-
Survival time models quantitatively can predict lethal
effects
of pulsed and different duration exposures to water-accommodated
fraction PAH from spilt oil
A Final Report Submitted to
The Coastal Response Research Center
Submitted by
Dr. Michael C. Newman Dr. Michael A. Unger
Department of Environmental and Aquatic Animal Health Virginia
Institute of Marine Science
College of William and Mary P.O. Box 1346
Gloucester Point, VA 23062
Project Period: February 1, 2005 to January 31, 2007
Submitted: August 10, 2007
This project was funded by a grant from NOAA/UNH Coastal
Response Research Center. NOAA Grant Number(s): V710640. Project
Number: 05-956
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Abstract The CRRC 2004 RFP identified the need to quantitatively
predict injury from realistic conditions including pulsed, short
term and long term exposures. The research described herein
addresses this 2004 priority area. Toxicity data derived from
conventional concentration-effect test designs predict effect at a
single exposure time. A few test durations might be used in some
tests to coarsely predict how mortality changes with exposure time.
Even in tests employing several exposure times, the selected
concentration treatments generate optimal data for only one of the
exposure durations. Predictions of effect at different durations
are unavoidably gross because mortality information was not
collected optimally during the entire exposure. Also, the
conventional concentration-effect approach does not quantify
mortality that potentially could occur after exposure stops. Such
post-exposure mortality can be quite high. These shortcomings can
be avoided by noting mortality in test treatments through time
including post-exposure mortality, and applying survival time
modeling to the resulting data. Survival time models allow
inclusion of covariates such as exposure concentration, resulting
in models that include both exposure concentration and duration. We
parameterized survival time models with data generated for six
representative polycyclic aromatic hydrocarbons (PAH) associated
with the water-accommodated fraction of oil. Survival models
incorporating exposure concentration and duration were produced for
the grass shrimp, Palaemonetes pugio, a common test species and an
ecologically important one in salt marshes and other coastal
environments. The absence of post-exposure mortality allowed use of
models based only on mortality during the actual exposure. The
results were then used to demonstrate a QSAR approach to predicting
survival time model parameters for untested PAH. Keywords: PAH,
survival, intermittent exposure, grass shrimp
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Acknowledgments Funding was provided by the NOAA/UNH Coastal
Response Research Center (V710640). Help with animal maintenance
and tests provided by John Carriger and Kyle Tom is much
appreciated.
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Table of Contents
1.0 Introduction …………………………………………………………………………………..6 2.0
Objectives …………………………………………………………………………………….7 3.0 Methods
………………………………………………………………………………………8 4.0 Results
………………………………………………………………………………………..9 5.0 Discussion and Importance
to Oil Spill Response/Restoration ……………………………..14 6.0 Technology
Transfer ………………………………………………………………………...10 7.0 Achievement and
Dissemination ..……………………………………………………….…15 8.0 References
...…………………………………………………………………………………16 Appendices
………………………………………………………………………………………17
Appendix A. ……………………………………………………………………………..18 Appendix B
……………………………………………………………………………...44
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List of Figures and Tables
FIGURES Figure 1. Cumulative mortality for amphipods (Hyalella
azteca) exposed for 48 hours to four copper concentrations (Zhao
and Newman 2004). Substantial post-exposure mortality occurred,
reflecting damage incurred during exposure. Conventional toxicity
test designs and associated metrics do not quantify post-exposure
mortality, leading to the possibility of a downward inaccuracy in
predicted mortality for field exposures. Figure 2. Exposure tanks
used in all shrimp survival time experiments. Figure 3. Survival
curves (bars indicate the estimated standard errors) from the
phenanthrene tests. With the termination of exposure (60 hr),
mortality quickly dropped to a minimum level in all concentration
treatments. Greenwood’s formula was used to produce the standard
errors shown as vertical bars for each exposure time. Figure 4. An
illustration of the ability of survival time models to predict the
mortality with different combinations of exposure time and
concentration. In this figure, contours for percentage of exposed
shrimp expected to die are shown. The 48 hr LC50 estimate is also
shown for comparison. Less than 5% of the mortality during the
entire test was post-exposure mortality. Figure 5. QSAR models for
48 hr LC50 (top panel) and parameter estimates (b1 and b2) for the
survival time models (middle and bottom panel). Figure 6.
Concentration-effect (log normal) model slopes for the six
compounds estimated with the Microtox7 toxicity assay. Brackets
indicate 95% confidence intervals. TABLES Table 1.
Concentration-effect models (48 hr exposure duration)(95% CI = 95%
confidence interval, SE = standard error) Table 2. Parameter
estimates for survival models. (95% CI = 95% confidence interval,
SE = standard error)
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1.0 Introduction
The CRRC 2004 RFP identified the need to quantitatively predict
injury from realistic exposures including pulsed, short term and
long term exposures. The research described here addresses this
priority area, illustrating an approach to combining exposure
duration and concentration into lethality predictions, and
generating information applicable nationwide to oil spills. The
general approach might also be relevant to predicting injury when
confounding factors change through time, e.g., changes in
PAH-induced mortality due to diurnal changes in UV light intensity
or seasonal temperature changes. Most toxicity data are derived
from conventional concentration-effect test designs that produce an
effect metric, such as the 96 hr LC50, at one set exposure time.
Occasionally, a few test durations might be used to coarsely
predict how mortality changes with exposure time. Gross prediction
is inevitable if mortality information was not collected throughout
the exposure. As a further complication in the conventional
concentration-effect approach, mortality potentially occurring
after exposure stops is not considered in predictions. In the few
studies that quantified such post-exposure (latent) mortality, it
was found to vary widely and could be quite high. Mosquitofish
exposed to a concentration of sodium chloride that killed 15% of
exposed fish by 144 hr, experienced an additional 44% mortality in
the days following the pulsed exposure (Newman and McCloskey 1996,
2002). Zhao and Newman (2004) measured high (15 to 35%; copper
sulfate; e.g., Figure 1) to minimal (2 to 5%; sodium
pentachorophenol) latent mortality of amphipods. The magnitude of
latent mortality appeared to depend on the mode of action,
consequent residual damage, and recuperative capacity of the
exposed individual. The conventional approach is limited relative
to predicting all mortality from pulsed exposures differing in
duration from conventional tests. These shortcomings are weighty
impediments to accurately predicting effects from spilt oil
exposures that vary in both duration and concentration through
time, and that require prediction of all mortality resulting from
exposure.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 20 40 60 80 100 120
Prop
ortio
n D
ead
Time (hours)
Exposure Post Exposure
Control
0.2 mg/L
0.3 mg/L
0.4 mg/L
0.6 mg/L
Figure 1. Cumulative mortality for amphipods (Hyalella azteca)
exposed for 48 hours to four copper concentrations (Zhao and Newman
2004). Substantial post-exposure mortality occurred, reflecting
damage incurred during exposure. Conventional toxicity test designs
and associated metrics do not quantify post-exposure mortality,
leading to the possibility of a downward inaccuracy in predicted
mortality for field exposures.
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2.0 Objectives The shortcomings just described can be avoided by
noting mortality in test treatments through time including any
potential post-exposure mortality, and applying survival time
models to the resulting data. We applied survival time methods to
data generated for representative PAH1 associated with weathered
oil. Survival models capable of incorporating exposure
concentration and duration, and post-exposure mortality were
produced using widely accepted methods from health science, social
sciences, engineering, and ecology (Allison 1995, Cox and Oakes
1984, Kalbfleisch and Prentice 1980, Muenchow 1986, Miller 1981,
Nelson 1972). Although underutilized in ecotoxicology, the PI
(Newman and Dixon 1996, Newman and McCloskey 1996, 2002, Zhao and
Newman 2004) and others (Crane et al. 2002, Lee et al., 2002)
applied this approach successfully to other ecotoxicity test data
and risk assessment information. We also began addressing issues
germane to predicting joint effects of the PAH in the WAF mixture.
As described in the references cited above, survival time models
can predict mortality during and after exposures of different
durations and concentrations. Models were developed for the grass
shrimp, Palaemonetes pugio, exposed to key PAH associated with
weathered oil. By conducting toxicity tests with these PAH, we were
later able to incorporate compound qualities (e.g., log Kow) into
predictive models. The selected PAH were 1-ethylnaphthalene,
2,6-dimethylnaphthalene, dibenzothiophene, fluorene, naphthalene,
and phenanthrene. The grass shrimp was chosen because it is a
common test species, and is an ecologically important one in salt
marshes and other coastal environments. Because of the areas it
inhabits, it would also be subject to periodic exposures of a
pulsed nature, e.g., compounds released from deposited weathered
oil during tidal cycles, or varying in exposure concentration and
duration due to spill characteristics or dispersant application.
Specific objectives were the following:
1. Produce predictive survival time models for grass shrimp
exposed to a pulse of six representative PAH in weathered oil WAF.
Models included predictions of pulse and post-pulse mortality under
realistic ranges of exposure duration and concentration. This
objective was met by generating models for each compound. The ease
of model generation was greatly improved by the observation of
minimal post-exposure mortality for shrimp exposed to these
narcotics.
2. Produce survival time models that incorporate molecular
qualities (e.g., log Kow) of the six PAH. Such a model will allow a
level of prediction by interpolation to other untested PAH in spilt
oil. Several chemical properties allowed generation of QSAR models
for conventional LC50 and survival time model parameters. In this
report, we use the conventional lipophilicity metric, log Kow, to
illustrate the QSAR models.
3. Produce predictive survival time models for grass shrimp
exposed to pulses of a PAH mixture. Models include predictions of
pulse and any potential post-pulse mortality at a range of
realistic exposure concentrations and durations. Several
experimental designs were assessed and a final design employing a
mixture of two substituted PAH
1 The term, PAH, is used for clarity throughout this report;
however, one compound (dibenzothiophene) was a heterocyclic
aromatic hydrocarbon.
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8
(ethylnaphthalene and dimethylnaphthalene) and the heterocyclic
aromatic hydrocarbon, dibenzothiophene, was executed. The
difficulties of simultaneously maintaining the dosing
concentrations for these three compounds resulted in concentrations
producing too low mortality in the single compound treatments so
the test of combining effects could not be done with the planned
conceptual approach.
3.0 Methods
Because specific methodological details are provided in the
attached manuscripts (Appendices A and B), only general
descriptions will be provided here. Grass shrimp were exposed
(Figure 2, 26 shrimp/tank) to a series of concentrations of each of
six PAH. The range of concentrations was determined during pilot
studies with the intent of encompassing concentrations above the
threshold of apparent lethal effect and below the PAH solubility in
saltwater. Shrimp were kept in separate glass tubes with free
exchange of exposure tank solutions. Triplicate tanks were randomly
placed on two adjacent water tables for each of five (including
controls) exposure concentration treatments. Tanks were not aerated
to reduce PAH volatilization. Exposure solutions were renewed every
12 hours.
Figure 2. Exposure tanks used in all shrimp survival time
experiments. At death or the end of the experiment, shrimp were
removed from tanks, weighed, and frozen. Body concentrations of the
exposure PAH were determined in subsets of shrimp with the intent
of estimating body burdens upon death, and fortuitously, estimating
elimination rate constants during the post-exposure period.
Exposure data for each PAH were fit to a log logistic survival
model,
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)()(lnln 321 WbionConcentratbbDeathtoTime ++=−− (1) where b1 =
the model intercept parameter estimate, b2 = model parameter
estimate for the effect of PAH concentration, and b3 = a scaling
parameter estimate. The W is associated with a specific proportion
dying. In this case of a log logistic model, W is the log odds,
i.e., ln(P/(1-P)) where P is the proportion of exposed individuals
dying. As an example, W for predicting median time-to-death (P=0.5)
produces a W = ln(0.5/(1-0.5) = 0. The units of concentration are
ug/L. Under the intense and relatively brief exposure conditions,
the mode of action for all six compounds was judged to be general
narcosis. Consequently, one could begin by assuming that models for
joint similar action could be applied. Such models assume a common
slope for all similar acting compounds so that the proportion of
exposed individuals dying during exposure to a specified mixture of
these compounds can be estimated with the separate probit models
for the individual toxicants:
)(log)(Pr AAA ionConcentratSlopeInterceptPobit += (2)
)(log)(Pr BBB ionConcentratSlopeInterceptPobit += (3)
For the conventional concentration-effect design, the log of
relative potency can be calculated.
SlopeInterceptInterceptLog ABB
−=
(ρ (4)
The combined effect of two similar acting toxicants (PA+B) would
then be predicted as the following,
))(log(log BBAABA ionConcentrationConcentratSlopeInterceptP
ρ++=+ (5) The assumption can be assessed by projecting this
reasoning to survival time models that similar acting PAH should
have similar b2 values. Also the survival time models for each PAH
in a mixture could be used together to predict the total proportion
dying at any time.
4.0 Results The survival time tests for the six PAH produced 48
hr LC50 estimates in addition to parameterized survival time models
(Tables 1 and 2). The conventional concentration-effect model (with
natural mortality) used to estimate 48 h LC50 values was the
following:
)()1( 1021 ionConcentratLogaaPPP BBDead +Φ−+= (6)
where PDead = predicted proportion dying by 48 h, PB = estimated
proportion dead at 48 h due to background mortality, a1 and a2 =
estimated model intercept and slope parameters, and M() = the
normal cumulative distribution function.
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Table 1. Concentration-Effect Models (48 hr exposure duration).
(95% CI = 95% confidence
interval, SE = standard error)
The 48 hr LC50 estimates and associated 95% confidence intervals
are provided in the left side of Table 2. These 48 H LC50 estimates
were consistent with the literature as described below in
discussions of QSAR models. The background mortality in the
exposure experiments averaged 4%: roughly 1 of 25 shrimp that died
in an exposure tank died due to nontoxicant-related reasons during
the 48 to 60 hour experiments. Table 2. Parameter estimates for
survival time models (95% CI = 95% confidence interval, SE =
standard error)
Initial survival modeling was designed to either model survival
during exposure, or survival during and after (48 h post-exposure)
exposure if warranted. Because of the narcotic mechanism for action
at these PAH concentrations and the test durations, latent effects
were minimal. This permitted the most parsimonious modeling to
occur: latent mortality was minimal for the
Compound PB (SE) a1 (SE) 95% CI a2 (SE) 95% CI Naphthalene
0.01
(0.01) -60.3 (8.2)
-76.5 - -44.2 18.2 (2.5)
13.3 – 23.1
Fluorene 0.03 (0.02)
-33.3 (3.6)
-40.3 – -26.2 11.9 (1.3)
9.4 – 14.4
Dibenzothiophene 0.06 (0.02)
-35.4 (5.3)
-45.8 – -25.0 14.8 (2.2)
10.6 – 19.1
Ethylnaphthalene 0.06 (0.05)
-31.4 (10.7)
-52.4 – -10.4 12.7 (4.2)
4.4 – 21.0
Dimethylnaphthalene 0.06 (0.03)
-20.4 (3.6)
-27.5 – -13.8 7.6 (1.3)
4.9 – 10.2
Phenanthrene 0.02 (0.01)
-14.5 (2.4)
-19.2 – -9.8 5.7 (1.0)
3.8 – 7.5
Compound LC50 (µg/L)
95% CI
b1 (SE) 95% CI
b2 (SE)
95% CI
b3 (SE) 95% CI
Naphthalene 2111 2057-2161
52.2 (2.1)
48.0-56.3
-6.3 (0.3)
-6.8- -5.8
0.64 (0.03)
0.60-0.70
Fluorene 616 593-637
22.1 (1.6)
18.9-25.2
-2.8 (0.2)
-3.3- -2.3
0.41 (0.02)
0.37-0.46
Dibenzothiophene 242 228-253
17.4 (0.6)
16.1-18.7
-2.4 (0.1)
-2.6- -2.2
0.31 (0.02)
0.28-0.35
Ethylnaphthalene 295 162-331
15.3 (1.0)
13.4-17.3
-2.0 (0.2)
-2.4- -1.7
0.26 (0.01)
0.23-0.29
Dimethylnaphthalene 500 463-535
14.9 (1.2)
12.5-17.3
-1.8 (0.2)
-2.2- -1.4
0.34 (0.03)
0.30-0.40
Phenanthrene 360 333-402
13.0 (1.1)
10.7-15.2
-1.5 (0.2)
-1.9- -1.2
0.38 (0.03)
0.33-0.45
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modeled toxicant exposure scenarios and only mortality during
exposure required estimation. Figure 3 demonstrates this using the
phenanthrene survival data.
00.10.20.30.40.50.60.70.80.9
0 20 40 60 80 100
Hours
Pro
porti
on d
ead
153 µg/l
224 µg/l
302 µg/l
389 µg/l
Post-exposureExposure
Figure 3. Survival curves (bars indicate the estimated standard
errors) from the phenanthrene tests. With the termination of
exposure (60 hr), mortality dropped quickly to a minimum level in
all concentration treatments. Greenwood’s formula was used to
produce the standard errors shown as vertical bars for each
exposure time. The models defined in Table 1 predict the amount of
mortality expected with a specified pairing of exposure
concentration and duration as illustrated for phenanthrene in
Figure 4. The first objective of the project was completed
successfully with the generation of these models.
TTD PHEN = e12.9659 e- 1.5409 ln C e0.3828W0
10
20
30
40
50
60
300 350 400 450 500 550 600
Water concentration (µg/L)
Tim
e to
dea
th (h
ours
)
5%10%
30%50%
70%
90%
Figure 4. An illustration of the ability of survival time models
to predict the mortality with different combinations of exposure
time and concentration. In this figure, contours for percentage of
exposed shrimp expected to die are shown. The 48 hr LC50 estimate
is also shown for comparison. Less than 5% of the mortality during
the entire test was post-exposure mortality.
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The second goal of assessing whether QSAR models could be
generated for these data was also successfully achieved (see Figure
5).
y = 4E+06e-2.1064x
R2 = 0.9379
1
10
100
1,000
10,000
100,000
2 2.5 3 3.5 4 4.5 5
LC 5
0 (u
g/L)
Unger et al, in prep
Tatem, 1975
Unger et al, 2007
y = -33.211x + 162.63
0
10
20
30
40
50
60
2 2.5 3 3.5 4 4.5 5
Surv
ival
Mod
el E
stim
ate
b 1
Unger et al, in prep
Unger et al, 2007
y = -3.9292x + 19.381
0
1
2
3
4
5
6
7
2 2.5 3 3.5 4 4.5 5
Log KOW
Sur
viva
l Mod
el E
stim
ate
b 2
Unger et al, in prep
Unger et al, 2007
Figure 5. QSAR models for estimates of the 48 hr LC50 (top
panel) and survival time model parameter estimates (b1 and
b2)(middle and bottom panel). The LC50 data generated from this
study were consistent with those of Tatem (1975) (Figure 5, top
panel). Toxicity increased for these oil-related PAH with
increasing lipohilicity (as quantified with the log Kow).
Similarly, the decrease in survival model parameter estimates (b1
and b2) with increasing lipophilicity indicates that time-to-death
decreases with increasing
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lipophilicity. The ability to accurately predict survival time
model parameters based on data from these six compounds could be
greatly enhanced by adding estimates for more key PAH (3.5
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intent of the authors to continue exploring alternative models
to better analyze these data, e.g., a survival model using all data
and log Kow values for the six compounds to predict survival during
exposure to mixtures. Another option is to combine the relative
potency approach embedded in Equations 4 and 5 for the
concentration-effect approach into survival time models. Relative
potencies would be included and the resulting predictions compared
to those generated during the three-compound mixture experiment. A
final option is to explore the models described by Olmstead and
LeBlanc (2005) and Barata et al. (2005).
5.0 Discussion and Importance to Oil Spill
Response/Restoration
The survival time modeling approach was successful in that
models were produced that can be applied immediately to predict
lethal consequences of PAH exposures differing in concentration and
duration. Such a predictive capacity extends well beyond that
afforded by the conventional LC50 approach. It is also needed to
make predictions about realistic exposures that will vary in
concentration and duration. Clearly, post-exposure mortality can be
ignored during general predictions. This statement is based on the
minimal post-exposure mortality seen in the six compound toxicity
exposure tests. This greatly simplifies modeling and consequent
predicting of lethal consequences. The minimal post-exposure
mortality experienced by shrimp exposed to these general narcotics
is similar to that reported for mosquitofish exposed to sodium
pentachlorophenol, a toxicant with a reversible mode of action
(uncoupling of oxidative phosphorylation). The minimal
post-exposure contrasts with that associated with sodium chloride
exposed mosquitofish (Newman and McCloskey 2000) or the
copper-exposed amphipods shown in Figure 1 that experienced
extensive damage during exposure. The extensive damage required
significant time and energetic resources to repair. Like sodium
pentachlorophenol, the six compounds used in these tests also were
eliminated very quickly when shrimp were moved to clean water (see
Appendix A for elimination rate constants). Likely, this also
contributed to the rapid recovery and minimal post-exposure
mortality. In the absence of direct evidence for each relevant PAH,
it is a reasonable assumption at this point that minimal
post-exposure mortality will occur for untested PAH components
under exposure scenarios in which narcosis is the dominant mode of
lethal action. Relative to a sequence of pulsed exposures, one
additional factor emerges as relevant, the individual tolerance or
individual effective dose theory (Zhao and Newman 2007). Briefly,
this theory holds that an individual has a unique or characteristic
dose (or concentration) below which it will survive but at or above
which it will die: the distribution of effective
doses/concentrations are assumed to be log normally distributed in
populations of exposed individuals. This theory is pervasive in the
ecotoxicology literature but it remains poorly tested. The
alternative, stochasticity theory (Newman and McCloskey 2000)
assumes that all individuals are equally sensitive and which die
during an exposure is a matter of chance. The stochastic process
can be modeled with a skewed (e.g., log normal) distribution. With
repeated exposures as might happen with a tide-dominated movement
of PAH into and out of a marsh, which theory is correct becomes
important to predictions of population consequences (Zhao and
Newman 2007). Based on the research of Zhao and Newman (2007), and
Newman and McCloskey (2000) the conservative assumption of the
stochastic theory seems most advisable at this time. The
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methods of Zhao and Newman (2007) can also be applied to
assessing whether shrimp sensitivity to the lethal effects of PAH
exposure changes significantly with repeated exposures.
6.0 Technology Transfer
The results of this study were presented annually at NOAA CRRC
meetings and, upon request, at NOAA-related workshops. For example,
three presentations were made in 2006 at NOAA’s request. One
(August 15-16) was given at the CRRC PAH Toxicity Summit in
Seattle, WA. Another was given in September at the CRRC Coastal
Modeling for Decision Makers meeting in Durham, NH. A final
presentation was made at another Durham meeting, CRRC Submerged Oil
Workshop (December). These opportunities allowed for researchers
and oil spill responders to interact and discuss the utility and
applications of the CRRC research projects. Much discussion at the
Toxicity and Modeling workshops was centered on time dependence as
a factor influencing PAH toxicity and how dispersants and physical
factors may affect the exposure duration and concentration of
toxicants in the water-soluble fraction from oil. The time to death
approach of evaluating PAH toxicity was of great interest to
managers as a mechanism to better understand and predict population
mortality under changing conditions to help facilitate oil spill
response. Correctly so, these models were also seen as a viable
approach to couple chemical fate and toxicity models, a long-term
goal of NOAA oil spill responders.
7.0 Achievement and Dissemination Beyond the contributions to
NOAA sponsored workshops, the results of this study have been
disseminated in a variety of formats including national and
international meetings, and peer-reviewed publication. Presentation
at national and international (The Hague, The Netherlands) meetings
provided wide exposure of the environmental science community to
the techniques and results. Written transfer of the study
techniques and findings was achieved primarily in the peer-reviewed
literature. The first part of the study was published in 2007
(Unger et al. 2007) as cited in the reference section of this
report. The second part of the research project is appended to this
final report as an “in preparation” manuscript that will soon be
submitted to the international journal, Environmental Toxicology
and Chemistry. The third part of the study involving mixture
effects requires further analysis and possibly more
experimentation. Although it is difficult to anticipate the
outcome, the results will minimally be presented at Society of
Environmental Toxicology and Chemistry annual meetings in
combination with the rest of the CRRC-funded study.
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16
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2147-2153.
Zhao, Y. and M.C. Newman. 2006. Effects of exposure duration and
recovery time during pulsed exposures. Environ. Toxicol. Chem.
25:1298-1304.
Zhao, Y. and M.C. Newman. 2007. The theory underlying
dose-response models influences predictions for intermittent
exposures. Environ. Toxicol. Chem. 26:543-547.
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17
Appendices
Appendix A. Unger, M.A., M.C., Newman, and G.G. Vadas. In prep.
Predicting survival of grass shrimp (P. pugio) for naphthalene,
fluorine and dibenzothiophene exposures differing in concentration
and duration. Environ. Toxicol. Chem. Appendix B. Unger, M.A., M.C.
Newman, and G.G. Vadas. 2007. Predicting survival of grass shrimp
(P. pugio) during ethylnaphthalene, dimethylnaphthalene, and
phenanthrene exposures differing in concentration and duration.
Environ. Toxicol. Chem. 26: 528-534.
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18
PAH TOXICITY TO GRASS SHRIMP
Michael A. Unger
Department of Environmental and Aquatic Animal Health
Virginia Institute of Marine Science
College of William and Mary
P.O. Box 1346, Gloucester Point, VA 23062-1346, USA
804-684-7187
804-684-7793 FAX
[email protected]
Total words: 4,535
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19
PREDICTING SURVIVAL OF GRASS SHRIMP (P. pugio) EXPOSED TO
AROMATIC
COMPOUNDS DERIVED FROM OIL
Michael A. Unger*, Michael C. Newman, and George G. Vadas
*Virginia Institute of Marine Science, College of William and
Mary, P.O. Box 1346, Gloucester
Point, VA 23062-1346, USA
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20
* To whom correspondence may be addressed ([email protected])
This paper is contribution XXXX of the Virginia Institute of
Marine Science, College of
William and Mary.
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21
Abstract- The composition and persistence of dissolved
polycyclic aromatic hydrocarbons
(PAH) released to the water column during oil spills are altered
by weathering, tidal transport
and the addition of dispersants. Conventional toxicity effect
metrics such as the LC50 are
inaccurate predictors of mortality from all toxicant exposure
duration/concentration
combinations likely to occur during spills. In contrast,
survival models can predict the
proportions of animals dying as a consequence of exposures
differing in durations and
intensities. Extending previous work with ethylnaphthalene,
dimethylnaphthalene and
phenanthrene, survival time models were developed that include
exposure duration and
concentration to predict time-to-death for grass shrimp,
Palaemonetes pugio, exposed to two
additional PAH (naphthalene, fluorene) and a heterocyclic
aromatic hydrocarbon
(dibenzothiophene). Preliminary explorations of these models
confirmed that QSAR models
were possible for predicting survival model parameters from
compound characteristics.
Conventional 48h LC50 values were also calculated for the
compounds and were combined with
published LC50 values to predict relative PAH toxicity to P.
pugio based on octanol:water
partitioning.
Keywords- Survival analysis, Oil spill, Polycyclic aromatic
hydrocarbons, Toxicity, Grass
shrimp
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22
INTRODUCTION
Tides, currents, weathering, and dispersant application alter
both the exposure
concentration and duration an organism is exposed to
water-soluble compounds from an oil spill
[1-3]. Conventional toxicity tests use a concentration-effect
design to produce a metric such as
an LC50 at a set exposure time, e.g., 96 h, that is not well
suited to predict toxic effects for
varied exposure scenarios. Although underutilized by
environmental toxicologists, survival
models can include both exposure duration and concentration.
Survival models have been
applied to predict the effects of a variety of contaminants
[4-8] and potentially provide managers
a tool with which to make more effective response and
remediation decisions about the potential
impacts from spills. In addition to predicting mortality under
varying exposure time and toxicant
concentrations, the models are easily expanded to include
post-exposure (latent) mortality effects
or other cofactors, such as organism size or sex, temperature or
salinity [9].
The main objectives of this study were to add three more
parametrized models to the
three built earlier [8] and then explore the possibility of
building QSAR models for survival
model parameters. QSAR interpolation would permit prediction for
exposures to untested oil
spill-related aromatic compounds. Exposed shrimp were also
analyzed post mortem for
contaminant body burdens to determine if accumulated
contaminants were consistent with a
critical body burden at time of death.
Acute toxicity experiments were done with the grass shrimp,
Palaemonetes pugio,
exposed to two PAH (naphthalene, fluorene) and the heterocyclic
aromatic compound
dibenzothiophene. Combined with the previous models for
dimethylnaphthalene,
ethylnaphthalene and phenanthrene [8], the resulting six models
represent the range of aromatic
compounds thought to be important contributors to the toxic
effects of oil spills [2]. Results from
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23
these exposure experiments were modeled with survival time
methods [10] to produce models
for each compound that predicted the proportion of individuals
dying during, and potentially,
after an exposure of specified duration and concentration.
Survival models for six compounds,
representative of those found in the water soluble fraction
derived from oil, were used to
examine the relationship between the toxicant octanol:water
partitioning behavior and its
toxicity.
MATERIALS AND METHODS
Grass shrimp collection and maintenance
Grass shrimp collected locally from the York River (Virginia,
USA) were maintained in
the laboratory in filtered York River water (salinity 19-20
0/00) for at least 2 weeks prior to being
used in the exposures. Shrimp were fed Tetramin® Tropical flake
food (Tetra Holding,
Blacksburg, VA) daily. Individual shrimp with no outward signs
of damage or disease were
gently placed into glass tubes and the exposure aquaria one day
before exposures began.
PAH survival analysis experiments
The survival analysis experiments were conducted in August
(naphthalene 1 and
dibenzothiophene), September (fluorene), and October
(naphthalene 2) of 2006. The first
naphthalene exposure resulted in more mortality than anticipated
by 24 hours so an additional
exposure experiment was conducted in October at lower
concentrations. After graphical
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24
comparison for consistency, the data from both tests were
combined to develop a single
naphthalene survival model and to estimate the 48 h LC50. For
each experiment, three replicates
of four PAH concentrations and a control were prepared from
saturated solutions generated with
filtered York River water by techniques described in detail
previously [8]. Briefly, a 7.5 cm x
59.2 cm generator column was used to produce the large volume of
saturated PAH solutions
required to avoid the use of solvent carriers for the
experiments.
To minimize toxicant volatilization, exposure chambers were not
aerated during the experiment
and solutions were renewed every 12 h. All experiments were
conducted under constant light
from standard fluorescent light fixtures that were approximately
1.5 m above aquaria.
Experimental chambers were constructed from 25 cm x 50 cm x 58
cm glass aquaria with glass
lids and were randomly assigned to positions on a wet table. A
water-tight glass partition was
installed down the center of each aquarium to create tandem, 30
L exposure chambers 25 cm x
25 cm x 58 cm tall. This design reduced the surface area to
volume ratio to minimize volatile
losses of the PAH. One chamber of each aquarium was used to
expose the test organisms and the
other was used to prepare the new test solution every 12 h. The
tandem 30 L test chambers
permitted the filling of one side with new exposure water and
transferring of the shrimp in racks
from old to renewed water with minimal disturbance to the test
organisms.
Individual grass shrimp (26 shrimp/replicate) were placed in 2.8
cm x 10.8 cm (40 ml)
glass vials with an open ended screw cap fitted with a stainless
steel mesh screen. Shrimp were
not sexed but berried females were excluded from the tests. The
vials were suspended in
randomly assigned exposure chambers on aluminum racks to
facilitate monitoring the test
organism’s condition and to allow easy transfer of the test
organisms to newly prepared solutions
every 12 h. Shrimp were monitored for mortality every 4 h and
scored as dead if unresponsive to
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25
repeated prodding and no appendage movement was apparent. All
dead shrimp were removed,
weighed, and frozen. Shrimp surviving the exposure period
(naphthalene 1, 24 h; naphthalene 2,
36 h; fluorene 60 h; and dibenzothiophene, 48 h) were
transferred to filtered and aerated York
River water. The water was renewed every 12 h and shrimp were
routinely monitored for post-
exposure mortality for 48 h, or until no further mortality was
apparent. All shrimp were weighed
and frozen at the completion of the test (48 h
post-exposure).
Water chemistry
Old and fresh test solution water chemistries were measured
every 12h and included
temperature, dissolved oxygen, salinity, and pH. A HydrolabTM
Surveyor 4a (Hydrolab Corp.,
Austin, TX) was used for these measurements. Unfiltered water
samples were also collected and
frozen for ammonia analyses (phenol method).
PAH Analysis of water and tissue samples
Exposure waters were analyzed for PAH concentrations using a
HPLC with a
fluorescence detector by methods described in detail previously
[8]. Briefly, calibration
standards were made in York River water from stock solutions of
internal standard (1-methyl
naphthalene) and the PAH analyte of interest. Calibration
standards were injected on the HPLC
and the method calibrated for the specific analyte over a range
from its quantitation limit to near
its aqueous solubility prior to running samples. The HPLC
calibration was verified again with
fresh standards once the analyses for a particular exposure were
completed. Shrimp tissues were
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26
analyzed by GC-MS using selective ion monitoring (GCMS-SIM).
Individual shrimp were
weighed, rinsed with DI water and placed into a 50 mL Teflon
centrifuge tube containing 2.0 mL
of concentrated HCL and 500 ng of deuterated PAH surrogate
standards. The shrimp were
homogenized with a spatula and ultrasonicated for 10 m. The
aqueous homogenate was
sequentially extracted with two aliquots of hexane (2.0 mL
each), centrifuging between
extractions to separate the layers. The combined hexane extracts
were reduced to 0.1 mL under
dry nitrogen and 0.6 ug of p-terphenyl internal standard was
added before analysis on a Varian
CP-3800 Gas Chromatograph with a Saturn 2000 GC/MS/MS ion trap
mass spectrometer
operated in electron ionization mode (EI). Analytes and ions
monitored were: p-terphenyl I.S.
[152+230], naphthalene-d8 [108+135+136], acenaphthene-d10
[160-165], phenanthrene-d10
[187-189], naphthalene [128+127+102], fluorene [163-166] and
dibenzothiophene [184+139].
Six point calibration curves were generated for each analyte and
identifications were based on
retention time and matches to library spectra. Shrimp tissue
concentrations were corrected for
surrogate recovery relative to the internal standard
(p-terphenyl). Surrogate standards were
naphthalene (naphthalene-d8), fluorene (acenaphthene-d10), and
dibenzothiophene
(phenanthrene-d10).
Calculating LC50 values
The measured PAH exposure concentrations, number of dead shrimp
at 48 h and total
exposed shrimp were fitted by maximum likelihood estimation
(MLE) to a log normal model
with the PROBIT procedure in the SAS software (SAS Corp., Cary,
NC). Spontaneous mortality
was included in the log normal model because low levels of
mortality (≤ 6%) occurred in the
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27
control aquarium shrimp during exposures. The 48 h LC50 values
and associated 95% fiducial
limits were estimated.
Survival models
Survival time was modeled as a function of PAH concentration
using mean
concentrations in each exposure tank and the SAS LIFEREG
procedure (SAS Corp., Cary, NC).
The general approach was that described in detail in previous
publications, e.g., [8, 10]. Survival
times noted at 4 h intervals were used directly in the model
instead of incorporating interval
censoring because the fineness of the sampling used in these
experiments minimized any
inaccuracies arising from the discreteness of the sampling [10].
The log logistic model was
chosen to predict survival based on exposure concentration (see
[8] for details of model
selection),
TTD e e eb b Concentration b W= 1 2 3(ln )
where TTD = the predicted time-to-death for a specified
proportion of the exposed shrimp, b1 =
intercept (MLE estimate) , b2 = the coefficient for the
influence of ln of PAH concentration on
time-to-death (MLE estimate), b3 = the scale parameter (MLE
estimate), and W = the response
metameter for the model distribution associated with the
proportion dying (P) of the exposed
shrimp for which prediction is being made. The W can be
generated by special functions within
most statistical or spreadsheet software, or taken from tables
such as Appendix Table 7 in [9].
By changing W, the various combinations of exposure
concentration and duration can easily be
found that result in P of the exposed shrimp dying. However,
prediction is only recommended
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28
within the range of concentrations and durations used in the
tests from which these data were
generated.
RESULTS
Water chemistry
Temperature, salinity, dissolved oxygen, pH and ammonia were
monitored during the
three exposure experiments for both freshly prepared solutions
and 12 h-exposed test solutions.
Measured parameters remained within a narrow range for all
exposure experiments. During the
two naphthalene exposure experiments temperature in the various
treatments averaged 19.6-21.8
°C, salinity 18.8-21.2 ppt, dissolved oxygen 6.8-7.2 mg/L, pH
7.76-7.95, and ammonia 0.14-0.17
mg/L. During the fluorene exposure experiment temperature
averaged 19.4-20.6 °C, salinity
18.4-19.8 ppt, dissolved oxygen 6.7-8.7 mg/L, pH 7.8-8.9, and
ammonia 0.14-0.90 mg/L. During
the dibenzothiophene exposure experiment, temperature averaged
20.7-21.2 °C, salinity 21.3-
21.7 ppt, dissolved oxygen 6.6-7.1 mg/L, pH 7.6-7.7, and ammonia
0.14-0.17.
Toxicant concentrations
The measured toxicant concentrations are summarized in Table 1.
Saturated solutions
prepared by the generator column technique had the following
mean concentrations: naphthalene
20,100 ug/L, fluorene 884 ug/L and dibenzothiophene 504 ug/L.
These values are approximately
50-60% of the published values for the same compounds in fresh
water at 25 oC [11, 12]. The
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29
decreased solubility at lower temperature (20 oC) and high
salinity (20 ppt) is expected based on
results with similar PAH and conditions [8, 13]. The PAH
concentrations varied because they
were measured in both the newly prepared and the 12 h old
solutions. Although tanks were not
aerated in order to minimize surface exchange losses, there was
still some decrease of the lightest
compound, naphthalene. Biodegradation during the 12 h period
might have contributed because
the rate of loss increased as the experiment progressed. Control
PAH concentrations were below
detection limits (1 ug/L) during all exposures.
Survival analysis
Shrimp rapidly revived when placed in clean water and there was
very little apparent
latent mortality. Conventional LC50 values and 95% fiducial
limits were calculated,
naphthalene 2111 µg/L (2057-2161), fluorene 616 µg/L (593-637)
and dibenzothiophene 242
µg/L (228-253). Survival data were fitted to accelerated failure
time models with the log logistic
distribution [8]. Contours were developed from the models to
depict shrimp mortality for various
combinations of exposure times and concentrations (Figure
2A-2C). Compound-specific models
and conventional LC50 values with 95% fiducial limits are also
included in Figure 2. Estimated
values for model parameters (b1,b2,b3) for each compound tested
are presented in Table 2 along
with those from previous work with three other PAH [8].
Tissue concentrations Toxicant concentrations (wet weight) were
measured in select whole shrimp that died
during the exposure experiments. Body burden concentrations at
time of death spanned a
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30
relatively wide range during each experiment. Mean
concentrations are presented in Table 3. The
large range in concentrations was, in part, likely due to
increasing body burdens with increasing
dose. Tissue concentrations measured in individual shrimp from
the 300 µg/L and 500 µg/L
dibenzothiophene treatments are presented in Figure 2 for
comparison. There is a trend of
increasing body burdens with increasing time of exposure and
with increasing exposure
concentration.
Naphthalene and fluorene were eliminated rapidly from the shrimp
after the exposures
ended. The dibenzothiophene was two-fold slower than the PAH to
decrease in concentration. In
our previous study [8], phenanthrene body burdens were measured
in exposed shrimp that died
during the depuration phase and they showed an exponential
decrease in concentration with time.
Based on this earlier work with similar compounds, we assumed a
first order decrease and
calculated elimination rate constants for each compound from the
slope of the log transformed
concentration data. The elimination rate constants are shown in
Table 3 with PAH data from the
previous study [8] included for comparison. Corresponding
half-lives for the six compounds
ranged from 3.7 -17 h.
DISCUSSION
Conventional LC50 values calculated for P. pugio were
naphthalene 2111 µg/L (2057-
2161), fluorene 616 µg/L (593-637) and dibenzothiophene 242 µg/L
(228-253). Tatem [14]
reported a 48 h LC50 concentration of 2600 µg/L (2340-2890) for
P. pugio exposed to
naphthalene that is in general agreement with our results for
naphthalene (2111 µg/L). There was
a trend of increasing toxicity with increasing molecular weight
or lipophility. While individual
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31
PAH toxicity data for P. pugio are not numerous in the
literature, enough data were available
from two additional studies [8, 15] to examine the relationship
between octanol-water
partitioning (Kow) and LC50 for the grass shrimp (Figure 3a).
Data include LC50 values for
diverse compounds ranging form the single ring aromatic
compound, benzene, to the three ring
compound, phenanthrene, and includes alkylated PAH as well as
the heterocyclic compound,
dibenzothiophene. The results indicate good relationship between
log Kow and LC50 over a range
of log Kow that includes the most abundant aromatic components
found in the water-soluble
fraction of oil. Similarly, we looked at the relationship
between log Kow and the estimates of the
survival model parameters (b1,b2) (Figure Xb, Xc). While
survival data is currently too limited to
develop a robust relationship, the available data suggests that
a similar approach could be used to
develop models for untested PAH compounds. Due to the increased
predictive capability of
survival models over standardized LC50 values, additional work
is warranted to develop the
QSAR approach further.
The PAH concentrations measured in whole shrimp at time of death
showed a great deal
of variability and appears dependent on exposure concentration
and duration. This is consistent
with the results from our earlier study examining the acute
toxicity of dimethylnaphthalene,
ethylnaphthalene and phenanthrene to grass shrimp [8]. The high
toxicant concentrations and
short exposure durations used in these acute experiments likely
contribute to the variability in
body burdens at time of death. Lower doses with long-term
exposures result in steady state body
burdens and less variance in tissue concentrations at time of
death. Landrum et al [16] have
shown that critical body residues of various PAH are similar on
a molar basis (7.5 ± 2.6 µmol g-
1) when amphipods (Diporia sp.) were exposed for 28 days. When
the mean lethal body burden
concentrations for P. pugio are compared on a molar basis there
is a range of critical body
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32
residues from 0.41 -1.2 µmol g-1 (Figure 4). There is much
better agreement for the four
unsubstituted compounds tested (1.0 ± 0.19 µmol g-1) and this
may be the result of the higher
toxicity of akylated PAH resulting in lower body burdens at time
of death
Unlike the single LC50 value, the survival time models developed
here can predict what
proportion of an exposed population would be killed for
different exposure scenarios generated
from oil spill field measurements or computer simulations. The
toxicants tested were eliminated
rapidly from the organisms post-exposure and little
post-exposure mortality was observed,
allowing simplified survival models that just included mortality
occurring during the exposure
period. Additional work is needed to better understand how the
sub-lethal narcotic effects of
PAH exposure can affect survival of prey organisms due to
increased predation under natural
conditions. The acute toxicity (LC50) of PAH to P. pugio is
proportional to the log Kow of the
compound and provides the ability to predict, by interpolation,
the toxicity of untested
compounds which may be present in the water soluble fraction
derived from oil. Preliminary data
suggests that survival models can also be constructed for
untested compounds based on their
partitioning behavior.
ACKNOWLEDGMENT
We thank John Carriger, Kyle Tom, Chris Prosser, J. Greene, and
E. Harvey, for laboratory
assistance. Funding for this research provided by the Coastal
Response Research Center
(www.crrc.unh.edu).
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33
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8. Unger MA, Newman MC, Vadas GG. 2007. Predicting survival of
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dimethylnaphthalene, and phenenathrene
exposures differing in concentration and duration. Environ
Toxicol Chem 26:528–534.
9. Newman MC. 1995. Quantitative Methods in Aquatic
Ecotoxicology. CRC/Lewis
Publications, Boca Raton, FL, USA. 137-153.
10. Dixon, PM and MC Newman. 1991. Analyzing toxicity data using
statistical models for
time-to-death: an introduction. In: Newman MC and McIntosh AW
(Eds.). Metal
Ecotoxicology. Concepts & Applications. CRC/Lewis
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242.
11. Lide DR. 2005, CRC Handbook of Chemistry and Physics,
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http://www.hbcpnetbase.com, CRC Press, Boca Raton, FL, USA.
12. Pearlman, RS, Yalkowsky, SH, Banerjee S. 1984. Water
solubilities of polynuclear aromatic
and heteroaromatic compounds. J. Phys. Chem. Ref. Data, Vol. 13,
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13 . Gordon JE, Thorne RL. 1967. Salt effects on non-electrolyte
activity coefficients in mixed
aqueous electrolyte solutions-II. Artificial and natural sea
waters. Geochem Cosmochem Acta
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14. Tatem, HE, Anderson JW. 1973. The toxicity of four oils to
Paleomonetes pugio (Holthuis)
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15. Tatem, HE. 1975. Ph.D. Thesis, Texas A&M University,
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16. Landrum, PF, Lotufo GR, Gossiaux DC, Gedeon ML, Lee JH.
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36
Figure legends
Figure 1. Response surfaces predicting mortality levels for P.
pugio exposed to two polycyclic
aromatic hydrocarbons (PAH), A) naphthalene, B)fluorene, and the
heterocyclic C)
dibenzothiophene. The survival models from which predictions are
made are also given in the
figure. Lines indicate different proportions dying predicted
with the models for different
combinations of exposure concentration and duration. The 48-h
median lethal concentrations
(LC50) and the 95% fiducial limits are shown for comparison.
TTD= time to death,
NAP=naphthalene, FLUOR=fluorene, DBT=dibenzothiophene.
Figure 2. Individual whole body concentrations (µg/g wet weight)
of dibenzothiophene measured
in P. pugio at time of death. Shrimp from two exposure
concentrations (300 µg/L and 550 µg/L)
are shown for comparison.
Figure 3. Acute toxicity of PAH to P. pugio predicted from
compound log Kow, A) LC50, B)
survival model parameter b1= the maximum likelihood estimation
(MLE)-estimated intercept, C)
survival model parameter b2 = the estimated coefficient for the
influence of ln of PAH
concentration on time-to-death.
Figure 4. Mean molar body burden concentrations measured in P.
pugio at time of death.
Average unsubstituted compound concentrations (1.0 ± 0.19 µmol
g-1) compared to akyl-
substituted PAH. NAP=naphthalene, FLUOR=fluorene,
DBT=dibenzothiophene,
PHEN=phenenthrene, DMN=dimethylnaphthalene,
ENAP=ethylnaphthalene.
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37
Table 1. Measured toxicant concentrations in exposure
aquariaa
Toxicant concentration (µg/L)
Treatment Replicate Naphthalene 24h Naphthalene 48h Fluorene
Dibenzo-thiophene
Conc. 1A 2430 ± 260, n=7 1430 ± 650, n=10 520 ± 110, n=8 180 ±
20, n=8
Conc. 1B 2330 ± 250, n=7 1400 ± 710, n=10 520 ± 140, n=10 190 ±
20, n=8
Conc. 1C 2430 ± 250, n=7 1330 ± 680, n=10 520 ± 130, n=8 190 ±
20, n=8
Conc. 2A 2710 ± 210, n=7 1600 ± 670, n=10 590 ± 110, n=9 250 ±
30, n=8
Conc. 2B 2540 ± 290, n=5 1590 ± 740, n=10 570 ± 120, n=7 240 ±
40, n=8
Conc. 2C 2620 ± 210, n=4 1550 ± 730, n=10 620 ± 150, n=8 280 ±
40, n=8
Conc. 3A 3050 ± 220, n=5 1630 ± 720, n=10 720 ± 110, n=8 330 ±
60, n=8
Conc. 3B 3030 ± 130, n=6 1700 ± 650, n=10 730 ± 90, n=9 330 ±
40, n=8
Conc. 3C 2950 ± 180, n=5 1700 ± 710, n=10 690 ± 80, n=8 330 ±
50, n=8
Conc. 4A 3290 ± 280, n=3 1870 ± 590, n=10 780 ± 120, n=9 430 ±
70, n=8
Conc. 4B 3390 ± 270, n=3 1770 ± 810, n=10 830 ± 110, n=9 440 ±
100, n=8
Conc. 4C 3310 ± 240, n=3 1930 ± 520, n=10 810 ± 80, n=7 440 ±
70, n=8 a values presented as the mean ± standard deviation, number
of samples)
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38
Table 2. Survival models to predict time to death for P.
pugio
Survival Models TTD=eb1eb2 (ln Conc)eb3W
Compound b1 (SE) b2(SE) b3 (SE) Naphthalene 52.2 (2.1) -6.3
(0.3) 0.64 (0.03)Fluorene 22.1 (1.6) -2.8 (0.2) 0.41
(0.02)Dibenzothiophene 17.4 (0.6) -2.4 (0.1) 0.31
(0.02)Ethylnaphthalenea 15.3 (1.0) -2.0 (0.2) 0.26
(0.01)Dimethylnaphthalenea 14.9 (1.2) -1.8 (0.2) 0.34
(0.03)Phenanthrenea 13.0 (1.1) -1.5 (0.2) 0.38 (0.03)TTD = the
predicted time-to-death for a specified proportion of the exposed
shrimp, b1 = MLE-
estimated intercept, b2 = the estimated coefficient for the
influence of ln of PAH concentration
on time-to-death, b3 = the estimated scale parameter, and W =
the response metameter for the
model distribution associated with the proportion dying (P) of
the exposed shrimp for which
prediction is being made. (SE) standard error for the calculated
parameter
adata from Unger et al [8]
-
39
Table 3. Body burdens of toxicants in P.pugio at time of
death
Compound Cb mean ± s.d. (µg/g)
Cb mean ± s.d. (µm/g)
Kelim (SE) T1/2 (hours)
Naphthalene 110 ± 50, n=12 0.82 ± 0.4, n=12
0.19(0.012), n=8
3.7
Fluorene 150 ± 50, n=19 0.91 ± 0.3, n=19
0.090(0.005), n=7
7.7
Dibenzothiophene 220 ± 150, n=15 1.2 ± 0.8, n=15
0.041(0.021), n=7
17
Ethylnaphthalenea 65 ± 50, n=12 0.41 ± 0.3, n=12
0.093(0.0066), n=6
7.8
Dimethylnaphthalenea 70 ± 30, n=11 0.45 ± 0.2, n=11
0.13(0.017), n=10
5.3
Phenanthrenea 210 ± 180, n=15 1.2 ± 1.0, n=15
0.12(0.0087), n=6
5.8
Cb measured body burden concentration on a wet weight basis ±
standard deviation, number of individuals analyzed Kelim calculated
elimination rate constant (standard error), number of individuals
analyzed T1/2 half-life for body burden concentration aData from
Unger et al [8]
-
40
TTD NAP = e.52.2 e- 6.31 ln C e0.643W
0
10
20
30
40
50
60
1800 2000 2200 2400 2600 2800 3000
5%10%
30%50%
70%
90%
95%
TTD FLUOR = e22.078 e- 2.816 ln C e0.4103W
0
10
20
30
40
50
60
500 550 600 650 700 750 800
Tim
e to
dea
th (h
ours
)
5%10%
30%
50%
70%
TTD DBT = e17.399 e- 2.405 ln C e 0.3138W
0
10
20
30
40
50
60
200 250 300 350 400 450 500
5%10%
30%
50%
70%
90%
Concentration (µg/L)
A
B
C
-
41
Dibenzothiophene
0
100
200
300
400
500
600
700
12 24 36 48 72 96
Time (H)
Bod
y B
urde
n C
once
ntra
tion
(ug/
g)
Post-exposureExposure
300 ug/L500 ug/L
-
42
y = 4.4 x 106e-2.1x
R2 = 0.94
100
1,000
10,000
100,000
2 2.5 3 3.5 4 4.5 5
log Kow
LC 5
0 (u
g/L)
This study
Tatem [15]Unger et al [8]
y = -33.211x + 162.63
0
10
20
30
40
50
60
2 2.5 3 3.5 4 4.5 5
Toxi
city
(b1)
This study
Unger et al [8]
y = -3.9292x + 19.381
0
1
2
3
4
5
6
7
2 2.5 3 3.5 4 4.5 5Log KOW
Toxi
city
(-b 2
)
This studyUnger et al [8]
A
B
C
-
43
0
0.5
1
1.5
2
2.5
120 130 140 150 160 170 180 190Molecular Weight
Tiss
ue C
onc.
(µm
/g w
et w
eigh
t) This StudyUnger et al [8]
NAP
DMNENAP
FLUOR
PHEN DBT
-
44
Appendix B
-
45
-
46
-
47
-
48
-
49
-
50
-
51