Technical Memorandum

15375 SE 30th Place, Suite 250 October 2015

Bellevue, WA 98007

Review of: Acute Toxicity of Runoff from Sealcoated

Pavement to Ceriodaphnia dubia and
Pimephales promelas

Authors: Barbara J. Mahler, Christopher G. Ingersoll, Peter C. Van Metre, James L. Kunz,
and Edward E. Little

Reference: Environmental Science & Technology, 2015, 49(8):50605069

Key Points
The study by Mahler et al. (2015) shows that simulated runoff from test plots
sealed with either refined tar sealer (RTS) or an asphalt/refined tar blend
(ARB) caused mortality, which was enhanced by UVA exposure in acute
aquatic toxicity tests. However, the methodological flaws and the lack of
environmental relevance of the simulated runoff from test plots limit the
applicability of these data to conditions in natural surface waters.
While a 10% dilution of RTS and ARB runoff is more environmentally
realistic than undiluted runoff, the dilution water did not mimic natural
waters, which contain organic carbon and suspended sediment that quickly
bind sealcoat chemicals, which reduces bioavailability to biota.
Undiluted runoff samples were analyzed for total PAHs, but diluted samples
were reported as nominal concentrations. Because these unfiltered samples
are heterogeneous mixtures of particulate and dissolved PAHs, the accuracy
of nominal concentrations and corresponding LC50s are likely to be highly
uncertain.
Toxicity is expected to be most closely related to the concentration dissolved
PAHs. Particles in unfiltered samples can act as source or as a sink of
dissolved PAHs. Thus concentrations of total PAHs and associated levels of
toxicity reported in this study may not be comparable to environmental
concentrations. Lack of information in this study on the actual bioavailability
of PAHs limits the usefulness of these results.
Photo-enhanced toxicity was observed in this study, but only two replicates
were used to assess the effects of UVA exposures. This low number of
replicates hinders the ability to conclude whether UVA cause significant
differences in toxicity.

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Overview
Using standard acute laboratory toxicity tests with a cladoceran (Ceriodaphnia dubia) and
fathead minnow (Pimephales promelas), Mahler et al. (2015) evaluated the acute toxicity of
undiluted (100%) and diluted (10%) simulated runoff from types of pavement sealers, refined
tar sealer (RTS) and an asphalt/refined tar blend (ARB), with and without subsequent exposure
to ultraviolet A radiation (UVA). The experiment compared RTS and ARB, but post-application
sampling and analysis suggested that RTS was also present in the ARB product.

The study reported high toxicity to both species after exposure to undiluted RTS runoff
collected as much as 36 days after sealcoat application. Exposure to UVA exposure resulted in
100% mortality to both species exposed to RTS runoff collected as much as111 days after
sealcoat application. However, the assumption that aquatic life is exposed to undiluted sealant
runoff is not environmentally realistic. Diluted (10%) RTS runoff samples without UVA
exposure were not acutely toxic to either species; however, with UVA exposure, high mortality
(70%100%) was observed for C. dubia out to 111 days after sealcoat application. While a 10%
dilution of RTS runoff is more environmentally realistic than undiluted runoff, the dilution
water did not mimic natural waters that contain organic carbon and suspended sediment that
quickly bind sealcoat chemicals, rendering them unavailable to biota.

Acute toxicity of ARB runoff samples was less than RTS runoff samples, which is expected
since the concentrations of PAHs measured in undiluted ARB runoff samples (2163
PAH16/L) were an order of magnitude lower than undiluted RTS runoff samples (167476 g
PAH16/L). ARB runoff was only moderately toxic to P. promelas during one collection day
(no toxicity was observed for C. dubia). The addition of UVA exposure to ARB runoff
treatment resulted in high mortality (80%100%) to P. promelas following the first 4 days after
ARB application, and to C. dubia out to the final collection day (36 days after sealcoat
application). Diluted (10%) ARB runoff was toxic to C. dubia only at days 2 and 3 following
application of the sealcoat with subsequent UVA exposure. Diluted (10%) ARB runoff was not
acutely toxic to P. promelas at any collection time after application, with or without UVA
exposure.

While the toxicity tests used in this study followed standard EPA protocols, methodological
issues related to the tests and the methods used to generate simulated runoff (described below)
limit the application of the studys findings to real world environments. In addition, no
statistical test results are provided in the paper or the supplemental materials. However, the lack
of statistical testing in this study may not change the overall findings given the high mortality
observed.

Critique of Methods
Field Methods: Generation of Simulated Runoff
Simulated runoff was generated for three types of test plots: unsealed asphalt, RTS-covered
asphalt, and ARB covered asphalt. The study plan was to use RTS and an asphalt based sealer.
Chemical analysis suggested it contained some refined tar, so was described as ARB. The
authors estimate the blend contains 7% RT, but its actual source and concentration is unknown.

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Traffic and parking were allowed after the recommended 24-hr curing period; therefore, the
unsealed asphalt plot was used as a control for traffic and parking activities. Runoff was
simulated by sprinkling control water (a mixture of well water and deionized water from the
toxicity-testing laboratory) across the plot and collecting it at the downslope end of the
pavement. A new area of the plot was sampled for each time point. Simulated runoff from RTS
was collected at various time points, ranging from 5 hrs up to 111 days after application.
Simulated runoff of ARB was collected at 4 hrs and up to 36 days after application. Simulated
runoff from the unsealed asphalt plot was sampled once seven days after the collections at other
plots began.

Simulated runoff from sealcoated and control (unsealed) pavement plots was not replicated at
each timepoint, thus excluding possibly relevant environmental variation in treatments. This
lack of replication is practiced in laboratory exposure experiments where solutions are created
under uniform conditions. In this case, however, variability in environmental exposure should
have been characterized with analysis of replicates.

Simulated runoff was collected on the plots for 7 days until a 2-day rain event occurred.
However, no samples of the runoff from this actual rain event were collected. This omission
would seem to represent a missed opportunity to validate the simulated runoff samples. In
addition, it is not clear how much washing occurred due to that natural rain event. Runoff
from the plots after subsequent rain events would be a more realistic measure of the decrease in
mobilized PAHs. Receiving streams would likely observe a high-PAH pulse after the first rain
event, followed by decreased PAH concentrations from subsequent rain events.

The control water, which was used as artificial rain to simulate runoff and also used as the
dilution water, consisted of a mixture of well water and deionized water. Water quality criteria
of control water (e.g., turbidity and dissolved organic carbon) are quite different from the runoff
to which aquatic organisms would be exposed in natural receiving waters. In addition, traffic
and parking were allowed on these plots during the course of the experiment. While a runoff
control (unsealed asphalt pavement) was used to account for introduced contamination from
parking and traffic, such a runoff control is not actually an appropriate control for the sealcoated
pavements. Auto-related contamination (e.g., oil) would likely be absorbed by or adhere to
unsealed pavement to a greater extent than to sealed pavement.

Laboratory Methods
Chemical Analysis
Unfiltered water samples were analyzed for 16 U.S. Environmental Protection Agency Priority
Pollutant PAHs, 7 N-heterocycles (quinoline, isoquinoline, acridine, phenanthridine, carbazole,
benzo[c]-cinnoline, and 2,2-biquinoline), and three other compounds (4H-cyclo-penta[def]-
phenanthrene, 1-methylphenanthrene and nitrobenzene). However, the authors state that some
metals and metal mixtures can cause synergistic toxicity with PAHs (Gauthier et al. 2014). The
test plots were subjected to vehicle traffic and parking after RTS application, which can release
a variety of heavy metals, but samples were not analyzed for metals. Those unmeasured and
unrelated chemicals could have contributed to the observed toxicity.

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Over the course of the study, the concentration of total (unfiltered) 16 priority pollutant PAHs
(PAH16) in RTS runoff samples fluctuated between approximately 200 and 457 g/L. The
PAH16 in day 1 and day 111 runoffs were similar at about 335 7 g/L. The ratio of the
lighter to heavier PAHs decreased from more than 2 to about 0.16, indicating that weathering
and possibly further curing occurred. The sum of the seven measured azaarenes decreased from
over 900 to 24 g/L. Concentrations of total (unfiltered) PAH16 in ARB runoff samples varied
between 21 and 43 g/L, except for the day 111 sample, which was 63 g/L. The ratio of the
lighter to heavier PAHs decreased from about 4 to approximately 0.24 g/L, while the total
azaarenes decreased from 30 to 6 g/L.

The authors analyzed undiluted runoff samples for total PAHs, but diluted samples were
reported as nominal concentrations. Because these unfiltered samples are heterogeneous
mixtures of particulate and dissolved PAHs, the accuracy of nominal concentrations are likely to
be highly uncertain. This in turn, may lead to overestimating or underestimating toxicity.

Toxicity is expected to be most closely related to the concentration of dissolved PAHs. Particles
in unfiltered samples can act as a source or as a sink of dissolved PAHs.1 Therefore,
concentrations of total PAHs and associated levels of toxicity reported in this study may not be
comparable to environmental concentrations. Lack of information in this study on the actual
bioavailability of PAHs limits the usefulness of these results.

Toxicity Tests
The toxicity tests used in this study followed standard EPA protocols with commonly used
freshwater test organismsa fish (P. promelas) and an invertebrate (C. dubia), both of which
are water column organisms. Four replicates were used in the initial acute toxicity tests of the
sealcoat runoff and runoff control samples, but only two replicates were used to assess the
effects of subsequent UVA exposures. This low number of replicates hinders the ability to
conclude whether UVA cause significant differences in toxicity.

The intensity (UVB: 7.5 W/cm2; UVA: 454 W/cm2; visible light: 604 W/cm2) and duration
(4 hrs) of subsequent exposure of test organisms to UVA could be environmentally realistic, but
some degree of cloud and tree canopy cover should have been considered.

Two concentrations of each runoff sample were testedundiluted (100%) and diluted (10%).
This approach does not yield sufficient data to calculate a dose-response curve or effect
concentrations (e.g., LC50 [median lethal concentration]). However, the authors did calculate
phototoxic LC50 values for each species in this study by combining results from the two
experiments. They calculated phototoxic concentrations for UVA-exposed runoff samples using
relative photodynamic activity (RPA), which is an index of potency of a given compounds
phototoxicity. RPAs were available for P. promelas (Oris and Giesy 1987), but not for C. dubia.
RPAs were available for another cladoceran, Daphnia magna (Newsted and Giesy 1987), and

1
In studies with mixtures of water and crude oil, entrained oil droplets can be transferred to subsequent dilutions
following preparation of initial stock solution. The presence of entrained oil droplets in diluted samples adds
additional dissolved hydrocarbons to the diluted samples, and thus exerts an additional toxic effect (Redman
2015; Redman et al. 2012). These studies demonstrate the importance of measuring concentrations of dissolved
PAHs in exposure systems.

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these were used as surrogate RPAs for C. dubia. The RPAs for ten measured compounds2 were
used to calculate phototoxic concentrations for C. dubia, and RPAs for five measured
compounds3 were used to calculate phototoxic concentrations for P. promelas. They also used
those samples with 100% toxicity prior to exposure to UVA; thus, a phototoxic response of
100% was assumed. Given that phototoxicity has been demonstrated to be an additive effect, the
phototoxic concentrations in a given sample were summed to create phototoxic equivalents
(PAHeq). Dose-response curves for each species were constructed using the PAHeq and
observed mortality for the two experiments. While this approach seems reasonable, the authors
did not use every sample in the dose-response curves, and it is not clear how samples were
chosen for that exercise. It is also not clear whether they used both undiluted and undiluted
samples. In addition, water turbidity and water depth, which can significantly reduce UVA
penetration, were not accounted in this study. Environmental factors such as these could
significantly reduce photoenhanced toxicity of PAHs (Ankely et al 2003).

ARB runoff samples had lower PAHeq than did the RTS runoff samples. However, the authors
state, Where the range of PAHeq for a species was similar, however, for a given PAHeq
samples of AS/CT-blend4 runoff appeared to provoke greater mortality, indicating that there
might be compounds in the AS/CT-blend product other than those measured that are
contributing to toxicity. Alternatively, automobile-related contamination from traffic and
parking that occurred during the study could have affected these runoff samples. This suggests
that the chemical characterization of the samples was inadequate.

Caution should be used when applying the LC50 values presented in this paper, because the
simulated runoff samples do not represent conditions in natural surface water. For instance,
natural surface water will likely have higher suspended sediment loads and dissolved organic
carbon, which will quickly scavenge PAHs and other sealer-related organic compounds as they
enter the receiving water. In addition, cloud and tree canopy cover in natural environments can
reduce sunlight penetration, thus reducing phototoxicity. Therefore, the effective concentrations
in natural waters would likely be much higher than those predicted in this study.

Statistical Analysis
No statistical comparisons were made in this study between the test-plot treatments in terms of
dilution or collection date. The authors were careful not to state whether the observed mortality
was significant. Given the high mortality observed in this study, including statistical evaluations
may not change the overall findings.

Conclusions
The study by Mahler et al. (2015) shows that RTS and ARB caused high mortality and was
enhanced with UVA exposure in acute laboratory toxicity tests with aquatic biota. However, the
methodological flaws and the lack of environmental relevance of the simulated runoff from test

2
Anthracene, fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[k]fluoranthene, benzo[a]pyrene,
benzo[g,h,i]perylene, dibenzo[a.h]anthracene, and acridine.
3
Anthracene, pyrene, benz[a]anthracene, benzo[a]pyrene, and acridine.
4
Referred to herein as ARB.

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plots limit the applicability of these data to conditions in natural surface waters. Additional
studies are needed to address these shortcomings.

References
Ankely, G.T., L.P. Burkhard, P.M. Cook, S.A. Diamond, R.J. Erickson, and D.R. Mount. 2003.
Assessing risks from photoactivated toxicity of PAHs to aquatic organisms. In PAHs; An
Ecotoxicological Perspecitve, Douben, P.E.T., editor. John Wiley & Sons Ltd, West Sussex,
England, pp 275-296.

Gauthier, P.T., W.P. Norwood, E.E. Prepas, and G.G. Pyle. 2014. MetalPAH mixtures in the
aquatic environment: A review of co-toxic mechanisms leading to more-than-additive outcomes.
Aquat. Toxicol. 154: 253-269.

Mahler, B.J., C.J. Ingersoll, P.C. Van Metre, J.L. Kunz, and E.E. Little. 2015. Acute toxicity of
runoff from sealcoated pavement to Ceriodaphnia dubia and Pimephales promelas. Environ.
Sci. Technol. 2015, 49(8):50605069

Newsted, J.L., and J.P. Giesy. 1987. Predictive models for photoinduced acute toxicity of
polycyclic aromatic hydrocarbons to Daphnia magna, Strauss (Cladocera, Crustacea). Environ.
Toxicol. Chem. 6:445461.

Oris J.T., and J.P.J. Giesy. 1987. The photo-induced toxicity of polycyclic aromatic
hydrocarbons to larvae of the fathead minnow (Pimephales promelas). Chemosphere
16(7):13951404.

Redman, A.D., J.A. McGrath, W.S. Stubblefield, A.W. Maki, and D.M. Di Toro. 2012.
Quantifying the concentration of crude oil droplets in oil-water preparations. Environ. Toxicol.
Chem. 31(8):1814-1822.

Redman, A.D. 2015. Role of entrained droplet oil on the bioavailability of petroleum substances
in aqueous exposures. Mar. Pollut. Bull. 97:342-348.

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