Mediterranean coast (Chouvclon et al.. 2019). Finally, Pb accumulation was higher in
invertebrates compared with higher trophic level species (fish) in an aquatic food web in Liaodong Bay,
China (Radomvski et al.. 2018).
In summary, studies published since the 2013 Pb ISA support findings in the ISA that Pb
generally decreased with increasing trophic level in coastal and marine food webs, although some studies
found evidence of bioaccumulation.
11.4.3. Environmental Concentrations of Pb in Saltwater Biota in the United
States at Different Locations and Over Time
Studies of aquatic bivalves in coastal ecosystems can be used to reconstruct historical records of
Pb concentrations. The NOAA Mussel Watch program has monitored pollutant trends since 1986 via
periodic sampling of bivalve tissue (Mytilus species and C. virginica oysters) and sediment along the U.S.
coastline (Kimbrough et al.. 2008). In general, the highest concentrations of Pb are in bivalves in the
vicinity of urban and industrial areas, and Pb is, on average, three times higher in coastal mussels than in
oysters. Metal concentrations in Mytilus californianus were sampled at long-term biomonitoring sites off
the coast of California from 1977 to 2010 (specific years vary by site) as part of the National Mussel
Watch (NSW) (n = 35 sites) and California State Mussel Watch (CSMW) (n = 21 sites) (Melwani et al..
2014). Decreasing trends were observed at 11 NMW sites and 8 of the CSMW sites; no significant trends
were found at the remaining sites. These observations show that Pb inputs to coastal aquatic ecosystems
from runoff have decreased significantly, especially at sites off the coast of southern California near large
municipal wastewater treatment facilities.
Quantification of chemical variation in relative presence of Pb and of other elements taken up and
deposited in shells of marine organisms (sclerochronology) provides a temporal record ofPb deposition
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inputs to coastal environments. In a 2005 study of Mercenaria shells collected off the coast of Cape
Lookout, North Carolina in 1980, 1982, 2002 and 2003, annual average Pb/Ca ratios were estimated from
1949-2002 using concentration measurements milled between the mollusk shell growth lines, which
provide corresponding chronological measurements (Gillikin et al.. 2005). Although high variability
between samples was observed, overall Pb/Ca ratios in shells peaked near 1980 and decreased until the
conclusion of the sampling in 2003. This study provides an indicator that reductions in Pb pollution
resulted in decreased Pb inputs to aquatic ecosystems through runoff on the east coast. Elemental analysis
of shell carbonate of the long-lived bivalve Arctica islandica collected off the coast of Virginia revealed a
pattern of continuous increase in Pb concentration after 1910, reaching a peak in 1979 and declining after
that date to pre-1930 values after 2000 (Krausc-Nchring et al.. 2012). The elevated shell Pb corresponded
to the period of peak leaded gasoline use in the United States, with Pb deposition to the offshore site
including atmospheric transport by easterly winds. Cariou et al. (2017) synthesized data from 15 studies
from different geographic locations that quantified Pb in marine bivalve shells. They found that shell
concentration had a strong relationship with the environmental level of local contamination; values in the
shells, which ranged from 0.08 mg Pb/kg to 2 mg Pb/kg, were associated with environments with distant
Pb sources including atmospheric deposition.
In addition to bivalve tissue and shell, heavy metals in horseshoe crab (L. polyphemus) eggs
collected from breeding grounds on beaches along Delaware Bay provide some historical data for trend
analysis. Horseshoe crab eggs collected in 1993, 1994, 1995, 1999, 2000, and 2012 showed a decline in
Pb overtime in a comparison of compiled data from the earlier surveys (1993, 1994, 1995)
(x = 0.289 ± 0.068 mg Pb/kg) and to the data from 1999 to 2000 (x = 0.0353 ± 0.00496 mg Pb/kg)
(Burger and Tsipoura. 2014). Some of the individual resampled sites showed a clear temporal decrease in
Pb from 1993 to 2012, while at other locations, the temporal Pb concentration trend was more variable.
A study of migratory shorebird species in Delaware Bay compared feather Pb concentrations
from the 1990s with samples from 2011 and 2012 (Burger et al.. 2015). The decline of shorebirds
migrating through Delaware Bay over the study period has been widely attributed to the reduced size of
horseshoe crab populations, whose eggs the migratory birds feed on. Declining populations have been
observed elsewhere in the shorebirds' ranges, and the authors investigated heavy metals as a driver of
those declines. Across the time period studied, Pb concentrations increased in red knots (Calidrus
canutus), decreased in semipalmated sandpipers (Calidrus pusilla), and did not change significantly in
sanderlings (Calidris alba) (Burger et al.. 2015). The authors noted that Pb concentrations observed in
this study were below the known adverse effect risk levels for similar species.
In a decade-long biomonitoring study of metals in the muscle tissue of dolphinfish (Coryphaena
hippurus) in the southern Gulf of California from 2006-2015, Gil-Manrique et al. (2022) found no
temporal trend in Pb concentrations. However, a negative correlation was identified between sea surface
temperature and Pb concentrations in dolphinfish during the decade-long biomonitoring study. Summary
statistics of dolphinfish sampled in Gil-Manrique et al. (2022) are included in Table 11-1.
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In long-term biomonitoring studies of saltwater ecosystems, there is some evidence of declining
Pb concentrations, particularly in studies which began sampling before the 1990s. However, other studies
document mixed results, with some observations of insignificant change or even increases in Pb
concentrations.
11.4.4. Effects of Pb in Saltwater Systems
Saltwater taxa included in this section are broadly grouped into vegetation, microbes,
invertebrates, and vertebrates. The biological effects of Pb in the 2013 Pb ISA and in this appendix are
generally presented from suborganismal responses (i.e., enzymatic activities, changes in blood
parameters) to endpoints relevant to the population level and higher (growth, reproduction, and survival)
up to effects on ecological communities and ecosystems. Exposure-response studies that report
toxicological dose descriptors (e.g., LC50, EC50, LOAEL) for effects on growth, reproduction or survival
endpoints are reported in Section 11.4.5.
11.4.4.1. Effects on Saltwater Microbes
Microbial communities in saltwater ecosystems were not reviewed in detail in the 2006 Pb
AQCD (U.S. EPA. 2006a) or the 2013 Pb ISA (U.S. EPA. 2013). More recent experimental and
observational studies reviewed here examine the relationship between Pb concentration in the sediment
and saltwater and the effects on marine microbial communities. Pb was largely negatively or not
associated with microbial community structure and abundance, although a few studies found positive
associations between sediment Pb concentrations and microbial abundance.
Pb negatively affected microbial diversity and structure in rhizosediments of sea rush (Juncus
maritimus) and the common reed (P. australis) collected from the Lima estuary, Portugal (Mucha ct al..
2013). Rhizosediments colonized with Juncus maritimus (a phytostabilizer that retains metal in the roots
and rhizomes) and P. australis (a phytoextractor, which accumulates metals in the aboveground tissue)
were analyzed for heavy-metal concentrations, physicochemical properties, and microbial abundance.
Sediments were then exposed to a nominal gradient of Pb (0 mg Pb/kg, 218 mg Pb/kg, 2180 mg Pb/kg or
10900 mg Pb/kg and incubated for 7 days using artificial seawater). Rhizosediments collected initially
contained 63 ± 2 mg Pb/ kg (mean ± S.D.) and 70 ± 11 mg Pb/ kg for P. australis and J. maritimus,
respectively. Only 0.02-1.3% of the initially added Pb remained in solution at the end of the experiment.
At the end of the experiment, bacterial total cell count (cells/g wet sediment) was higher in the
218 mg Pb/kg treatment than in the control for J. maritimus, while total cell count was unaffected by Pb
exposure for P. australis. Juncus maritimus rhizosediment exhibited lower OTU number, diversity,
evenness, and dominance in Pb-exposed sediments relative to the control. Similarly, P. australis
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rhizosediments exposed to Pb exhibited lower OTU number, diversity, and evenness than the control,
whereas dominance was unaffected by Pb exposure. Both rhizosediment microbial community structures
under 218 mg Pb/kg and 2180 mg Pb/kg were dissimilar to the controls and to one another.
Sediment Pb concentration was correlated with bacterial richness and evenness along a gradient
of metal pollution in estuaries on the southeast Australian coast (Sun et al.. 2012). The relationships
between sediment heavy-metal content (Pb, Cr, Cu, Fe, Mn, Ni, Pb, and Zn), organic contaminants
(polycyclic aromatic hydrocarbons), physicochemical variables (silt content and OM), water column
environmental parameters (temperature, pH, and salinity) and bacterial community structure were
explored using Automated Ribosomal Intergenic Spacer Analysis profiles across six sites with different
degrees of anthropogenic disturbance. High collinearity existed between silt content and Cr, Ni, Zn, and
Pb; therefore, only latitude, salinity, temperature, pH, %silt, Cu, and Zn were included in the analysis of
bacterial community structure. Silt, which was highly correlated with Pb concentration, was the main
driver of bacterial community structure, followed by temperature. Although only a subset of variables was
used in the analysis of bacterial community structure, all sediment and water column predictors were used
for the bacterial community diversity analysis. Pb and Cu sediment concentration were the most
important predictors for bacterial diversity. Out of all 28 predictors used in boosted regression tree (BRT)
models, Pb explained the highest relative proportion of variance in bacterial diversity (16.1% explained
by Pb followed by 14.5 % by Cu and 7.5% by silt), and bacterial diversity decreased with increasing Pb
and Cu sediment concentration.
The relative abundances of certain bacterial groups were negatively correlated with the Pb
sediment concentration of mangroves in southern China (Meng et al.. 2021). Sediments were collected
and analyzed for heavy metals, bacterial communities were analyzed using 16S rDNA sequencing, and
potential functional genes associated with heavy-metal transport and elimination were examined using
GeoChip analysis. Sediment Pb concentrations ranged from 0.142 ± 0.094 mg Pb/kg to
3.257 ± 0.094 mg Pb/kg (mean ± S.D.) across seven sites, and the mean Pb concentrations in surface
sediments (0-5 cm) were higher than those in deep sediments (25-30 cm). Pb was significantly correlated
with Zn sediment concentration. The abundance of the genus Fusibacter was negatively correlated to Pb,
Zn, Cu, Co, Ni, Cd, and Ag with statistical significance, while Syntrophorhabdus was positively
correlated with Pb. Among the >200 genera and functional genes involved in heavy-metal transportation,
most bacteria associated with Pb elimination and transport demonstrated lower abundances compared
with other genera, and few Pb-transporting genes were found.
Although some studies report negative correlations between Pb sediment concentration and
bacterial community structure, other studies found no such relationship. For example, Pb sediment
concentration was not correlated with bacterial community structure in the Jiaozhou Bay, China (Yaoet
al.. 2017). Sediment samples were collected from inside Jiaozhou Bay and a site outside of the bay to
achieve an environmental gradient of water quality. Sediment heavy-metal concentration (V, Ni, U, Mo,
Zn, Se, antimony [Sb], Co, Cr, Cd, Pb, As, Cu, and Hg) and the bacterial abundance and community
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structure (polymerase chain reaction denaturing gradient gel electrophoresis fingerprinting) were
quantified. The concentration of Pb varied among the three sites (mean ± S.D. site Shilaoren Beach:
19.09 ± 1.86 mg Pb/kg, site Haibohe estuaries: 38.65 ± 9.26 mg Pb/kg and site Licunhe estuaries:
72.87 ± 17.56 mg Pb/kg). The concentrations of Co, Zn, Hg, As, and Se explained more of the variation
in bacterial community composition in this system, and Pb concentration was not one of the top three
strongest predictors of bacterial diversity at any site.
In eastern Guangdong, China, marine microbial community diversity was not correlated with Pb
content; however, Pb was significantly correlated with the abundance of a few dominant taxa (Zhuang ct
al.. 2019). Pb in the sediment from the Shantou coastline ranged from 4.9 mg Pb/kg to 95.7 mg Pb/kg,
with a mean value of 37.04 mg Pb/kg. The only significant correlations between bacterial diversity and
abundance and environmental variables were total OC and Cr, and the correlation between Pb and
microbial abundance and diversity was not significant. Although bacterial diversity and abundance were
not correlated with Pb, the metal was significantly negatively correlated with the abundance of
Nitrospirae and positively correlated with candidate phylum OD1. Additionally, sediment Pb
concentration was significantly negatively correlated with a few dominant classes, including Epsilon-
proteobacteria, Nitrospira, and Sva0725. Given there was a significant positive correlation between OD1
and all metals and a negative relationship between Nitrospirae and all metals and Pb was highly correlated
with other heavy metals (As, Hg, Cu, Zn, and total OC), it is difficult to disentangle the sole effects of Pb
on marine microbial communities.
Finally, Pb concentration in the water column did not affect bacterioplankton community
composition in the Toulon Bay, France (Coclet et al.. 2019). Mn, DOC, salinity, Cu, and Cd explained the
most variation in the bacterioplankton community composition (range in variance: 1.01%—1.22%), while
Pb concentration only explained 0.51% of the variance in bacterioplankton community composition.
Rhodobacteraceae, SARI 1 (Alphaproteobacteria), Balneola (Bacteroidetes), and Synechococcus
(Cyanobacteria) were negatively correlated with either Cd, Cu, Pb or Zn, while Candidatus aquilina
(Actinobacteria) was positively correlated with Pb.
In summary, several experimental and observational studies since the 2013 Pb ISA (U.S. EPA.
2013) reported negative relationships between sediment or saltwater Pb concentration and microbial
abundance and diversity (Mcng et al.. 2021: Mucha et al.. 2013). while other studies found no relationship
(Coclet et al.. 2019: Zhuang et al.. 2019: Yao et al.. 2017).
11.4.4.2. Effects on Saltwater Plants and Algae
In the 2013 Pb ISA, evidence was inadequate to infer a causal relationship between Pb exposure
and endpoints relevant to saltwater plants and algae (growth, survival, physiological stress) (Table 11-6).
Key studies in the 2013 Pb ISA included a 72-hour EC50 for growth inhibition reported in the marine
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algae Chaetoceros sp. at 105 |ig Pb/L (Debelius et al.. 2009). A study with the microalga Tetraselmis
suecica reported a statistically significant decrease in growth rate, total dry biomass and final cell
concentration between control cultures and algae cultured in 20 |ig Pb/L (Soto-Jimcncz et al.. 201IV Few
data are available in prior Pb reviews for saltwater plant and algal species. Effects in plants, in general,
are observed at concentrations of Pb that greatly exceed concentrations of this metal typically measured in
soils, water and sediment (Table 11-1).
No new information is available on the effects of Pb in saltwater algae at levels that are within the
concentrations of interest for this ISA (Section 11.1.1). There was, however, one new endpoint of note. In
a novel assay designed to assess the effects of toxicants on algal swimming behavior, Pb was shown to
inhibit motility in four saltwater algal motile species (Feng et al.. 2016). The lowest ECio for 2-hour algal
swimming inhibition was 2.36 (j,M (488 |ig Pb/L) in Platymonas subcordiformis; effects on the three other
algal species tested were found at higher exposures. All exposures at which effects on swimming behavior
were observed support previous findings of Pb toxicity to algae at concentrations that greatly exceed
concentrations of Pb encountered in the natural environment.
There are a few studies on the effects of seepweeds (plants in the genus Suaeda found in
saltmarshes) which support previous findings of Pb toxicity at higher exposures. For instance, significant
negative effects on the growth of S. heteroptera were observed at concentrations of Pb higher than
400 mg Pb/kg (He etal.. 2016). A study of metabolic biomarkers in S. salsa revealed that Pb exposure at
20 |ig Pb/L could induce osmotic stress and disturbances in energy metabolism after long-term exposure
for 1 month, whereas no effects were seen in the short term (1 week) (Wu et al.. 2012b). Growth effects
were not seen in a congeneric species, S. fruitcosa, even at exposures of 600 (j,M (125 mg Pb/L), though
the study affirmed the plant nutrient content and activity of antioxidant enzymes were affected by metal
stress at high levels of exposure (Bankaii et al.. 2016). As in freshwater plants, Pb is concentrated in root
tissue, but sensitivity is species-specific. In general, effects in saltwater plants are observed at much
higher Pb exposures than are found in the natural environment.
11.4.4.3. Effects on Saltwater Invertebrates
No studies reporting effects of Pb in saltwater invertebrates were reviewed in the 1977 Pb AQCD
or the 1986 Pb AQCD. In the 2006 AQCD, a few effects were noted in saltwater invertebrates including
gender differences in sensitivity to Pb in copepods, increasing toxicity of Pb with decreasing salinity in
mysids and effects on embryogenesis in bivalves (U.S. EPA. 2006a'). In the 2013 Pb ISA, available
evidence was sufficient to be suggestive of a causal relationship between Pb exposure and the endpoints
of physiological stress, hematological effects, reproduction, and development in saltwater invertebrates
(U.S. EPA. 2013). For all other effects, the evidence was inadequate to assess causality (Table 11-6). New
information for saltwater invertebrates since the 2013 Pb ISA includes additional studies that report
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physiological perturbations associated with Pb exposure, including a few observations in previously
untested taxa. Only a few of the many studies identified in the literature search on suborganism-level
responses to Pb exposure in saltwater invertebrates were conducted in the low |ig Pb/L range and hence
met the criteria for inclusion in the ISA (Section 11.1.1).
11.4.4.3.1. Suborganism-Level Response
The majority of studies in saltwater invertebrates do not link the effects reported at the molecular
and cellular levels to effects at the organism level of biological organization (e.g., survival, growth,
reproduction). One study in Tiger prawn (Penaeus monodon) exposed to a range of Pb concentrations (14
to 232 |ig Pb/L) in seawater for 30 days reported an increase in lipid peroxidation starting at the
56 |ig Pb/L exposure concentration. Chronic exposure yielded NOEC = 14 |ig Pb/L and
LOEC = 29 |ig Pb/L for survival in this species (Hariharan et al.. 2012).
For the endpoint of physiological stress, many studies from the 2013 Pb ISA, especially those that
considered enzymatic responses to Pb exposure, were conducted with nominal Pb concentrations in
mollusks. In several studies published since the 2013 Pb ISA, perturbations in biomarkers of oxidative
stress were observed at nominal concentration of 10 |ig Pb/L. In an 8-day exposure study, there were
changes in the activity of CAT, SOD and GST in the gill and digestive gland of oysters (Crassostrea
madrasensis) (Shenai-Tirodkar et al.. 2017). In Manila clam Ruditapes philippinarum (Aouini et al..
2018) (exposed 7 days nominally to 10 jxg Pb/L), biomarkers assessed in this species showed a response
at 10 (ig Pb/L including inhibition of GST and a significant increase of lipid peroxidation.
New information on molecular biomarker responses to Pb in an additional taxonomic group
(marine ciliate protozoans) has become available since the 2013 Pb ISA. Intracellular reactive oxidative
species and GSH content was quantified in Euplotes crassus, a single-celled eukaryote (Kim et al.. 2014)
following 8-hour nominal exposure to Pb ranging from 25 |ig Pb/L to 250 |ig Pb/L. ROS levels were
significantly increased at the lowest concentration tested and decreased to the same levels as the control at
100 |ig Pb/L. Total GSH was significantly induced at 25 |ig Pb/L and 100 |ig Pb/L, although the increase
was greatest at the lowest concentration. Concurrent gene expression of the glutathione-related genes
(glucocorticoid receptor and GPx) was observed with Pb exposure in ciliates.
Since the 2013 Pb ISA, additional studies have explored the mechanisms of Pb-induced
physiological stress in saltwater invertebrates by linking observed responses to changes in gene
expression. Over the course of a 4-week exposure of the mussel M. edulis to 111.68 |ig Pb/L (0.54 |iM).
transcripts involved in the unfolding protein response were differentially expressed with Pb, which
correlated with the bioaccumulation of Pb in gill tissue (Povnton et al.. 2014). In addition, a sequence of
unknown function showed a statistically significant relationship with Pb concentration in gill tissue, and
the authors proposed the sequence may be identified in the future as a dose-dependent Pb-specific
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biomarker in this species. In the marine polychaete Perinereis nuntia, the expression of three different
SOD genes was significantly upregulated following 48-hour nominal exposure to Pb (50 |ig Pb/L), and
the expression patterns differed from those observed with exposure to Ni, As and combinations of these
metals, suggesting the suitability of SOD genes as a molecular biomarker for in situ monitoring of sites
contaminated with multiple metals (Won et al.. 2014). Meng et al. (2018) conducted a detailed analysis of
C. gigas (pacific oyster) gene expression and physiological response in gill and digestive gland tissue
following 9-day nominal exposure to 5 |ig Pb/L. In both tissues, tumor necrosis factor alpha, a marker of
immune response, was significantly inhibited under Pb exposure. The mechanism of Pb toxicity in the gill
was altered Ca2+ homeostasis in the endoplasmic reticulum, which led to induced expression of stress
chaperones. In digestive glands, a significant increase in ROS, lipid peroxidation products and MDA
content compared with control suggested the primary mechanism of Pb toxicity was oxidative stress.
Physiological stress responses associated with Pb exposure were also observed in sediment
bioassays with saltwater invertebrates. Biomarkers of oxidative stress, cellular damage and genotoxicity
were measured in the benthic bivalve A. trapezia following 56-day exposure to Pb-spiked sediments
(analytically verified concentration of 100 and 300 mg Pb/kg) (Taylor and Maher. 2012). Pb
concentration in bivalves (1 mg Pb/kg for the low concentration and 12 mg Pb/kg for the high
concentration) suggested low bioavailability from sediment, especially at the lower concentration. The
total antioxidant capacity was statistically significantly reduced in both Pb treatments compared with the
control. Lysosomal stability in hepatopancreas of Pb-exposed bivalves was significantly decreased,
suggesting effects on cellular membrane integrity and function. In gill tissue, which is an important site
for Pb uptake in bivalves, there was a statistically significant increase in both treatment groups in
micronuclei frequency, a biomarker of genotoxicity. A similar suite of biomarkers was assessed in the
deposit-feeding bivalve T. deltoidalis, also exposed to Pb-spiked sediments (100 and 300 mg Pb/Kg)
(Taylor and Maher. 2014). In contrast to the filter-feeding A. trapezia, T. deltoidalis accumulated Pb to a
concentration equal to that of the spiked sediment over the course of the experiment (28 days). Exposed
T. deltoidalis individuals had significantly reduced total antioxidant capacity and significantly higher
lysosomal destabilization and micronuclei frequency compared with control organisms.
Evidence in the 2013 Pb ISA was suggestive of a causal relationship between Pb exposure and
hematological effects, primarily based on field studies that correlated ALAD activity to measured Pb
levels in bivalve tissue (Company et al.. 2011; Kalman et al.. 2008). Generally, these studies have noted
that Pb content varies significantly among species and is related to habitat and feeding behavior. A few
additional studies have reported inhibition of ALAD activity in Pb-exposed saltwater invertebrates;
however, the concentration at which enzyme activity is affected appears to be higher than the
concentration of Pb typically encountered in seawater (Table 11-1). In R. philippinarum gill tissue,
ALAD activity decreased significantly after 7-day nominal exposure to 10 (ig Pb/L, and activity did not
recover in a 7-day depuration period following the initial exposure (Aouini et al.. 2018). ALAD activity
was higher in the gill compared with the digestive glands. Duarte et al. (2020) demonstrated various
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sublethal biomarkers were activated during 28-day exposure at a low concentration of Pb (10 (ig Pb/L) in
the crab U. cordatus. This concentration was too low to inhibit ALAD activity in the crabs; however,
metallothioneins were induced and DNA damage occurred in exposed individuals. Various immunotoxic
endpoints were assessed in hemolymph of the marine crab Charybdis japonica during a 30-day exposure
to Pb (Xu et al.. 2019). At the lowest concentration, 0.066 (j,M (13.6 |ig Pb/L) immune responses were not
significantly different from control responses. At the next lowest concentration (0.132 (j,M, 27.2 |ig Pb/L),
there was an initial increase followed by a decrease in the total hemocyte count. Total hemocyte count
was significantly lower than control counts at the end of the exposure duration.
11.4.4.3.2. Organism-Level Response
Saltwater invertebrate studies that report effects on growth, reproduction and development, and
survival are primarily reviewed in the exposure-response section (Section 11.4.5). A few additional
studies that provide information on these endpoints are discussed here. Dietary exposure of sea cucumber
(A. japonicus) to a Pb-amended diet (100, 500 or 1000 mg Pb/kg dry weight) for 30 days resulted in no
significant effects on growth or survival; however, antioxidant enzyme activity was significantly lower in
the treatment groups compared with the control (Wang et al.. 2015a). In sea hare (A. californica) exposed
to Pb solely through diet over 2 or 3 weeks (green seaweed U. lactuca previously exposed to either
10 fxg Pb/L or 100 |ig Pb/L for 48 hours), growth was significantly lower in the treatment groups
compared with the control (Jarvis et al.. 2015).
Brine shrimp (Artemia franciscana) exposed nominally to Pb (8, 16, 32, 64 |ig Pb/L) for 20 days
(from recently hatched nauplii to adult lifestage) exhibited a significant decrease in mating behavior
(sexual couples assessed during the last 3 days of the experiment) at all concentrations, compared with
nonexposed shrimp (Frias-Espcricucta et al.. 2022). In the marine ascidian Ciona intestinalis, various
reproductive and developmental parameters were assessed at nominal concentration of 10, 20 and
100 |ig Pb/L (Gallo et al.. 2011). Hatching rate and embryo development were unaffected in all treatment
groups. Significant inhibition of oocyte voltage gated Na+ currents and postfertilization contraction were
observed at the two highest Pb concentrations, suggesting an effect on the mechanisms of fertilization.
11.4.4.4. Effects on Saltwater Vertebrates
In the 2013 Pb ISA, there was inadequate evidence to infer causality relationships between Pb
exposure and effects in saltwater vertebrates (Table 11-6). Few studies on saltwater vertebrates were
reviewed in the 2013 Pb ISA or in the previous Pb AQCDs, especially for reproduction, growth, and
survival (endpoints that may have relevance to the population level of biological organization and higher).
Studies reviewed in the exposure-response section (Section 11.4.5, Table 11-7) of this appendix include
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chronic toxicity data for growth and survival endpoints in saltwater fish species published since the 2013
Pb ISA. Summarized below are recent studies that report Pb perturbation on physiological endpoints in
fish and other saltwater vertebrates.
11.4.4.4.1. Fish
Most of the available studies in saltwater fish seek to identify molecular and cellular responses to
Pb exposure and do not report effects at the organism level of biological organization (e.g., survival,
growth, reproduction). Furthermore, studies since the 2013 Pb ISA that quantify effects on biomarkers in
saltwater and euryhaline fish are typically conducted at Pb concentrations considerably higher than
conditions found in natural environments. Nunes et al. (2014b) assessed the response of anadromous
European eel (A. anguilla) to Pb exposure down to 165 |ig Pb/L in 28-day aqueous exposure studies and
observed no statistically significant effects on the biomarkers of neurotoxicity or peroxidative membrane
damage. Only gill tissue GST activity was significantly increased at 165 (.ig Pb/L. and further increased
with higher Pb concentration. Similar 28-day chronic bioassays were performed in juvenile turbot
(Scophthalmus maximus). Very few significant effects were reported at the lowest concentration tested
(291 jig Pb/L). Hepatic CAT activity significantly decreased, liver GST significantly increased and no
measurable changes in biomarkers of neurotoxicity were observed (Nunes et al.. 2014a). Fernandez et al.
(2015) evaluated the suitability of ALAD as a biomarker for Pb exposure in wild-caught red mullet
(Mullus barbatus) along several locations of the Spanish coast. Pb concentration in muscle tissue was
low. However, there was a weak, but significant, inverse relationship with ALAD activity; ALAD activity
showed no statistically significant relationship to the condition factor, gonadosomatic index and
hepatosomatic index of the fish.
Since the 2013 Pb ISA, a series of studies have further elucidated the effects of Pb exposure via
diet on multiple physiological endpoints in saltwater fish, and these perturbations were linked to a
decrease in weight gain (growth) in one study. In juvenile Korean rockfish (Sebastes schlegelii),
biomarkers of oxidative stress (SOD, GST) were significantly increased, AChE was significantly
decreased in muscle (Kim et al.. 2017). and physiological stress indicators (heat shock protein 70 mRNA
gene expression and plasma Cortisol) were significantly increased (Kim and Kang. 2016). as were
hematological parameters (hemocrit, hemoglobin) (Kim and Kang. 2015) by 4-week dietary
Pb > 60 mg/kg in experimental diet formulation. This is consistent with dietary exposure in starry
flounder (Platichthys stellatus), in which the same hematological parameters as well as red blood cell
count were significantly decreased at 4-week dietary Pb exposure over 60 mg Pb/kg (Hwang et al.. 2016).
In rockfish, immune response was elicited at a higher dietary concentration (>120 mg Pb/kg) at 4 weeks
(Kim and Kang. 2016). A decrease in daily weight gain was observed in rockfish at >120 mg Pb/kg (Kim
and Kang. 2015). A dietary intake above 60 mg Pb/kg daily after 4 weeks of exposure to Pb appeared to
be the threshold for most effects.
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11.4.4.4.2. Other Saltwater Vertebrates
There are a few new studies of nonfish saltwater vertebrates that report BLLs and associated
effects. In a survey of blood Pb levels in common eider ducks (Somateria mollissima) at a breeding
colony in the northern Hudson Bay, birds with higher BLLs had lower body condition indexes (body
mass/head length) when they arrived at the breeding grounds (Provencher et al.. 2016). Birds with higher
BLLs arrived later at the breeding grounds. Birds that arrive later at the breeding grounds and with lower
body condition indexes are more likely to have lower reproductive success. A study of loggerhead sea
turtles (Caretta caretta) in Casey Key, Florida examined the connection between blood Pb concentrations
and hematological effects (Perrault et al.. 2017). Over a range of blood Pb levels (0.07-0.52 jxg/g dry
weight), there was a significant negative relationship between BLLs and albumin, a2-globulins, total
solids, and Fe.
11.4.5. Exposure and Response of Saltwater Species
Evidence regarding exposure-response relationships and potential thresholds for Pb effects on
saltwater biota can provide tools for quantitative analyses of risks for coastal saltwater ecosystems. No
exposure-response studies in saltwater algae or vertebrates, and very few studies on saltwater
invertebrates, were reported in the 1977, 1986 or 2006 Pb AQCDs. For saltwater invertebrates, available
evidence at the time of the 2013 Pb ISA was suggestive of a causal relationship between Pb exposure and
reproductive and developmental effects (U.S. EPA. 2013). Much of the evidence was from exposure-
response bioassays.
Since the 2013 Pb ISA, new toxicity data for saltwater algae, invertebrates and fish have been
reported based on analytically verified Pb concentration. This information reduces uncertainties identified
in the previous review in terms of a lack of exposure-response data for saltwater biota, especially for
chronic toxicity, and enables calculations of effect levels for saltwater biota based on experimental data
(Church et al.. 2017). The studies listed in Table 11-7 are those that report exposure-response values at
concentrations comparable to, or lower than, the most sensitive saltwater biota identified in the 2013 Pb
ISA or the 2006 AQCD (i.e., the most environmentally relevant studies). Exposure-response data from
previously untested taxonomic groups are also discussed in this section. In general, marine organisms are
tolerant of Pb at much higher concentrations than those encountered in uncontaminated natural
environments.
In studies reviewed in the 2013 Pb ISA, marine algae exhibited a range of sensitivity to Pb, with a
72-hour EC50 of 105 |ig Pb/L reported for Chaetorceros spp. Other tested species were considerably less
sensitive (72-hour EC50 = 740 |ig Pb/L or higher) (Debelius et al.. 2009). Exposure-response data for
marine algal species published since the 2013 Pb ISA greatly exceed environmental concentrations; for
example, in the marine alga Nannochloropsis oculata, the 72-hour IC50 = 1,810 fxg Pb/L for growth
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inhibition (Zam an i - Ah m adm ah m oodi et al.. 2020). Longer-term exposure studies assessing the population
growth rates of polar marine algal species have reported effects as low as 24-day ECio = 152 |ig Pb/L for
Cryothecomonas armigera (Koppel et al.. 2017) and a 10-day ICio = 260 |ig Pb/L for 1'haeocystis
antarctica (Gissi et al.. 2015).
In the 2013 Pb ISA, studies that reported effect concentrations in saltwater invertebrates included
a delay in reproduction onset in the marine amphipod, E. laevis, at 118 mg/Pb kg sediment, a
concentration the authors indicated was below the current marine sediment regulatory guideline for Pb
(218 mg Pb/kg sediment) (Ringcnarv et al.. 2007; NOAA. 1999). A 96-hour EC50 = 197 fxg Pb/L for the
growth of larvae and EC50 = 297 |ig Pb/L for embryogenesis inhibition was observed for the clam
Meretrix meretrix (Wang etal.. 2009). Another study reported a decrease in the fertilization rate of eggs
of the marine polycheate Hydroides elegans; in eggs pretreated with 48 |ig Pb/L, hatching decreased to
20% of control levels (Gopalakrishnan et al.. 2008). The lifestages of H. elegans varied in their sensitivity
to Pb, with the most sensitive period being larval settlement, with an EC50 of 100 |ig Pb/L. In the 2013 Pb
ISA, the most sensitive endpoint for growth in a saltwater invertebrate was LOAEL = 85 mg Pb/kg in
sediment in the polychaete Capitella sp. (Horng et al.. 2009). Other saltwater invertebrate exposure-
response studies in the 2013 Pb ISA reported effects at higher Pb concentrations. In the 2006 AQCD, the
most sensitive endpoint was a 48-hour EC50 = 221 |ig Pb/L and LOEC = 50 |ig Pb/L for embryogenesis in
the mussel M. galloprovincialis (based on nominal Pb concentration only) (Bciras and Albentosa. 2004).
Recent exposure-response data for saltwater invertebrates include reproductive and
developmental bioassay results based on analytically verified concentrations for mollusks and
echinoderms, with effects reported at lower concentrations than in studies included in the 2013 Pb ISA
(Table 11-7). Embryo development of the scallop Argopecten purpuratus was impaired with Pb exposure,
with the 48-hour EC50 reported as = 44 |ig Pb/L (Romero-Murillo et al.. 2018). The order of sensitivity of
10 marine bivalve species (based on the percentage of normal D-veliger larvae assessed at 48 hours of Pb
exposure) was oysters > mussels > scallops > cockles > clams (Markich. 2021). The oysters M gigas (48-
hour EC50 = 49.5 |ig Pb/L, 48-hour NEC = 9.9 |ig Pb/L) and S. glomerata (48-hour EC50 = 52.1 |ig Pb/L,
48-hour NEC = 10.1 |ig Pb/L) were most sensitive while the clam Irus crenatus (48-hour
EC50 = 196 jxg Pb/L, 48-hour NEC = 39.8 |ig Pb/L) was the least sensitive bivalve tested. In a series of
bioassays, Nadella et al. (2013) assessed Pb effects on embryo development in two mussels, M
galloprovincialis andM. trossolus, and the sea urchin S. purpuratus. Both mussel species exhibited
similar acute toxicity to Pb in 48-hour embryo-larval toxicity tests in 100% seawater (M
galloprovincialis-ECU,, = 63 |ig Pb/L, EC20 =19 jxg Pb/L; EC10 =10 jxg Pb/L, NOEC = 3.2 |ig Pb/L and
M. trossolus, EC50 = 45 |ig Pb/L; EC20 = 16 |ig Pb/L; EC10 = 9 jig Pb/L; NOEC = 3.4 |ig Pb/L). In the 72-
hour embryo-larval toxicity test in the sea urchin S. purpuratus, the EC50 = 74 |ig Pb/L,
EC20 = 31 |ig Pb/L, EC10 = 19 jxg Pb/L and NOEC = 2.7 |ig Pb/L. In a similar 72-hour larval development
toxicity test with the sea urchin Evechinus chloroticus, the EC50 = 52.2 |ig Pb/L, with skeletal
abnormalities observed in the lower range of concentrations (10 |ig Pb/L and 20 |ig Pb/L) (Rouchon and
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Phillips. 2017). However, Pb in the exposure water was not analytically verified in the study.
Developmental endpoints in oyster C. gigas were less sensitive to Pb, with EC50 = 660.3 |ig Pb/L for
embryo toxicity, 96-hour LC50 = 699.5 |ig Pb/L for larval mortality and LOEC = 96.7 |ig Pb/L for
significant increase of abnormal D-shaped larvae (Xic et al.. 2017). In a series of fertilization bioassays
with the marine polychaete broadcast spawner Galeolaria caespitosa, the EC10 for reproduction varied
with the density of sperm used in the bioassays and ranged from 65 to 910 (ig Pb/L. The toxicity of Pb
was significantly decreased at higher sperm density (Lockver et al.. 2019). The EC10 was calculated to be
30 |ig Pb/L at a sperm density required to achieve 50% of the maximum fertilization.
New exposure-response data on previously untested marine invertebrate taxa, including species of
corals and sea anemones, generally show that these organisms are tolerant to Pb at relatively high
concentrations. Hedouin et al. (2016) assessed survival in adult and larval stages of the Scleractinian coral
Pocillopora damicornis. Results from 96-hour acute toxicity testing in adults collected during two
seasons near Oahu, Hawaii (summer 96-hour LC50 = 742 |ig Pb/L, winter 96-hour LC50 = 477 |ig Pb/L)
and coral larvae tested in the laboratory at two temperatures (96-hour LC50 = 681 |ig Pb/L at 27°C, 96-
hour LC50 = 462 |ig Pb/L at 30°C) showed similar tolerance to Pb. In Cnidarian (sea anemone) Aiptasia
pulchella, the 96-hour LC50 values were 8,060 |ig Pb/L and 12,400 |ig Pb/L in two separate tests. In the
same species, the 6-hour EC50 = 2,610 |ig Pb/L and 12-hour EC50 = 1,740 |ig Pb/L for rapid tentacle
retraction, suggesting that anemones are tolerant to Pb, even at concentrations that greatly exceed that of
Pb in seawater (Howe et al.. 2014). In contrast, a 30-day exposure to Pb in the marine Tiger prawn P.
monodon yielded NOEC = 14 |ig Pb/L and LOEC = 29 |ig Pb/L for survival, suggesting that these
crustaceans are relatively sensitive to Pb (Hariharan et al.. 2012). A recent review of Pb effects on marine
invertebrates bv(Botte et al.. 2022) summarizes many of the effect concentrations and studies described
above.
For vertebrates, several studies published since the 2013 Pb ISA provide chronic toxicity data for
saltwater fish species, information that was previously lacking for evaluating the longer-term effects of Pb
on these organisms.Reynolds etal. (2018) conducted 28-day chronic toxicity tests with larval topsmelt A
affinis (a fish species native to the coast of the western United States) at two salinities (14 ppt and 28 ppt)
to represent conditions in estuarine and marine environments. In the larval fish, survival was affected to a
greater extent at the lower salinity (LC50 =15.1 jxg Pb/L, NOEC<13.8 |ig Pb/L) than at the higher salinity
(LC50 = 79.8 |ig Pb/L, NOEC = 45.5 |ig Pb/L) due to the higher fraction of Pb in the form of Pb2+ at lower
salinity. Growth effects (assessed as standard length) were reported in the same study, with greater
response observed at the lower salinity (EC10 = 16.4 |ig Pb/L) compared with the higher salinity
(EC10 = 82.4 (.ig/L). Tests conducted with juvenile topsmelt at 28 ppt (28-day LC50 = 167.6 |ig Pb/L)
showed that this lifestage was less sensitive to Pb than the larval stage (28-day LC50 = 79.8 |ig Pb/L). The
authors observed abnormal swimming and morphology, but these endpoints were not quantified.
Calculated chronic values for additional saltwater fish species include NOEC = 14 |ig Pb/L and
LOEC = 29 |ig Pb/L for grey mullet (M cephalus) fingerling survival and NOEC =11 jxg Pb/L and
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LOEC = 22 |ig Pb/L for Tiger perch (T. jarbna) fingerling survival following 30-day exposure to Pb
(Hariharan et al.. 2016). The 96-hour LC50 values in these species were 2,570 and 2,990 |ig Pb/L,
respectively.
Given the increased availability of toxicity data for saltwater biota since development of the
AWQC for Pb by the EPA Office ofWater in 1984 (U.S. EPA. 1985a) (Section 11.1.7.3), Church et al.
(2017) recently proposed updated U.S. saltwater acute AWQC of 100 |ig Pb/L (acute) and chronic
AWQC of 10 (ig Pb/L (chronic) based on genus mean toxicity values following EPA methodology (U.S.
EPA. 1985b). For the acute genus sensitivity distribution (Figure 11-6), data from 54 species and 49
genera were included. The proposed value of 100 |ig Pb/L is less than the current acute criterion of
210 (ig Pb/L due to toxicity data from relatively sensitive early lifestages of Echinodermata and Mollusca.
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The proposed Church et al. (2017) chronic value of 10 (.ig Pb/L for saltwater (based on EC20 or, in
some cases, EC50 data divided by a factor of two when EC20 data could not be calculated from available
data) is based on data for 21 species and 17 genera. The four lowest genus mean chronic values were
10 jug Pb/L for a mysid, 28 j.ig Pb/L for blue mussel (Mytilis spp.), 36 |ig Pb/L for purple sea urchin (S.
purpuratus), and 55 |ig Pb/L for topsmelt (A. affinis). In their derivations of acute and chronic values,
Church et al. (2017) included some non-North American species. If the analysis was limited to North
American biota, the proposed acute and chronic values would be 110 jxg Pb/L and 8.8 ^g Pb/L,
respectively. Comparison of chronic sensitivity distributions in saltwater biota for dissolved Pb following
EPA and European Union methods is shown in Figure 11-7. Following the publication of these proposed
values, Reynolds et al. (2018) conducted additional testing with topsmelt larvae (LC20 = 10.7 Lig Pb/L at
14 ppt salinity).
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30% H
20%
10% H
0%
Chasmagnathus
s op.
XL.
Phaeodactylum tricomutum
Tisbe battagliai
Dendraster excentricus
Paracentrotus lividus
Strongylocentrotus
pupuratus
Champia parvula ' l§
II
Americamysis bahia
Mytilus trossolus
1
Lo!igo/r\
/ 9 Dunaliella tertiolecta
Lithodes O „
A Crassostrea gigas
1
I
Neanthes arenaceodentata
II
Skeletonema costatum # .
ii
Mytilus galloprovincialis i
Atherinops affinis
O Crassostrea
O Tisbe
O Macrobrachium
^ O Callianassa
O Mercenaria
O Cancer
o Dendraster
O Cyprinodon
O Paracentrotus
O Neanthes
Atherinops
Strongylocentrotus
Mytilus
100
Diss. Pb, (jg/L
1 000
10 000
Species mean chronic values (European Union method) are shown in red circles; genus mean chronic values (U.S. EPA method)
are shown in open circles; solid red curve = Weibull distribution fitted to species mean chronic values; solid black curve = triangular
distribution fit to the four most sensitive genus mean chronic values; dashed red vertical line = median 5th percentile hazardous
concentration based on Weibull distribution; dashed black vertical line = criterion continuous concentration (proposed chronic
criterion); black text = genera associated with genus mean chronic values; red text = species associated with species mean chronic
values.
Source: Church et al. (2017)
Figure 11-7 Comparison of chronic sensitivity distributions in saltwater biota
for dissolved Pb following the U.S. EPA and European Union
methods.
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Table 11-7 Studies in saltwater biota with analytically verified Pb concentration that report an effect on
growth, reproduction, or survival comparable to, or lower than, the lowest effect concentrations
reported in previous Pb AQCDs or the 2013 Pb ISA.
Species
Concentration Exposure Method
Modifying
Factors
Effects on Endpoint
Effect Concentration
Reference
(Published
since the
2013 Pb ISA)
Invertebrates
Mussel
(Mytilus
galloprovincialis)
Mussel
(Mytilus trossulus)
Sea urchin
(Strongylocentrotus
purpuratus)
Nominally 3.2,
10, 32, 100,
320,1,000 |jg Pb/
L (concentrations
were measured
for each
individual assay)
Standard embryo
development acute
toxicity tests for
larvae of mussel (to
48-h postfertilization)
and sea urchin (to
72-h postfertilization)
conducted using
ASTM protocols in
100% sea water
DOC:
1.79 ± 0.02 mg/L
Additional toxicity
tests were
conducted with
added DOC
Salinity:
33 ppt
Developmental
assays conducted
over a range of
salinities from 15-
33 ppt
Temperature:
20°C ± 1°C
for mussels
15°C± 1°C
for sea urchin
Reproduction:
Development of larvae: The
percentage of embryos exhibiting
normal development was
assessed after 48-h (mussels) or
72-h (sea urchin) exposure to Pb
at varying concentration in
seawater. The acute toxicity of Pb
was similar between the two
species of mussel and sea urchin
M. galloprovincialis
48-h ECso = 63 pg Pb/L
48-h EC2o= 19 pg Pb/L
48-h EC10 = 10 pg Pb/L
48-h NOEC = 3.2 pg Pb/L
M. trossolus
48-h ECso = 45 pg Pb/L
48-h EC2o= 16 pg Pb/L
48-h EC10 = 9 pg Pb/L
48-h NOEC = 3.4 pg Pb/L
S. purpuratus
72-h ECso = 74 pg Pb/L
72-h EC20 = 31 pg Pb/L
72-h EC10 = 19 pg Pb/L
72-h NOEC = 2.7 pg Pb/L
Nadella et al.
(2013)
Scallop
(Argopecten
purpuratus)
7 (control), 25,
50, 100, 140,
570, 730,1000,
48-h embryo-larval
development assay
with Pb-nitrate
conducted in 100%
Salinity:
35 ppt
Reproduction:
Embryos exhibited abnormal
development (impaired D-larvae
Embryo:
48-h ECso = 44 pg Pb/L
Romero-
Murillo et al.
(2018)
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Species
Concentration Exposure Method
Modifying
Factors
Effects on Endpoint
Effect Concentration
Reference
(Published
since the
2013 Pb ISA)
1590 pg Pb/L
sea water. In
pH:
development) following Pb
Juvenile:
(measured)
addition, a 96-h acute
8.0
exposure.
96-h
toxicity test was
Survival:
LCso = 1,420 pg Pb/L
conducted with
juveniles (21 mm in
Temperature
Assessed in juvenile lifestage
only
shell length)
(embryo
exposure)
19°C ± 1°C
Oyster
(Magallana gigas)
Oyster
(Saccostrea
glomerata)
Mussel
(.Xenostrobus
securis)
Scallop
(Scaeochlamys
livida)
Cockle
(.Anadara trapezia)
Cockle
(Fulvia
tenuicostata)
Clam
(Hiatula alba)
Each test with
1.5 to 2-h-old
embryos (8-cell
stage) consisted
of a control and
12 metal
concentrations
(based on
preliminary
range-finding
tests).
Concentrations
were analytically
verified but not
reported
48-h embryo-larval
development assay
with Pb-nitrate
conducted in 100%
sea water. Test
waters were not
renewed, and
embryos were not
fed. Percentage of
normal D-veliger
larvae was
determined by direct
observation of 100
larvae (per replicate)
Salinity:
30 ppt± 0.5%
pH:
7.85 ± 0.05
Temperature:
21°C ± 1°C
DO:
80 to 95%
saturation
Reproduction:
Embryos exhibited abnormal
development (impaired D-larvae
development) following Pb
exposure. The order of sensitivity
of the bivalves to Pb was oysters
> mussels > scallops > cockles >
clams
M. gigas
48-h ECso = 49.5 pg Pb/L
48-h NEC = 9.9 pg Pb/L
S. glomerata
48-h ECso = 52.1 pg Pb/L
48-h NEC = 10.1 |jg Pb/L
X. securis
48-h ECso = 59.9 pg Pb/L
48-h NEC = 12 pg Pb/L
S. livida
48-h ECso = 67.2 pg Pb/L
48-h NEC = 13.7 pg Pb/L
A. trapezia
48-h ECso = 84.9 pg Pb/L
48-h NEC = 16.8 pg Pb/L
F. tenuicostata
48-h ECso= 108 pg Pb/L
48-h NEC = 22.3 pg Pb/L
Markich
(2021)
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Reference
Species Concentration Exposure Method Effects on Endpoint Effect Concentration (Published
Factors since the
2013 Pb ISA)
Clam
(Barnea
australasiae)
H. alba
48-h ECso= 129 pg Pb/L
48-h NEC = 24.8 pg Pb/L
Clam
(Spisula trigonella)
Clam
(Irus crenatus)
B. australasiae
48-h ECso= 140 pg Pb/L
48-h NEC = 28 pg Pb/L
S.trigonella
48-h ECso= 177 pg Pb/L
48-h NEC = 36.7 pg Pb/L
I. crenatus
48-h ECso= 196 pg Pb/L
48-h NEC = 39.8 pg Pb/L
Prawn
(Penaeus
monodon)
1.7 (control-lab
seawater used in
bioassays), 14,
29, 56, 108,
230 pg Pb/L
(measured)
Post larvae were
exposed to Pb
acetate for 30 days in
a continuous flow-
through system.
Prawns fed twice
daily and Pb
concentrations
measured every
10 days
Salinity:
27.7 ± 0.5 ppt
Temperature:
25.4 ± 0.7°C
DO:
6.3 ± 0.6mg/L
Survival:
Survival of P. monodon was
significantly decreased at the
higher exposure concentrations
30-d NOEC = 14 pg Pb/L
30-d LOEC = 29 pg Pb/L
Hariharan et
al. (2012)
pH: 7.1 ± 0.5
Vertebrates
Topsmelt Measured:
(Atherinops affinis) Mean ± SD
Larval fish (< 3 day
old) were tested in
two different salinities
Low Salinity
larval fish:
Survival:
Pb was consistently more toxic to
larva fish at the lower salinity
28-d survival of larval fish
at 14 ppt salinity
Reynolds et
al. (2018)
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Species
Concentration Exposure Method
Modifying
Factors
Effects on Endpoint
Effect Concentration
LC5 = 7.7 |jg Pb/L
LC10 = 8.3 |jg Pb/L
LC15 = 9.9 |jg Pb/L
LC20 = 10.7 |jg Pb/L
LC25 = 11.5 |jg Pb/L
LC40 = 13.6 |jg Pb/L
LCso = 15.1 |jg Pb/L
NOEC = <13.8 |jg Pb/L
LOEC = 13.8 |jg Pb/L
28-day survival of larval
fish at 28 ppt salinity
LC5 = 36.6 |jg Pb/L
LC10 = 43.4 |jg Pb/L
LC15 = 48.8 |jg Pb/L
LC20 = 53.5 |jg Pb/L
LC25 = 58.0 |jg Pb/L
LC40 = 70.8 |jg Pb/L
LCso = 79.8 |jg Pb/L
NOEC = 45.5 |jg Pb/L
LOEC = 89.9 |jg Pb/L
28-day survival of juvenile
fish at 28 ppt salinity
LC5 = 105.3 |jg Pb/L
LC10 = 110.9 |jg Pb/L
LC15 = 116.8 |jg Pb/L
LC20 = 123 |jg Pb/L
LC25 = 129.5 |jg Pb/L
Reference
(Published
since the
2013 Pb ISA)
Low salinity,
larval fish:
Total Pb
BDL,
17 ± 1 |jg Pb/L,
34 ± 1 |jg Pb/L,
69 ± 4 |jg Pb/L,8
5± 15 |jg Pb/L,
127 ± 16 |jg Pb/L
Dissolved Pb
BDL,
14 ± 1 |jg Pb/L,
27 ± 2 |jg Pb/L,
51 ± 3 |jg Pb/L,
80 ± 7 |jg Pb/L,
117 ± 19 |jg Pb/L
High salinity,
larval fish:
Total Pb
BDL,
58 ± 9 |jg Pb/L,
107 ±20 |jg Pb/L
200 ± 14 |jg Pb/L
386 ± 43 |jg Pb/L
563 ± 45 |jg Pb/L
Dissolved Pb
BDL,
46 ± 10 |jg Pb/L,
90 ± 20 |jg Pb/L,
171 ±22 |jg Pb/L
(14 ppt and 28 ppt) in
28-day exposures to
Pb nitrate
administered in a
flow-through test
system set to replace
the total volume of
synthetic seawater in
each 2-L exposure
chamber replicate
once every 12 h. In
addition, a 28-d
exposure was
conducted with
juvenile fish (2.5 mo
old) at 28ppt at
higher Pb
concentration
(control, 100 and
200 |jg Pb/L)
Salinity
14.1 ±0.1 ppt
Temperature:
18.2 ± 0.3°C
Alkalinity:
58 ± 5 mg/L as
CaC03
pH:
7.96 ± 0.17
DO:
7.58 ± 0.39
High Salinity
larval fish:
Salinity
28.1 ± 0.6 ppt
Temperature:
18.1 ± 0.2°C
Alkalinity:
105 ± 8 mg/L as
CaC03
pH:
7.92 ± 0.07
DO:
6.88 ± 0.60
(14 ppt) compared with the higher
salinity and larvae were more
sensitive than juvenile fish at
28 ppt. Free Pb2+ ion
concentrations, the most
bioavailable form of Pb, were
higher in the lower salinity water
based on Pb speciation
calculations.
Growth:
Growth effects in larval fish
(assessed as standard length)
were more pronounced at the
lower salinity
(EC10 = 16.4 |jg Pb/L) compared
with the higher salinity
(EC10 = 82.4 |jg Pb/L)
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Species
Concentration Exposure Method
Modifying
Factors
Effects on Endpoint
Effect Concentration
Reference
(Published
since the
2013 Pb ISA)
259 ± 24 pg Pb/L
LC40 = 151.2 pg Pb/L
,
LCso = 167.6 pg Pb/L
435 ± 48 pg Pb/L
28-d EC10 for larval
High salinity
growth (standard length)
juvenile fish:
at 14ppt
Total Pb
salinity = 16.4 pg Pb/L
BDL,
28-d EC for larval growth
154 ±67 pg Pb/L
(standard length) at
,
28 ppt
239 ± 98 pg Pb/L
salinity = 82.4 pg Pb/L
Dissolved Pb
BDL,
100 ±21 pg Pb/L
190 ± 30 pg Pb/L
Grey mullet
(Mugil cephalus)
Tiger perch
(Terapon jarbua)
7, 16, 34, 65,
136 |jg Pb/L (M.
cephalus)
7,15,29,60,118 p
g Pb/L (7.
jarbua)
Wild-caught
fingerlings (3.0-4.5
cm in size) were
acclimated to
laboratory conditions
then exposed to Pb
as Pb acetate in a
continuous flow-
through system for
30d
Salinity:
33.5 ± 1.4 ppt
Temperature:
23.5 ± 0.9°C
pH:
7.8 ± 0.5
DO:
6.5 ± 0.6
Survival:
Survival of M. cephalus and T.
jarbua decreased with the
increase in exposure
concentrations
Grey mullet:
30-d NOEC = 14 |jg Pb/L
30-d LOEC = 29 pg Pb/L
Tiger perch:
30-d NOEC = 11 |jg Pb/L
30-d LOEC = 22 pg Pb/L
Hariharan et
al. (2016)
54 species and 49
genera of
invertebrates and
fish (acute toxicity
data included in
derivation of
proposed updated
acute saltwater
Pb was
analytically
verified in all
studies
U.S. EPA guidelines
(U.S. EPA. 1985b)
were used to identify
acceptable studies.
Acute: All included
assays were embryo-
larval toxicity studies
reporting 48 to 96-h
ECsos. The four
Acute toxicity end points
included survival,
immobilization, and embryo-
larval development
The proposed updated acute
criterion is lower than the current
EPA acute saltwater criterion of
210 pg Pb/L due to embryo-larval
Proposed Saltwater Acute Church et al.
Water Quality Criterion: (2017)
100 pg Pb/L. Limiting the
derivation to North
American species, the
proposed criterion is
110 pg Pb/L.
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Species
Concentration Exposure Method
Modifying
Factors
Effects on Endpoint
Effect Concentration
Reference
(Published
since the
2013 Pb ISA)
quality criterion for
Pb)
21 species and 17
genera of
invertebrates and
fish (chronic toxicity
data included in
derivation of
proposed updated
chronic saltwater
quality criterion for
Pb)
lowest genus mean
acute values
(Strongylocentrotus
purpuratus = 75 |jg P
b/L; Mytilus
spp = 123 |jg Pb/L;
Paracentrotus
lividus = 363 |jg Pb/L;
and Dendraster
excentricus = 371 |jg
Pb/L) and a total of
49 genus mean
values were used to
determine a final
acute value of
203.6 |jg Pb/L. This
value was divided by
2 to derive the
proposed acute
criterion based on
U.S. EPA methods.
toxicity tests with sensitive
echinoderm and mussel species.
Chronic toxicity endpoints
included survival, growth,
development, and reproduction
The proposed updated chronic
criterion is greater than the
current U.S. EPA acute saltwater
criterion of 8.1 |jg Pb/L.
Uncertainty in the derivation of
the chronic criterion has
decreased due to increased
availability of studies; an acute-to-
chronic ratio was not used.
Proposed Saltwater
Chronic Water Quality
Criterion: 10 |jg Pb/L.
Limiting the derivation to
North American species,
the proposed criterion is
8.8 |jg Pb/L
Chronic: Based on
EC20 from life cycle
tests or ECso data
divided by a factor of
2 when EC20 data
could not be
calculated and
augmented with 48-h
toxicity data in some
cases. The four
lowest genus mean
chronic values
(Americamysis
bahia =10 |jg Pb/L;
Mytilus
spp. = 28 |jg Pb/L;
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Reference
Species Concentration Exposure Method Effects on Endpoint Effect Concentration (Published
Factors since the
2013 Pb ISA)
Strongylocentrotus
purpuratus = 36 |jg P
b/L; Atherinops
affinis = 55 |jg Pb/L)
and a total of 17
genus mean values
were used to identify
a chronic 5th
percentile of
10 |jg Pb/L following
EPA guidelines
ASTM = American Society for Testing and Materials; BDL = below the method detection limit; CaC03 = calcium carbonate; DO = dissolved oxygen; DOC = dissolved organic carbon;
EC = effect concentration; LC = lethal concentration; LOEC = lowest observed effect concentration; mo = months; NEC = no-effect concentration; NOEC = no observed effect
concentration; Pb = lead.
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11.4.6. Saltwater Community and Ecosystem Effects
As discussed in the 1986 Pb AQCD (U.S. EPA. 1986). the 2006 Pb AQCD (U.S. EPA. 2006a)
and the 2013 Pb IS A (U.S. EPA. 2013). the body of evidence was inadequate to infer a causal relationship
between Pb exposure and saltwater community- and ecosystem-level effects. Observations from field
studies in the 2006 Pb AQCD and the 2013 Pb ISA found either negative or null relationships between Pb
and species abundance, richness, and diversity in saltwater macroinvertebrates; however, Pb was not the
only contaminant in most observational studies, making it difficult to separate the effects of Pb from those
of other metal pollutants. New mesocosm and observational studies published since the 2013 Pb ISA
examined the relationship between Pb in sediment and saltwater as well as the community effects. Several
reported negative or null relationships between sediment Pb concentrations and foraminiferal abundance
and community structure, while others reported positive associations.
Reductions in benthic foraminiferal and meiofaunal community richness, diversity, and the
abundance of certain taxa under Pb exposure were supported by a mesocosm study conducted in Italy
(Frontalini et al.. 2018). Sediment was collected from a relatively undisturbed coastal area and placed in
mesocosms with artificial seawater exposed to nominal concentrations of 0, 10, 100, 200, 500, 1000,
5000, or 10000 |ig Pb/L. Geochemical parameters, meiofaunal communities, and foraminiferal
communities were sampled after 1, 2, 3, 4, 6, and 8 weeks. Meiofaunal density, richness, and Nematoda
abundance decreased overtime at higher concentrations of Pb. Although most meiofaunal taxa abundance
and richness decreased in a dose-dependent manner with increasing Pb concentrations in the sediment,
Ostracoda exhibited an increase in abundance with increasing Pb sediment concentration. Pb water
concentration was negatively correlated with Bivalvia abundance, while positive correlations were found
between Pb water content and Gastropoda, Copepoda, and Polychaeta. Finally, the abundance of most
benthic foraminiferal species, the Shannon-Weiner diversity index, and Pielou's evenness, were
negatively correlated with Pb concentrations in the sediment, whereas positive correlations were observed
with Ammonia tepida and Bolivana spathulata.
Foraminiferal diversity and community structure via changes in the abundance of certain taxa
have been found to vary with sediment Pb along environmental gradients in various locations including in
the Pearl River estuary, China (Li et al.. 2013). the Ria de Aveiro lagoon, Portugal (Martins et al.. 2011).
the San Jose Bay estuary, Puerto Rico (Martinez-Colon et al.. 2018). the Gulf of Milazzo, Sicily, Italy
(Cosentino et al.. 2013). the Strait of Malacca, Malaysia (Minhat et al.. 2020) and Chilika lagoon in India
(Barik et al.. 2022).
In the Pearl River estuary, surface sediment OC, grain size and benthic foraminifera communities
were assessed (Li et al.. 2013). Mean ± S.D. sediment Pb concentrations in the study area were
36.98 ± 11.18 mg Pb/kg (range: 13.5-62.9 mg Pb/kg). Trace metal concentrations in the sediment of Pb,
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Cu, Co, Cr, Ni, V and Zn were negatively correlated with the Shannon-Weaver index, Fisher a index,
species richness, and abundance of certain foraminiferal species. The CCA demonstrated that Pb
explained 7.5% of variation in the foraminiferal community.
Some foraminifera taxa were found to positively correlate with bioavailable Pb in the channels of
Ria de Aveiro, Portugal, but diversity was unaffected by bioavailable Pb (Martins et al.. 2011). The
concentrations of Pb in the sediment in the resistant mineralogical phase, adsorbed by clay minerals, and
associated with OM ranged from about 20 mg Pb/kg to 180 mg Pb/kg. There was a positive correlation
between total bioavailable concentrations of Pb in the sediment (the fraction absorbed by clay and OM
and coprecipitated with carbonates) and the abundance of miliolids, and bioavailable Pb was not
significantly correlated with the abundance of Ammonia tepida, Bulimina spp., Bolivina spp., Haynesina
germanica, Elphidium spp., agglutinated spp., and Shannon diversity index. CCA indicated that miliolids
and agglutinated species were correlated with Pb and Al. Principal components analysis suggested that
higher bioavailable concentrations of Pb in addition to As, Cd, Cu, Ni, and Zn generally lead to less
diverse foraminifera communities and that the agglutinated foraminifera and miliolids were more tolerant
to Pb than other taxa examined. The authors noted that agglutinated foraminifera and miliolids were
typically concentrated near the lagoon mouth where Pb concentrations were higher.
In another example, Pb was negatively correlated with the abundance of certain foraminiferal
taxa, but not to diversity metrics in the San Jose Bay estuary, Puerto Rico (Martinez-Colon et al.. 2018).
Sediment Pb concentration ranged from 2-38 mg Pb/kg in the lagoon. Pb was significantly negatively
correlated with the relative abundance of Amphistegina gibbosa, Archaias angulatus, Asterigerina
carinata, Discorbis, Elphidium crispum, Heterostegina depressa, Miliolinella, Quinqueloculina
agglutinans, and Triloculina bicarinata and positively correlated with the relative abundance of
Triloculina and Quinqueloculina agglutinans. Pb sediment concentration was not significantly correlated
with any of the other foraminiferal abundances or diversity indices such as species diversity, Shannon's
index, Equitability Index, foraminiferal density, or the relative abundances of Ammonia.
Pb enrichment factors were slightly positively correlated w ith Ammonia spp. (Ammonia beccarii,
A. gaimardii, A. tepida, and A. parkinsoniana) and low-oxygen foraminiferal assemblages in the Gulf of
Milazzo, Italy, but not to total deformed foraminifera, foraminiferal density, or the abundance of other
foraminiferal taxa (Cosentino et al.. 2013). Pb concentrations in the sediment ranged from 4.75 to
49.19 mg Pb/kg. Finally, Pb and Al were negatively correlated with foraminiferal abundance across a
gradient of sites in the Strait of Malacca, Malaysia (Minhat et al.. 2020). with Pb showing the greatest
enrichment among all metals, with values ranging from 8.8-29.2 mg Pb/kg. Overall, dissolved oxygen,
depth, Al, and Pb concentrations explained the most variation in foraminiferal species distributions. The
abundance of Ammonia tepida, which was the highest, was not significantly correlated with Pb sediment
concentration, while those of Bulimina marginata, Pararotalia ozawai, and Nonion subturgidum were
negatively correlated with Pb.
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Foraminiferal abundance and diversity were correlated with certain bioavailable Pb sediment
concentrations in Chilika, which is the largest brackish water lagoon in Asia (Barik et al.. 2022). The
concentrations of Pb in the sediment were 68.27 ± 22.14 mg Pb/kg (mean ± S.D.) across 22 sampling sites
(range: 22.14-107.57 mg Pb/kg). Pb was statistically significantly positively correlated to the
concentrations of Co. In addition to Pb concentrations in the sediment, bioavailable fractions of Pb and
other heavy metals were determined. Specifically, Pb in the first fraction is the Pb bound to carbonates,
the second fraction includes Pb bound to FeMn oxides, the third is bound to OM, and the fourth is bound
to silicate. Pb concentration was significantly negatively correlated to the percentage of Pb in the second
(reducible) and third (oxidizable) fractions and positively correlated to the percentage of Pb in the fourth
(residual) fraction. Pb concentration alone was not correlated to the total number of live and dead
abundance, diversity, or species richness, while the percentage of Pb in the first fraction was positively
correlated to the abundance of dead foraminifera per gram sediment and negatively correlated to the
diversity of dead foraminifera. The diversity, measured by the Shannon diversity index, of live and dead
foraminifera was negatively correlated to the percentages of Pb in the second and third fractions and
positively correlated to the percentage of Pb in the fourth fraction. Finally, live and dead foraminiferal
species richness was significantly negatively correlated to the percentage of Pb in the third fraction.
In summary, some mesocosm and observational studies published since the 2013 Pb ISA found
reductions in foraminiferal and meiofaunal community richness, diversity or abundance associated with
higher concentrations of Pb in sediment and water (Barik et al.. 2022; Minhat et al.. 2020; Frontalini et
al.. 2018; Martinez-Colon et al.. 2018). Other studies found positive or null correlations (Barik et al..
2022; Martinez-Colon et al.. 2018; Cosentino et al.. 2013; Martins et al.. 2011).
11.5 References
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