45. Ren, L., Zeiler, L.F., Dixon, D.G. and Greenberg, B.M. 1996 ...

ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 33, 73–80 (1996)
ARTICLE NO. 0008
Photoinduced Effects of Polycyclic Aromatic Hydrocarbons
on Brassica napus (Canola) during Germination
and Early Seedling Development
LISHA REN, LORELEI F. ZEILER, D.GEORGE DIXON, AND BRUCE M. GREENBERG 1
Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
Received July 21, 1995
Nikolaou et al., 1984). The properties of PAHs can be altered
It has recently been demonstrated that light dramatically en- either abiotically (e.g., photochemically) or biotically (e.g.,
hances the toxicity of polycyclic aromatic hydrocarbons (PAHs) cytochrome P450-mediated monooxygenation) (Yang et al.,
to the duckweed Lemna gibba L. G-3 (L. Ren, X.-D. Huang, B. J. 1976; Neff, 1979; Katz et al., 1979; Zepp and Schlotzhauer,
McConkey, D. G. Dixon, and B. M. Greenberg, 1994, Ecotoxicol. 1979; Nikolaou et al., 1984; Shimada and Nakamura, 1987;
Environ. Saf. 28, 160–171). To extend this research to terrestrial
Harvey et al., 1988; Huang et al., 1993; Ren et al., 1994).
plants, Brassica napus L. (oil seed rape) seeds were germinated in
Photochemical activation occurs when the extensive p-orthe
presence of three PAHs; anthracene (ANT), benzo[a]pyrene
(BAP), and fluoranthene. The chemicals were applied both in in- bital systems of PAHs absorb light in the ultraviolet-B (UVtact
form and following photomodification in UV-B radiation; toxnm)
regions of the solar spectrum (Suess, 1976; Cook et al.,
B; 290–320 nm) and/or the ultraviolet-A (UV-A; 320–400
icity was assessed in simulated solar radiation (SSR), a light source
with a visible light:UV-A:UV-B ratio similar to that of sunlight. 1983; Nikolaou et al., 1984; Huang et al., 1993; Ren et
Germination efficiency, root and shoot growth, and chlorophyll al., 1994). The compounds are reactive following photon
content, measured after 6 days of exposure, were used as toxicity absorbance, which can result in photomodification of the
endpoints. Intact and photomodified PAHs had little impact on PAH, defined here as photooxidation and/or photolysis; the
shoot fresh weight or chlorophyll content, but markedly inhibited process is rapid under environmentally relevant levels of
root fresh weight, with the photomodified PAHs having greater actinic radiation (Katz et al., 1979; Zepp and Schlotzhauer,
impacts than the intact PAHs. The decline in root fresh weight was
1979; Nikolaou et al., 1984; Huang et al., 1993, 1995; Ren
not attributable to a decline in germination frequency or delayed
et al., 1994). Moreover, PAHs have high quantum yields for
germination. However, the seedlings produced shorter roots in the
presence of either intact or photomodified PAHs. To explore the triplet-state formation and are active type II photosensitizers
role of actinic radiation on PAH toxicity, seedlings were incubated (Morgan et al., 1977; Newsted and Giesy, 1987; Schoeny
in SSR, visible light and darkness with either intact or photomodiat
promoting the formation of biologically damaging singlet
et al., 1988; Foote, 1991). As such, they are highly efficient
fied PAHs. Inhibition of root growth was only achieved by the
intact chemicals if actinic radiation was present. However, with oxygen (Foote, 1991) in the presence of light.
photomodified ANT or photomodified BAP, root fresh weight ac- It has been demonstrated that the toxicity of PAHs to
cumulation was inhibited in SSR, visible light and darkness. Thus, microbes, animals, and aquatic plants is enhanced by actinic
intact PAHs are hazardous to terrestrial plants in the presence of radiation (Oris and Giesy, 1987; Newsted and Giesy, 1987;
light, but once the compounds are photomodified, actinic radiation Huang et al., 1993; Ankley et al., 1994; Ren et al., 1994).
is no longer an absolute requirement for phytotoxic activity.
Recent work with the higher aquatic plant Lemna gibba (a
1996 Academic Press, Inc.
duckweed) has demonstrated that PAHs are photomodified
by actinic radiation to a complex, unidentified mixture of
photoproducts that are more toxic than the parent compounds
(Huang et al., 1993; Ren et al., 1994). For all species tested,
INTRODUCTION
except L. gibba, the relative contributions to toxicity of
PAHs applied in intact form and photomodified form have
Polycyclic aromatic hydrocarbons (PAHs) are a group of
not been evaluated. Furthermore, the toxicity of PAHs to a
ubiquitous environmental contaminants consisting of two or
terrestrial plant species has not yet been explored.
more fused benzene rings (Suess, 1976; Cook et al., 1983;
Terrestrial plants are subjected to pollutants in a variety
of ways, including contact with contaminated water. For
1
To whom correspondence should be addressed. Fax: (519) 746-0614. instance, rainwater carries environmental contaminants in-
E-mail: greenber@biology.watstar.uwaterloo.ca.
cluding PAHs, as does irrigation water from exposed lakes
73 0147-6513/96 $18.00
Copyright 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.

74 REN ET AL.
and ponds (Suess, 1976; Basu and Saxena, 1978; Neff, 1979;
Cook et al., 1983; Nikolaou et al., 1984; Eadie, 1984). To
further examine the photoinduced phytotoxicity of PAHs,
the effects of intact and photomodified PAHs on the terrestrial
plant Brassica napus (canola) during germination and
early seedling growth were probed. Significantly, germination
and early growth assays can be performed in the light
or dark, making it possible to test for the absolute requirement
of photoinduction with respect to the toxicity of intact
and photomodified PAHs. Experiments were completed in
simulated solar radiation (SSR; a light source with a visible
light:UV-A:UV-B ratio equivalent to that of sunlight),
visible light (vis) and darkness with intact and photomodified
anthracene (ANT, pmANT), benzo[a]pyrene
(BAP, pmBAP), and fluoranthene (FLA, pmFLA).
MATERIALS AND METHODS
The toxicity tests reported here were based on following
the germination and early growth of B. napus L. cv Topas
(canola) seeds. A general guide for this type of toxicity
test can be found in the ASTM Standard Practice for Early
Seedling Toxicity Tests (ASTM, 1994). In preparation for
each test, B. napus seeds were washed 10 times with filter- FIG. 1. Spectral distribution of the simulated solar radiation (SSR)
purified (ultrafiltration) water (FP-H 2 O) and placed on seed source. (A) Emission spectra of the SSR source filtered through a standard
trays (Wang, 1993) in petri dishes with 30 ml of the test polystyrene petri dish top. (B) Emission spectra of the SSR source filtered
solution. All toxicity tests were performed with 12 seeds per through a polystyrene petri dish top and a polystyrene seed tray. All spectra
are given in mmol m 01 s 01 nm 01 . The spectra provided are for a source
dish and were replicated 9 times. Germination and early with a total fluence rate of 100 mmol m 01 s 01 . A source with a total fluence
growth assays were conducted for 6 days. Light conditions rate of 250 mmol m 01 s 01 and the same spectral distribution was also used.
and endpoints of chemical impact are described below. Concentrations
for a 50% effect, EC 50 , were derived from the
dose–response curves. Statistical analyses (calculations of
means and standard errors) were performed for all applicable practical to further monitor the concentration of the PAH.
experiments.
To compensate, the test solution was replenished every 48
Germination tests on B. napus seeds were performed on hr by static renewal.
polystyrene seed trays (Wang, 1993) in 100 1 15-mm polystyrene
During the seed germination tests irradiation was provided
petri dishes. To form the test solutions, ANT, BAP, as either visible light (cool-white fluorescent) or SSR (Huang
and FLA (Sigma Chemical Co., St. Louis, MO) were dissolved
et al., 1993; Ren et al., 1994; Greenberg et al., 1995). Photon
in DMSO to concentrations ranging from 0 to 10 g fluence rates are given in the figure legends. The spectral
liter 01 and delivered to FP-H 2 O by 1000-fold dilution to final output of the SSR source following passage through a poly-
PAH concentrations ranging from 0 to 10 mg liter 01 . At 2 styrene petri dish cover (Fig. 1A) had a visible light:UVmg
liter 01 , solutions of ANT, BAP, and FLA are 11.0, 8.0, A:UV-B ratio of 100:10:1, based on the number of photons
and 9.7 mM, respectively. Due to their low solubilities in (Huang et al., 1993; Greenberg et al., 1995). While this
water, PAHs were delivered in DMSO to achieve concentrations
spectral output does not precisely replicate sunlight, the ratio
that allow for full-scale dose–response curves (Neff, of visible light:UV-A: UV-B is parallel to that of terrestrial
1979). The final DMSO concentration in solution (0.1% v/ sunlight from midspring to midfall in latitudes corresponding
v) does not interfere with availability of PAHs nor does it to southern Canada and the northern United States
affect plant growth (Huang et al., 1993; Ren et al., 1994). (Greenberg et al., 1995). The spectrum of the SSR source
Control seeds were always exposed to this concentration of below the seed tray was also measured (Fig. 1B). Since the
DMSO. Accuracy of PAH delivery to water was determined seed tray only slightly altered the spectrum, removing some
spectroscopically as previously described (Huang et al.,
1993). Once a toxicity test is started, photomodification and
bioaccumulation of the PAHs begins; it then becomes im-
of the UV-B, the seedling roots were subjected to irradiation
with characteristics similar to that received by the shoots.
Toxicity was assessed on the basis of inhibition of accu-

PHOTOINDUCED TOXICITY OF PAHs TO B. napus
75
mulation of root or shoot fresh weight relative to control
seedlings. The germination frequency was also quantified on
Days 1, 2, 3, and 6 of the test; it is defined as a visible
cracking of the seed coat, even if there was not measurable
root or shoot production. As well, the total number of roots
over 1 mm in length were scored and their lengths were
measured. Those seeds where the seed coat broke, but produced
roots under 1 mm, were not included in statistical
calculations of root length. To further quantify the level of
toxicity in the shoots the extent of chlorosis was determined
(Greenberg et al., 1991).
Toxicity of photomodified PAHs was assessed by root
and shoot growth, and chlorosis as per the intact chemicals.
Photomodified PAHs were generated by delivery of intact
chemicals in DMSO to a final concentration of 8 mg liter 01
in FP-H 2 O and incubated in sealed containers under UV-B
radiation (25 mmol m 02 from 290 to 320 nm) for 5 days for
ANT and 7 days for BAP and FLA. Half-lives of intact
chemicals under these conditions are: ANT, 3 hr; BAP, 33
hr; and FLA, 20 hr. Photomodification was judged to be
complete on the basis of absorbance criteria as previously
described (Huang et al., 1993; Ren et al., 1994). Because
more than 20 photoproducts are generated from each PAH,
and most of these are unidentified at present, the concentra-
tions of the photomodified PAHs were based on amount of
the intact PAHs (Huang et al., 1993; Ren et al., 1994). It
was previously demonstrated that toxicity of photomodified
PAHs can be assessed based on the concentration of the
parent compounds (Huang et al., 1993, 1995; Ren et al.,
1994). The chemical concentration was adjusted by dilution
with FP-H 2 O and applied to the seeds.
FIG. 2. Toxicity of intact ANT, BAP, and FLA to Brassica napus.
The PAHs were applied hydroponically to the plants at the concentrations
indicated. The light source was SSR (250 mmol m 01 s 01 ). Squares represent
relative root growth (fresh weight accumulation) as a percentage of control
growth. Triangles represent relative shoot growth (fresh weight accumulation)
as a percentage of control growth. Circles represent relative chlorophyll
(Chl) content as a percentage of chlorophyll in the control plants. Error
bars are SEM (n Å 9).
tion can, of course, occur in the environment prior to contact
with roots (Huang et al., 1993; Ren et al., 1994). Conse-
RESULTS
quently, ANT, BAP, and FLA were irradiated with UV-
B prior to application to the plants to study the effects of
To extend the work on the photoinduced toxicity of PAHs photomodification on the toxicity of PAHs to B. napus. The
to the aquatic higher plant L. gibba, the effects of ANT, impact of the photomodified PAHs was primarily observed
BAP, and FLA were tested on B. napus in the presence of in the root system, as measured by fresh weight accumula-
SSR. The order of toxicity of the three PAHs, applied in the tion, with the order of toxicity being pmANT ú pmBAP ú
intact form, was ANT ú BAP ú FLA (Fig. 2). Under 250 pmFLA (Fig. 3). Under 250 mmol m 02 s 01 of SSR, pmANT
mmol m 02 s 01 of SSR, ANT had a impact threshold of 0.5 mg had a threshold impact of 0.25 mg liter 01 and an EC 50 at
liter 01 and an effective concentration for 50% diminishment about 0.5 mg liter 01 , 50% lower than the EC 50 for intact
(EC 50 ) of root fresh weight accumulation between 1 and 2 ANT (cf. Figs. 2 and 3). Photomodified BAP and pmFLA
mg liter 01 . Conversely, FLA did not affect root growth below also exhibited marked increases in toxicity relative to the
2 mg liter 01 and plateaued at 50% lower root fresh weight intact compounds. The impact thresholds of pmBAP and
at 4 mg liter 01 . The PAHs had a greater impact on root pmFLA were 0.25 and 1 mg liter 01 , respectively, versus 1
growth than on either shoot fresh weight accumulation or and 2 mg liter 01 for the intact compounds (cf. Figs. 2 and
chlorophyll content, reflecting the hydroponic route of application
3). Again, the effects of the chemicals on shoot growth and
and poor transport of PAHs from the roots through chlorosis were minimal, suggesting that photomodified
the plant vascular systems (Edwards, 1983).
PAHs are also not transported well in the plants.
Because roots in soil are not exposed to sunlight, intact The effect of PAHs on root fresh weight accumulation
PAHs will probably have a limited effect on them. However, could be derived from delayed germination, inhibition of
it has been demonstrated that PAH toxicity to L. gibba in- growth, or both. Therefore, the germination frequency of B.
creases following photomodification. PAH photomodifica- napus in the presence of the PAHs was assayed in SSR

76 REN ET AL.
cordingly, the chemicals must be inhibiting root growth by
altering the ability of the roots to accumulate fresh weight.
The number of roots produced and/or the size of the indi-
vidual roots could be affected by the PAHs. First, the total
number of roots produced in 6 days was quantified. For the
intact PAHs there was no effect on the total number of roots
produced; approximately 90% of control and treated seeds
produced a measurable root in 6 days (defined here as 1 mm
or more in length, Fig. 4). However, photomodified ANT
caused a moderate decrease in the number of roots produced;
about 60% of the seeds produced a measurable root (Fig.
4). Photomodified BAP only slightly diminished the number
of measurable roots produced, while pmFLA had no effect
on the number of roots produced. Therefore, in general, the
effects of PAHs on B. napus cannot be attributed to diminished
root production.
Day a
Another possible effect of the chemicals is on root elongation.
Control seedlings in SSR had an average root length
1 2 3 6 of 18.2 mm (Table 2) and exhibited a distribution of roots
over a 1–60 mm range, with the number of roots in each
FIG. 3. Toxicity of pmANT, pmBAP, and pmFLA to Brassica napus.
The photomodified chemicals were applied at the concentrations indicated.
The concentrations are based on the amount of intact chemical prior to
photomodification. The light source was SSR (250 mmol m 01 s 01 ). Squares
represent relative root growth (fresh weight accumulation) as a percentage
of control growth. Triangles represent relative shoot growth (fresh weight
accumulation) as a percentage of control growth. Circles represent relative
chlorophyll (Chl) content as a percent of chlorophyll in the control plants.
Error bars are SEM (n Å 9).
(Table 1). Neither the efficiency nor the rate of germination
were affected by the intact PAHs. The photomodified PAHs
also had no impact on germination (data not provided). Ac-
TABLE 1
Efficiency of Brassica napus Seed Germination (Defined as
Cracking of the Seed Coat) in the Presence of Intact PAHs under
SSR (250 mmol m 01 s 01 )
FIG. 4. Fraction of Brassica napus seeds producing roots in the presence
of intact and photomodified PAHs. Histograms represent the percentage
(SEM, n Å 9) of seeds producing a root 1 mm or more in length after
6 days in SSR (250 mmol m 01 s 01 ). Chemicals are listed below the histograms.
Gray histograms are for the intact chemicals; black histograms are
for the photomodified (pm) chemicals. The control (open histogram) is the
same for intact and photomodified chemicals.
Control 73 (3.6) b 88 (2.7) 94 (1.5) 96 (1.1)
ANT 70 (4.7) 85 (3.0) 94 (2.2) 96 (1.9) successive 10-mm interval exhibiting a gradual decline (Fig.
BAP 77 (3.7) 96 (2.2) 98 (1.1) 98 (1.1) 5). Conversely, for intact ANT and BAP, the distribution in
FLA 68 (4.6) 87 (3.6) 94 (1.8) 98 (1.7) root lengths is skewed toward shorter lengths, with mean
root lengths of 8.9 and 12.5 mm, respectively (Fig. 5A). In
Note. The percent of seeds that germinated in the presence of no PAHs particular, in the presence of ANT the vast majority of roots
(control), ANT (10 mg liter 01 ), BAP (10 mg liter 01 ), or FLA (10 mg liter 01 )
at various times during a toxicity experiment.
are shorter than 10 mm. On the other hand, plants grown in
a
Days after application of chemical.
the presence of FLA had an average root length (22.1 mm)
b
Percentage germination (SEM in parentheses, n Å 9).
and a distribution of root lengths very similar to the control

PHOTOINDUCED TOXICITY OF PAHs TO B. napus
77
TABLE 2
With the finding that PAHs affect root fresh weight accu-
Average Root Length of Brassica napus Seedlings in the mulation and elongation in SSR, we determined if PAHs also
Presence of Intact or Photomodified PAHs
have negative impacts on the seedling roots in the absence of
Control ANT BAP FLA
UV radiation or in darkness. B. napus seeds were germinated
in SSR (100 mmol m 02 ), visible light (100 mmol m 02 ), and
Intact 18.2 (0.8) 8.9 (0.9) a 12.5 (0.7) 22.1 (1.3) darkness for 6 days in the presence of intact PAHs. None
Photomodified 18.2 (0.8) 5.0 (0.4) 14.6 (1.1) 19.7 (1.2) of the intact PAHs had an effect on root fresh weight in
darkness (Fig. 6). Inhibition of root fresh weight accumula-
Note. All experiments were performed in SSR (250 mmol m 01 s 01 ).
tion for BAP and FLA was similar in SSR and visible light
Chemical concentrations were based on intact chemicals. ANT, BAP, FLA,
pmBAP, and pmFLA were applied at 4 mg liter 01 and pmANT was applied (Fig. 6). However, for ANT root fresh weight accumulation
at 1 mg liter 01 .
was markedly depressed in SSR but there was no effect in
a
Average root length in mm (SEM in parentheses, n Å 9). Calculations visible light. Thus, for all three chemicals in intact form
included only those seeds which produced a root 1 mm or longer.
there was a requirement for light, but only in the case of
ANT was there an absolute requirement for UV radiation.
seedlings. Nonetheless, FLA does cause lower fresh weight To determine if photomodified PAHs require visible light
accumulation in roots, implying that these plants have thin or UV radiation to be hazardous, seeds were germinated in
roots or roots that are not retaining water as well as the SSR (100 mmol m 02 ), visible light (100 mmol m 02 ), and
control seedlings.
darkness for 6 days. Photomodified ANT exhibited similar
The effect of the photomodified PAHs on the length of effects on root fresh weight accumulation under all three
roots was similar to the intact PAHs, but more pronounced lighting conditions (Fig. 6), indicating the toxicity of
for pmANT (Fig. 5B). The longest root lengths for pmANT pmANT is not dependent on actinic radiation. Photomodified
were in the 11 to 20-mm range and the mean root length BAP diminished root fresh weight accumulation by the same
was only 5.0 mm. Interestingly, there was no discernible amount in the dark and visible light (75% of control); how-
difference in the effects between intact BAP and pmBAP or ever, growth was much slower in SSR (only 40% of control).
between intact FLA and pmFLA, with each pair of chemicals This indicates that the toxicity of pmBAP was enhanced
affecting the distribution of root lengths to the same degree by UV radiation. Nonetheless, photomodified PAHs derived
(Fig. 5). As well, the mean root length for each pair was from both ANT and BAP have an impact on the plants
virtually identical (Table 2).
without further photoactivation. In contrast, pmFLA did not
FIG. 5. Length distribution of Brassica napus roots. B. napus seeds were germinated in the presence of ANT, BAP, FLA, pmANT, pmBAP, or
pmFLA in SSR (250 mmol m 01 s 01 ). All chemical concentrations were 4 mg liter 01 except pmANT which was 1 mg liter 01 . Each histogram represents
the average number of roots per petri dish in successive 10-mm length intervals for the control and chemically treated seeds. (A) Intact PAHs; (B)
photomodified PAHs.

78 REN ET AL.
EC 50 range of 1–4 mg liter 01 for intact ANT, BAP, and FLA
is consistent with the levels of PAHs required to inhibit L.
gibba growth by 50% (Huang et al., 1993; Ren et al., 1994).
This is also consistent with the levels of PAHs found in
contaminated soils and sediments (Suess, 1976; Cook et al.,
1983; Nikolaou et al., 1984; Jones et al., 1989; Ankley et
al., 1994).
Photomodified PAHs exerted toxic effects similar to the
intact compounds but at lower concentrations. This is also
consistent with the work done with L. gibba (Huang et al.,
1993; Ren et al., 1994), where the photomodified PAHs had
an EC 50 range of one-half that of the intact PAHs. ANT,
whether intact or photomodified, was invariably the most
toxic of the three PAHs tested, which is also consistent with
the work with L. gibba (Huang et al., 1993; Ren et al.,
1994). The levels of photomodified PAHs that caused subacute
toxicity are within the range of environmental loadings
of intact PAHs (0.05–0.5 mg liter 01 ) in many industrialized
regions (Basu and Saxena, 1978; Neff, 1979; Cook et al.,
FIG. 6. Toxicity of intact and photomodified PAHs to Brassica napus 1983; Eadie, 1984).
under various lighting conditions. Seeds were incubated for 6 days in the The toxic effects of the PAHs to B. napus was first evident
presence of intact ANT, BAP, and FLA (10 mg liter 01 ) or pmANT, pmBAP,
in the root systems of the plants. Only at high concentrations
or pmFLA (4 mg liter 01 ). The light conditions were: SSR (100 mmol m 01
s was an effect in shoot development and chlorophyll content
), visible light (100 mmol m 01 s 01 ), or dark. Histograms represent root
fresh weight accumulation as a percentage of control. Dark histograms, observed. This is consistent with the uptake of the toxicants
ANT; medium histograms, BAP; light histograms, FLA. Error bars are by the roots of the germinating seedlings during hydroponic
SEM (n Å 9).
application of the PAHs to the roots. Because PAHs are
hydrophobic and known to partition into lipid membranes
(Neff, 1979; Edward, 1983), it is expected that they will
inhibit root growth in the dark (Fig. 6), but did inhibit root accumulate in the membranes of the root system where the
fresh weight accumulation in SSR and visible light. Furtherconcentrations
chemicals first contact the plants. Only at extremely high
more, inhibition by pmFLA in SSR was greater than in
(4–10 mg liter 01 ), when the roots are satu-
visible light, indicating that the toxicity of pmFLA is enthe
rated with the chemicals, can movement of the PAHs through
hanced by UV radiation, as was the case for pmBAP.
plant to the shoots be assumed (Edwards, 1983). Addi-
tionally, at high PAHs concentrations the ability of the roots
DISCUSSION
to assimilate water and nutrients would be compromised,
which would impact on shoot growth and photosynthesis.
ANT, BAP, and FLA in their intact and photomodified Germination of the seeds was unaffected by the PAHs
forms are toxic to the terrestrial plant B. napus. Similar to the whether applied in intact or photomodified form. Thus, the
toxicity of PAHs against the aquatic plant L. gibba (Huang et decline in root fresh weight accumulation was not attributal.,
1993; Ren et al., 1994), the toxicity of the intact PAHs able to a delay in germination or to a decrease in the germina-
to B. napus was photoinduced. Additionally, the toxic activroot
growth (fresh weight accumulation). In the presence of
tion frequency. The effects can be traced chiefly to inhibited
ity of the chemicals was enhanced following photomodification
of the parent compounds. Interestingly, actinic radiation ANT, BAP, pmANT, or pmBAP, dramatically shorter roots
is not absolutely required to induce the toxicity of pmANT were produced by the seedlings. Furthermore, pmANT
or pmBAP. The toxic effect of both intact and photomodified caused the germinated seeds to produce fewer roots relative
PAHs was manifested as inhibition of root fresh weight accument
to the control seedlings. At the early stages of plant developmulation.
This was attributed to shorter roots in the presence
examined here, root growth is due primarily to cell
of intact or photomodified ANT and BAP. However, intact expansion and not cell division (Taiz and Zeiger, 1991).
and photomodified FLA inhibited root fresh weight accumucould
Therefore, cell expansion is probably being impeded, which
lation without affecting root lengths.
occur, for example, by inhibition of hormone
action
(e.g., auxin) or interference with cellular metabolism (e.g.,
mitochondrial function). Interestingly, it was recently dem-
onstrated that photomodified PAHs inhibit cytochrome c oxi-
The toxicity of PAHs to B. napus was dependent on the
concentration of the intact PAH in solution, exhibiting an
approximately log-linear response to concentration. The

PHOTOINDUCED TOXICITY OF PAHs TO B. napus
79
dase (Huang, 1995), the terminal oxidase in mitochondrial (Fig. 6). On the other hand, pmFLA has an absolute dependence
electron transport.
on light to exert toxicity. This is consistent with the
On the other hand, FLA seemed to inhibit root fresh work done with L. gibba (Ren et al., 1994), where the toxicweight
accumulation by a different mechanism. Both FLA ity of FLA and pmFLA were both found to be strongly
and pmFLA had no effect on the length of the roots or enhanced by actinic radiation. Photomodified BAP moder-
number of roots; however, root fresh weight accumulation ately inhibited root growth (25%) in the dark and visible
was clearly inhibited relative to the control. This phenomegrowth
light, while in SSR, UV activation increased inhibition of
non could be attributable, for example, to FLA causing a
to about 60%. Thus, only part of the toxic potential
mechanical disruption to the membranes which would of pmBAP is realized in the absence of UV radiation. Inter-
weaken the fragile cellular structure of the new roots (Taiz estingly, pmANT is the strongest inhibitor of root growth
and Zeiger, 1991). This could diminish the capability of the with a level of inhibition (75%) independent of lighting con-
cells to retain water, leading to a decline in root fresh weight ditions. Thus, the photomodification products of ANT are
accumulation without necessarily disturbing the elongation toxic without further photochemistry.
process.
In previous work with L. gibba the role of fluence rate
CONCLUSION
and spectral quality on the photoinduced toxicity of PAHs
was investigated (Huang et al., 1993; Ren et al., 1994). The toxicity of PAHs to a terrestrial plant species (B.
Given that vegetative growth of L. gibba is light dependent, napus) is strongly enhanced by actinic radiation when the
these experiments could not be performed in the dark to compounds are applied in intact form. Once PAHs are photo-
determine if there is an absolute requirement for actinic radicompounds.
modified, however, they become more toxic than the parent
ation to activate the toxicity of PAHs. However, terrestrial
Strikingly, photomodified PAHs are not abso-
plants can be germinated in the absence of light. During the lutely dependent on actinic radiation for inhibition of root
6-day dark germination experiments in 10 mg liter 01 of a growth. Because PAHs frequently enter the environment via
given PAH (highly toxic levels for each in SSR) no inhibitomodification
atmospheric and aquatic disposition, the probability of photory
affect on root growth was observed (Fig. 6). This was
in the environment is quite high. The photo-
in marked contrast to the inhibition seen in the presence of modified PAHs can then migrate to environmental compart-
SSR and visible light. In SSR, ANT (10 mg liter 01 ) inhibited ments that are not exposed to sunlight and exert toxic effects.
root fresh weight accumulation by 75%, while in the preswater
Furthermore, the photomodified products of PAHs are more
ence of visible light it did not inhibit growth. This is consismore
soluble than the parent compounds and therefore are
tent with ANT’s strong absorption in the UV region and lack
readily dispersed throughout the biosphere. In the past
of absorption in the visible region of the spectrum (Huang et intact PAHs were considered to be eliminated by exposure
al., 1993). Clearly, UV induced sensitization and/or photochemicals
to sunlight. This exposure, however, photomodifies the
modification reactions are necessary for intact ANT to intial
to numerous products that are themselves poten-
duce toxicity in plants. Both BAP and FLA (10 mg liter 01 )
environmental hazards. Thus, not only is it imperative
inhibited root fresh weight accumulation by 50% in SSR to identify the photomodification products from PAHs, it
and visible light. BAP absorbs both in the UV and in the is crucial to determine their toxicity and half-lives in the
visible regions of the spectrum (Huang et al., 1993), and so environment.
photosensitization and photomodification reactions can occur
in both of these spectral regions. The case with FLA is
ACKNOWLEDGMENTS
not as clear. In organic solvents, FLA does not absorb
strongly in the visible spectral region (Ren et al., 1994), yet The authors are grateful to Xiao-Dong Huang, Cheryl Duxbury, Bob
Gensemer, Mike Wilson, Brendan McConkey, and other members of the
the degree of root fresh weight inhibition was the same in Greenberg and Dixon labs for fruitful discussions. This research was sup-
SSR and visible light. It is possible that FLA exerts toxicity ported by a grant from the Canadian Networks of Toxicology Centres and
through a different mechanism than ANT and BAP, which NSERC Strategic and Research Grants to B.M.G. and D.G.D.
would be consistent with its different symptoms of stress
vis à vis root elongation. Additionally, the absorbance spec-
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