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Environmental Pollution 156 (2008) 332e340

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Effects of air pollution from road transport on growth and

physiology of six transplanted bryophyte species
Keeley L. Bignal*, Mike R. Ashmore1, Alistair D. Headley
Department of Geography and Environmental Science, University of Bradford, Bradford BD7 1DP, UK
Received 13 August 2007; received in revised form 5 February 2008; accepted 10 February 2008

Motor vehicle pollution has significant effects on the growth, membrane leakage,
chlorophyll and nitrogen content of bryophytes.
Abstract
Motor vehicles emit a cocktail of pollutants; however, little is known about the effects of these pollutants on bryophytes located in roadside
habitats. Six bryophyte species were transplanted to either a woodland or a moorland site adjacent to a motorway, and were monitored over
seven months from autumn through to spring. All species showed an increase in one or more of the following near the motorway: growth, membrane leakage, chlorophyll concentration, and nitrogen concentration. The strongest effects were observed in the first 50e100 m from the motorway: this was consistent with the nitrogen dioxide pollution profile, which decreased to background levels at a distance of 100e125 m. It is
hypothesised that motor vehicle pollution was responsible for the effects observed, and that nitrogen oxides had a key influence. The observed
effects may lead to changes in vegetation composition with significant implications for nature conservation and management of roadside sites.
2008 Elsevier Ltd. All rights reserved.
Keywords: Air pollution; Motor vehicles; Nitrogen oxides; Bryophytes; Transplant

1. Introduction
Air pollution from road transport is of continuing concern,
despite measures to control emissions, as traffic volumes continue to increase. Motor vehicles emit a complex mixture of airborne pollutants, many of which may have ecological effects.
These include nitrogen oxides (NOx), volatile organic compounds (VOCs), polycyclic aromatic hydrocarbons (PAHs),
metals, and particulates. Ammonia (NH3) and nitrous acid
(HONO) are also potentially important, particularly at the roadside (Truscott et al., 2005). Whilst there is evidence of effects of

* Corresponding author: Department of Biology and Environmental Science,

University of Sussex, Falmer, Brighton BN1 9QG, UK. Tel.: 44 0 1273
678389.
E-mail addresses: k.bignal@sussex.ac.uk (K.L. Bignal), ma512@york.ac.
uk (M.R. Ashmore), alistairheadley@aol.com (A.D. Headley).
1
Present address: Environment Department, University of York, York YO10
5DD, UK.
0269-7491/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2008.02.011

motor vehicle pollution on roadside verges (e.g. Truscott et al.,

2005), there is uncertainty about the effects on vegetation on adjacent sites of conservation interest (Bignal et al., 2004). Many
sites in urban areas or close to motorways are of conservation
value (NEGTAP, 2001); for example, an estimated 5.4% of Sites
of Special Scientific Interest (SSSIs) in England have areas
within 200 m of a major road (Weigert, 2004).
Evidence that pollution from road transport has the potential to adversely affect nearby ecosystems comes mainly
from field and fumigation studies on plants. These studies document effects on growth, physiology, enzyme activity, chemistry, senescence, leaf/needle surface wax degradation, and
planteinsect interactions (see review by Bignal et al., 2004).
Although most studies are on individual plants, some have
identified impacts in the field at the habitat scale. These include adverse effects on tree health in beech and oak woods
(Bignal et al., 2007) and changes in species composition in
blanket bog, heathlands and coniferous forests (Bignal et al.,
2007; Bernhardt-Romermann et al., 2006; Angold, 1997).

K.L. Bignal et al. / Environmental Pollution 156 (2008) 332e340

Many bryophyte species have been reported to be absent in

urban areas or close to pollution sources with high concentrations of smoke and sulphur dioxide; for example, in the 1960s
bryophyte diversity in Newcastles centre was one third of that
in rural areas 16 km away (Gilbert, 1968). The effects of urban
pollution mixtures dominated by emissions from motor vehicles are, however, unknown, although there is some evidence
of bryophyte sensitivity to NOx. Exposure to 35 ppb nitrogen
dioxide (NO2) or nitric oxide (NO) over 21 days affected
nitrate reductase activity and/or oxygen evolution in four bryophyte species, but the effect was species-specific and varied
over time (Morgan et al., 1992). Another study observed stimulated and then reduced growth in Polytrichum formosum exposed to 60 ppb NO2 for 37 weeks (Bell et al., 1992). Effects
of other individual motor vehicle pollutants on bryophytes
have not been assessed. Vehicular emissions may affect current bryophyte distributions through direct toxic effects, or
by increasing susceptibility to environmental stresses. Increasing traffic on existing roads or construction of new roads could
pose a risk for bryophytes in adjacent sites. Bryophytes are of
ecological importance and effects on this component of the
flora could have wider implications for the habitat with which
they are associated.
This paper presents a transplant study that was undertaken
to determine the responses of bryophytes to exposure to motor
vehicle pollution in the field. The transplant technique is an
established tool for biomonitoring pollution, and moss transplants have been used near roads or in urban areas as biomonitors because they accumulate metals, PAHs, and other
gaseous or particulate pollutants from the atmosphere (e.g.
Johnsen et al., 1983; Orlinski, 2002; Viskari et al., 1997). Little attention, however, has been paid to the biological effects
of air pollution on bryophyte transplants.
This study aimed to assess whether air pollutants from a major road had significant effects on growth and physiology of
six bryophyte species, and, if so, to assess the distance from
the road at which significant effects could be detected. The
effects on transplanted bryophytes were assessed in two different habitats by selecting sites adjacent to a motorway in the
north of England of contrasting habitat types: an oak woodland and a moorland. The species tested are sensitive to atmospheric pollution and may, therefore, be sensitive to vehicle
pollution. They include epiphytic and terricolous species, as
well as pleurocarpous and acrocarpous species.
2. Materials and methods
2.1. Site descriptions
The section of motorway (M62) that passes the sites was constructed from
1966e1972. The vehicular flow is average for a UK motorway: in 2002, there
was a mean of 74 000 and a maximum of 130 000 vehicles/day (DfT, 2003).
The woodland site, Bradley Wood, is adjacent to the northern edge of the
M62 between junctions 24 and 25, southeast of Brighouse, West Yorkshire
(NGR SE 154212). The site is w150e300 m wide and 1 km long, with
16.2 ha of secondary broad-leaved deciduous woodland and 2.4 ha of mesotrophic grassland. The woodland is dominated by mature Quercus petraea with
Acer pseudoplatanus and Betula pendula, and an understorey of Rhododendron

333

ponticum, Rubus fruticosus, Pteridium aquilinum, Hyacinthoides non-scripta

and Holcus mollis. The vegetation is Quercus roburePteridium aquilinume
Rubus fruticosus woodland (W10) of the National Vegetation Classification
(NVC). The limited epiphytes are confined to the base of trunks and rain tracts.
The moorland site, Moss Moor, is part of the South Pennine Moors Special
Area of Conservation and is on the southern edge of the motorway, near junction 22 (NGR SD 992142). The study area covered w275  365 m of blanket
bog dominated by Eriophorum vaginatum. The only other vascular plants of
note are E. angustifolium and Deschampsia flexuosa in this species-poor
sub-community of the Eriophorum vaginatum blanket and raised mire community (M20) of the NVC. Polytrichum commune dominates the bryophyte flora
and there are few lichens (mainly Cladonia/Cladina species). The area is
grazed by sheep.

2.2. Nitrogen dioxide concentrations

NO2 concentrations were monitored at both sites using diffusion tubes exposed for 14  3 days along three or four 200e250 m long transects perpendicular to the motorway. Further details are provided by Bignal et al. (2007).

2.3. Species collection

Bryophyte material was collected from sites in Scotland with relatively
low air pollution. Racomitrium lanuginosum, Rhytidiadelphus loreus, Hylocomium splendens and Pleurozium schreberi were collected from a blanket bog
on Ben Hulabie, Isle of Lewis, Scotland (NGR NB 3634). Isothecium myosuroides was collected from Alnus glutinosa trees by the River Broom, Ross-shire,
Scotland (NGR NH 1884). Dicranum scoparium was collected from Betula pubescens/pendula trees in a wood above Loch Garve in Easter Ross, Scotland
(NGR NH 408592). Material was stored in sealed poly-bags in the dark at
4  C for <5 weeks before transplantation; this was unavoidable due to the logistics of collection of the bryophytes and may have affected their physiological activity.

2.4. Transplant technique

Bryophyte material (30e35 replicates for each species) was transplanted to
both sites for seven months (autumn 2002 to spring 2003), 15e275 m from the
motorway, with the majority close to the road. The summer period was
avoided as it is difficult to maintain bryophyte transplants in warm, dry
weather when they are prone to desiccation. Additionally, bryophytes have
greater physiological activity in wetter conditions. Fifteen metres was sufficient to avoid the road verge, wind gusts from passing traffic, and limited
edgeeeffects of the Bradley Wood canopy, as transplants were within the
woods. At Bradley Wood, it was impractical to locate the transplants along
fixed linear transects; therefore, suitable host trees for each bryophyte species
were selected in a 400 m band across the site. At Moss Moor, transplants were
placed along three parallel transects 200e250 m long, 165 and 200 m apart,
orientated roughly northesouth (350 ), and at right-angles from the road.
At Bradley Wood, two squares (6  6 cm) of I. myosuroides were attached to
the north-facing trunk of each host oak tree, and two clumps of D. scoparium
were placed on the top of branches of each host sycamore tree. The choice of
host tree reflected the substrate requirements of the bryophyte species. D. scoparium required an approximately horizontal surface due to its size and upright
growth form and the sycamore trees provided sufficient accessible branches.
I. myosuroides is suited to a vertical substrate and is characteristically found
on oak trees. The transplants were 2.0e2.5 m high and secured with plastic
netting (10 mm square mesh) stapled to the trees. Young trees (with a DBH
<20 cm), and oak trees with an inclination of >10 , were rejected.
The Moss Moor transplants were placed on peat in perforated plastic
aquatic plant pots (120 mm top diameter, 90 mm basal diameter, 100 mm
deep) sunk into the ground. Perforations (2  3e5 mm) allowed water to
pass through the pots vertically and laterally. The pots were covered in plastic
garden netting (w15 mm diamond mesh, ACW) secured with stainless steel
pegs to prevent disturbance by grazing/foraging animals.

334

K.L. Bignal et al. / Environmental Pollution 156 (2008) 332e340

2.5. Measured parameters

Pigment concentrations and membrane leakage were measured half-way
through, and at the end, of the exposure period (i.e. after three and seven months).
Growth and nitrogen concentrations were measured at the end of the exposure
period, and visible pigment loss was assessed visually at monthly intervals.
Visible pigment loss (browning and/or bleaching) of the moss mat surface
was scored on material rewetted with deionised water on a scale of 0e7: 0,
none; 1, 1e10%; 2, 11e25%; 3, 26e50%; 4, 51e75%; 5, 76e90%; 6, 91e
99%; and 7, 100%.
Growth was measured in terms of shoot extension by tying coloured thread
to three shoots per transplant. The distance from the thread to the shoot tip was
measured at the start and after seven months.
Chlorophyll and carotenoids were extracted from five shoots from each
transplant combined to make one sample following the method of Raeymaekers (1986). The absorbance at OD 750, 665, 649, 435 and 415 nm was
determined using a UV-visible spectrophotometer (SP6-200 Spectrophotometer, Pye-Unicam). Wellburns equations (1994) were used to calculate chlorophyll and carotenoid concentrations. In addition, two ratios were calculated as
indicators of stress/vitality: the carotenoid index (CI) (OD 435:OD 665) and
the phaeophytinisation quotient (PQ) (OD 435:OD 415) (Martinez-Abaiger
and Nunez-Olivera, 1998). The CI expresses the relationship between brown
and green pigments and increases in stress conditions. Conversely, the PQ
decreases in stress conditions, and is indicative of the relative proportions of
chlorophyll a and phaeophytin a, and so is used to quantify chlorophyll a degradation (Martinez-Abaiger and Nunez-Olivera, 1998).
The nitrogen concentration of shoot tips of D. scoparium and R. loreus was
determined using a method adapted from Allen (1989), Grimshaw et al. (1989)
and Rorison et al. (1993). Approximately 0.1 g of oven-dried moss material
was accurately weighed and wet digested with 5 ml of 10% (w/v) salicylic
acid in N-free H2SO4 containing w1 g of catalyst (90% Li2SO4, 10%
CuSO4 and 1% Se powder) and 1 ml of 100 vol. H2O2. Digested samples
were made up to 25 ml with DI water before an aliquot (10 ml) was steamdistilled with w10 ml of 40% NaOH. The quantity of ammonium-N in the
distillate (plus 25 ml boric acid indicator solution) was determined by titration
with 0.01 N HCl(aq.).
Membrane leakage was determined in terms of loss of electrolytes and
potassium ions. Five shoots from each transplant were combined as one
sample and analysed following the method of Tarhanen et al. (1999).

2.6. Statistical analysis

Linear regressions were carried out between the distance from the motorway or log(distance from the motorway) and each of the measured parameters
(with the exception of visible pigment loss), separately for each species, in
SPSS. Potential outliers were identified through visual inspection of the scatterplots: points that were isolated from other points on the scatterplot and were
50% different from the regression line were excluded from the analysis. The
relationship between visible pigment loss and distance from the motorway
was tested with Spearmans rank correlation.

3. Results
At both sites NO2 concentrations were elevated near the
motorway: concentrations averaged almost 25 ppb and decreased exponentially to a background level of w15 ppb at
100e125 m (see Bignal et al., 2007). Roadside concentrations
were comparable to many UK urban centres, although the
background concentration was approximately double the average rural background level from the UK Automatic Monitoring Network in 2003 (NETCEN, 2003). At both sites NO2
levels alone exceeded the critical level for vegetation of
16 ppb NOx (NO plus NO2) as an annual mean, set in the
UKs Air Quality Strategy (Defra, 2007).

Table 1 shows the response of the transplants of each species

closest to the motorway (15 m) as a percentage of the response
at background levels (150 m away), calculated from the fitted
regression line. Figures greater than 100% indicate an increase
in the measured response near to the motorway, relative to background, and figures less than 100% indicate a decrease. By
quantifying the magnitude of the response, the relative sensitivity of the tested species can be assessed. All species showed
a significant effect of being transplanted different distances
from the motorway, but the responses differed between species
and over time (Table 1). All of the measured parameters, except
visible pigment loss and the CI, declined logarithmically or linearly with distance from the road (Figs. 1e4). In general, the
greatest effects were observed within 50e100 m of the road
(Figs. 1e4); this is consistent with the pollution profile in terms
of NO2 concentrations, which decreased to background levels
100e125 m from the motorway (Bignal et al., 2007).
All transplants showed some visible pigment loss, although
there was no clear relationship with distance from the motorway. It was evident as browning of the moss shoot tips, which
spread down the shoot over time, and in extreme cases led to
bleaching of the shoot tips.
Three of the four species transplanted to Moss Moor
showed a significant increase in growth close to the motorway
of 63e112% of that 150 m away, which is likely to be of biological significance (Table 1; Fig. 1). Growth was not significantly affected in the Bradley Wood transplants.
Chlorophyll and carotenoid concentrations increased near to
the motorway, in all species except R. lanuginosum, by 11e74%
relative to background concentrations 150 m away (Table 1).
However, a significant relationship was only evident at the first
harvest in P. schreberi and R. loreus for both chlorophyll and carotenoids, and in H. splendens for the carotenoid concentration
only. The magnitude of the effect lessened over time in all species except I. myosuroides. In general, the chlorophyll a:chlorophyll b ratio and the PQ decreased, whilst the CI increased, with
distance from the motorway in the Bradley Wood transplants of
D. scoparium and in the Moss Moor transplants of H. splendens,
P. schreberi and R. loreus. There was no significant relationship
between any of the pigment ratios and distance from the motorway in I. myosuroides (Bradley Wood) or R. lanuginosum (Moss
Moor). One species has been plotted to illustrate the nature of
the pigment data (Fig. 2); all species showed a similar pattern
where significant effects were detected.
The nitrogen concentration in the Bradley Wood transplant
of D. scoparium increased by 22% near the motorway relative
to background (Fig. 3); however, the nitrogen concentration in
the Moss Moor transplants of R. loreus was not significantly
affected (Table 1).
Membrane leakage, in terms of loss of electrolytes, was significantly greater close to the motorway in both species transplanted to Bradley Wood, and in H. splendens transplanted to
Moss Moor (Fig. 4). This occurred after three months in I. myosuroides and H. splendens, and after seven months in D. scoparium. No effects on membrane leakage were seen in the Moss
Moor transplants of R. lanuginosum, P. schreberi and R. loreus.
No significant effect on K leakage was observed.

K.L. Bignal et al. / Environmental Pollution 156 (2008) 332e340

335

Table 1
Index of response of the six bryophyte species transplanted to Moss Moor and Bradley Wood, next to the M62 motorway
Measurement

Time (months)

Response at 15 m relative to 150 m (%)

Bradley Wood

Growth
Chlorophyll a
Chlorophyll b
Total chlorophyll
Carotenoid concentration
Chl a:chl b
Phaeophytinisation quotient
Carotenoid index
N concentration
Membrane leakage:
initial conductivity
Membrane leakage:
relative conductivity
Membrane leakage:
initial K loss
Membrane leakage:
relative K loss

7
3
7
3
7
3
7
3
7
3
7
3
7
3
7
7
3
7
3
7
3
7
3
7

Moss Moor

Ds

Im

Hs

Ps

Ra l

Rh l

NS
172**
130*
148**
118*
174**
128*
163**
121*
107**#
NS
117**
NS
86**
95*
122**
NS
154*
NS
NS
NS
NS
NS
NS

NS
119*
158**
NS
128**
114*
149**
111*
125**
NS
NS
NS
NS
NS
NS
n/a
199**#
NS
146*
NS
NS
NS
NS
NS

189*#
135**#
163*
136**#
NS
149**#
149*
126**#
NS
NS
113*
NS
110**
NS
82*
n/a
336**
n/a
158**
n/a
NS
n/a
NS
n/a

212**
126*#
NS
NS
NS
117*#
NS
122*#
NS
NS
112*
NS
NS
NS
NS
n/a
NS
n/a
NS
n/a
NS
n/a
NS
n/a

163**
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
n/a
NS
n/a
NS
n/a
NS#
n/a
NS#
n/a

NS
118*
NS
NS
NS
114*
NS
116*
NS
NS
NS
103*
107**
95*
87**
NS
NS
n/a
NS
n/a
NS
n/a
NS
n/a

The response of the transplants closest to the motorway (15 m) is expressed as a percentage of the response at background levels (150 m away), calculated from the
fitted regression line. Values >100% denote an increase in the measured response next to the motorway and vice versa for values <100%. Asterisks (*) and (**)
indicate a significant relationship with distance from the motorway at the 5% and 1% levels, respectively; the hash (#) indicates that outliers were removed from the
regression analysis. NS, not significant; n/a, not applicable; Ds, D. scoparium; Im, I. myosuroides; Hs, H. splendens; Ps, P. schreberi; Rh l, R. loreus; Ra l, R.
lanuginosum.

4. Discussion
4.1. Effects on bryophyte growth, visible pigment loss
and physiology
Visible pigment loss has been observed in transplanted
bryophytes exposed to a range of atmospheric pollutants (Gilbert, 1968; LeBlanc and Rao, 1973; Sergio, 1987); however,
no studies have been conducted specifically in relation to motor vehicle pollution. Browning or dieback of transplanted
bryophytes can also occur without exposure to air pollution
(Hazell and Gustafsson, 1999; Rosso et al., 2001) and may
be caused by desiccation (Seel, 1991). The type of visible pigment loss observed was consistent with this, with greater
amounts in the Bradley Wood transplants.
Significantly increased growth near to the motorway was
found in three of the four species at Moss Moor but in neither
of the species at Bradley Wood. Bryophyte growth in response
to motor vehicle pollution in field conditions has not been
documented in the literature, although both relatively recent
invasions and extinctions of species from areas in the UK
have been attributed to increased traffic pollution and/or
NOx (Adams and Preston, 1992; Smith, 2001). The results
need to be considered in terms of the length of the study, as

exposure to NO2 under controlled conditions has been shown

to have varying effects through time on growth of the moss
Polytrichum formosum. Exposure to 60 ppb NO2 over
37 weeks initially stimulated growth, but subsequently old
shoot growth was reduced by 46% and new shoot formation
was reduced by 36% (Bell et al., 1992). The increased growth
of some species may result from a fertilisation effect of increased nitrogen deposition from NOx, HONO and/or NH3
emissions. The elevated tissue nitrogen concentration in
D. scoparium, and increased chlorophyll and carotenoid concentrations in five of the six species, is consistent with this
mechanism. However, despite increased nitrogen and pigment
concentrations at Bradley Wood, neither species showed a significant effect on growth.
Increases in chlorophyll concentration and degradation,
sometimes simultaneously, have been observed in lichens transplanted to urban areas and near roads (Carreras et al., 1998; Von
Arb et al., 1990). Chlorophyll degradation usually leads to a decrease in the chlorophyll a:chlorophyll b ratio. In this study, the
ratio increased by the motorway due to increased chlorophyll
a concentrations rather than loss of chlorophyll b. Similarly,
the increase in the PQ near the motorway (which usually decreases under stress/chlorophyll degradation) is likely to result
from the observed increase in chlorophyll a concentration,

K.L. Bignal et al. / Environmental Pollution 156 (2008) 332e340

336

H. splendens

Increase in shoot length (mm)

Increase in shoot length (mm)

y = 6.6 - 2.0log(x)
r = 0.40
p < 0.05

7
6
5
4
3
2
1
0

50

100

150

200

250

300

Distance from motorway (m)

Increase in shoot length (mm)

P. schreberi

12

y = 8.9 - 2.9log(x)
r = 0.48
p < 0.01

10
8
6
4
2
0

50

100

150

200

250

300

Distance from motorway (m)

R. lanuginosum

5.0

y = 4.9 - 1.3log(x)
r = 0.47
p < 0.01

4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0

50

100

150

200

250

300

Distance from motorway (m)

Fig. 1. Growth of (a) Hylocomium splendens, (b) Pleurozium schreberi, and (c) Racomitrium lanuginosum over seven months after transplantation at different distances from the M62 motorway on Moss Moor. The open symbol denotes an outlier that was excluded from the regression analysis.

rather than a decrease in phaeophytin a. Likewise, the decrease

in the CI (which usually increases in stress) can be explained by
the increase in carotenoids rather than a decrease in chlorophyll
(which was not observed). Hence it can be concluded either that
the bryophytes transplanted close to the motorway were less
stressed than those further away, or that these pigment indices
are not useful measures of stress induced by motor vehicle
pollution.
The increased nitrogen concentration in D. scoparium may
have derived from increased nitrogen in the stemflow and/or
throughfall of the host trees, in the vicinity of the motorway,
or from direct uptake of gaseous nitrogenous road transport
pollutants. Epiphyte tissue chemistry (including nitrogen concentration) responded to changes in rainfall and stemflow
chemistry in both a lichen (Lobaria pulmonaria) and a bryophyte (I. myosuroides) species in oak woodlands (Farmer
et al., 1991). Stemflow and throughfall chemistry was not
determined at Bradley Wood so this could not be investigated.
Elevated nitrogen concentrations have been observed at
roadsides in the epiphytic lichen Physcia adscendens (Gombert et al., 2003) and in bryophytes, including D. scoparium,
exposed to elevated atmospheric nitrogen deposition (Mitchell
et al., 2004; Bakken, 1995a,b; Baddeley et al., 1994). However, Pearson et al. (2000) did not find a correlation between
traffic density in London and tissue-N concentration of eight
moss species sampled from walls or roofs next to roads with
different traffic volumes, although traffic density does not

necessarily lead to increased rates of nitrogen deposition.

Although tissue-N concentration did not increase, there was
evidence that the bryophytes were taking up traffic-derived
nitrogen from the isotopic signature of the nitrogen in the
bryophyte tissue.
Elevated tissue-N may not be of benefit to the plant. Van
der Heijden et al. (2000) found that a nitrogen concentration
>1.5% was detrimental to photosynthesis, reduced water content and induced necrosis in Sphagnum recurvum. Increased
tissue-N may also lead to increased grazing pressure due to
improved palatability, as increased grazing and/or outbreaks
of herbivorous insects has been observed in roadside vascular
plants (e.g. Port and Thompson, 1980; Martel, 1995) and
epiphytic lichen and bryophytes (Glenn et al., 1993), and bryophytes are grazed by birds, animals and many small invertebrates (Richardson, 1981). A higher critical nitrogen
concentration of 2% was determined by Pitcairn et al.
(1998) for ecotohydric woodland mosses. The nitrogen concentration of R. loreus was generally <1.5%, while that of
D. scoparium was up to 3% near to the motorway. However,
the critical concentration for tissue-N is likely to differ
between species and may be related to the nutrient status of
the habitat in which they occur. Although there is limited
evidence of adverse effects in this seven-month study, it is possible that longer-term exposure could increase nitrogen concentrations near the motorway above critical thresholds for
adverse effects.

K.L. Bignal et al. / Environmental Pollution 156 (2008) 332e340

Total chl concentration (g

mg air d wt-1)

total chlorophyll

3.0
2.5
2.0
1.5
1.0

2.0
1.5
1.0

y = 2.9 - 0.3log(x)
r = 0.42
p < 0.05

0.5

0.5
0.0

chl a/chl b ratio

3.0
2.5

y = 2.1 - 0.5log(x)
r = 0.40
p < 0.05

Chl a/chl b ratio

50

100

150

200

250

0.0

300

50

Distance from motorway (m)

337

Carotenoid Index

5.0

100

150

200

250

300

Distance from motorway (m)

Phaeophytinisation Quotient

1.4

4.5
1.2

3.5
3.0
2.5
2.0
1.5

y = 2.1 + 0.6log(x)
r = 0.38
p < 0.05

1.0
0.5
0.0

50

100

150

200

250

300

OD 435/OD 415

OD 435/OD 665

4.0

1.0
0.8
0.6
y = 1.2 - 0.1log(x)
r = 0.46
p < 0.01

0.4
0.2
0.0

50

100

150

200

250

300

Distance from motorway (m)

Distance from motorway (m)

Fig. 2. Pigment ratios and chlorophyll concentrations in Hylocomium splendens seven months after transplantation at different distances from the M62 motorway
on Moss Moor. Chlorophyll a, chlorophyll b, and carotenoid concentrations in all species tested showed the same pattern as total chlorophyll where they were
statistically significant.

The increased membrane leakage of electrolytes near the

motorway in D. scoparium, I. myosuroides and H. splendens,
indicates that these species were suffering adverse effects
from exposure to the motor vehicle pollution. In addition,
changes in substrate chemistry near to the motorway arising
from 30 years of deposition of motor vehicle pollutants may
have adversely affected the transplants. Although no data are
available in the literature on bryophyte membrane leakage in
relation to motor vehicle pollutants, increased membrane
leakage in response to ozone have been observed in
3.5
y = 2.3 - 0.003x
r = 0.46
p < 0.01

3.0

N (%)

2.5
2.0
1.5
1.0
0.5
0.0

50

100

150

200

250

300

Distance from motorway (m)

Fig. 3. The nitrogen concentration in Dicranum scoparium shoots seven months
after transplantation to sycamore trees at different distances from the M62
motorway within Bradley Wood.

S. angustifolium and S. recurvum (Niemi et al., 2002; Potter

et al., 1996). Lichens collected from sites next to paved highways near urban centres always had higher conductivity values
of the leachate than those adjacent to dirt roads in rural areas
where the traffic levels would presumably be lower (Pearson,
1985). This indicates that the increase in conductivity close to
the motorway was due to leakage (probably from pollution
exposure) and not from washing of adhered particles from
the surface of the lichen. However, the lack of any significant
effect on potassium leakage does suggest that there is no leakage from within the cell.
Thus, all of the transplanted bryophyte species showed an
increase in one or more of the following close to the motorway: growth, chlorophyll/carotenoid concentrations, nitrogen
concentration, and membrane leakage. Apart from the lack
of growth response in the two epiphytic transplants, there
was no clear difference in the response of epiphytic and
terricolous transplants. There were differences between individual species, both in terms of the magnitude and type of
response observed. Racomitrium lanuginosum showed the
fewest number of responses to motor vehicle pollution in
terms of the parameters tested, as significant effects were
only seen on growth. However, it could be argued that
R. loreus was the least sensitive species as pigment concentrations were only affected at the first harvest, membrane leakage
was not affected, and growth was unaffected (Table 1).
Conversely, H. splendens, D. scoparium and I. myosuroides

K.L. Bignal et al. / Environmental Pollution 156 (2008) 332e340

338

I. myosuroides - initial conductivity after three months

4.5
4.0

y = 3.5 - 1.1log(x)
r = 0.56
p < 0.01

3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0

50

100

150

200

b
Relative conductivity (%)

Conductivity
(s cm-1 mg d wt-1)

250

300

I. myosuroides - relative conductivity after three months

70
60

y = 57.7 - 13.3log(x)
r = 0.40
p < 0.05

50
40
30
20
10
0

c
Conductivity
(s cm-1 mg d wt-1)

1.6

y = 1.3 - 0.5log(x)
r = 0.57
p < 0.01

1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0

50

100

150

200

250

300

Conductivity
(s cm-1 mg d wt-1)

1.4

D. scoparium - initial conductivity after seven months

1.2

y = 0.8 - 0.002x
r = 0.39
p < 0.05

1.0

100

150

200

250

300

H. splendens - relative conductivity after three months

35
y = 33.9 - 8.7log(x)
r = 0.55
p < 0.01

30
25
20
15
10
5
0

50

100

150

200

250

300

Distance from motorway (m)

Distance from motorway (m)

d
Relative conductivity (%)

H. splendens - initial conductivity after three months

1.8

50

Distance from motorway (m)

Distance from motorway (m)

0.8
0.6
0.4
0.2
0.0

50

100

150

200

250

300

Distance from motorway (m)

Fig. 4. Membrane leakage as measured by initial and relative conductivity of Isothecium myosuroides, Hylocomium splendens, and Dicranum scoparium shoots
three (aed) or seven months (e) after transplantation at different distances from the M62 to Bradley Wood or Moss Moor. The open symbol denotes an outlier that
was excluded from the regression analysis.

appear to be the most sensitive to motor vehicle pollution as

they showed the greatest number of responses, the effects on
pigments persisted until the final harvest, and the magnitude
of the responses was generally greater than for the other species tested.
For those species that showed increased growth near to the
motorway, it seems that proximity to the road is of overall benefit, at least over the seven-month period of this study. However, for those species that showed no clear growth response
it is unclear whether exposure to motor vehicle pollution
over the seven months is of overall advantage or disadvantage
as both positive (increases in chlorophyll) and potentially negative (increased membrane leakage) effects were observed.
Whether a species is able to utilise the extra nitrogen supply
in the vicinity of roads and repair membrane damage from pollution exposure, may be key to determining whether overall

increases or decreases in chlorophyll concentration and growth

occur.
4.2. Edgeeeffect distance
Effects on growth, physiology and visible injury have been
observed in trees, shrubs and herbaceous plants up to 100e
200 m from roads (Bignal et al., 2004); however, no data
have been published on bryophytes. In addition, little is known
regarding bryophyte distribution in relation to roads. At Moss
Moor the moss Polytrichum commune was found to be significantly more abundant up to w50 m from the road (Bignal
et al., 2007). Figs. 1e4 indicate that the edgeeeffect distance
of the road was similar for both epiphytic (Bradley Wood) and
terricolous (Moss Moor) transplants and extended to w100 m.
This is consistent with studies on plant health and species

K.L. Bignal et al. / Environmental Pollution 156 (2008) 332e340

distribution at these and other roadside sites in the UK and

Europe (Bignal et al., 2007; Angold, 1997; Bernhardt-Romermann et al., 2006).
Bignal et al. (2007) found increased defoliation and insect
damage in oak trees at Bradley Wood and poorer crown condition in beech trees adjacent to another UK motorway, up to
100 m from the road. At Moss Moor there was a greater cover
of nitrogen-tolerant vascular plant species up to 75 m from the
motorway. In Angolds study, effects on dry heathlands in
southwest England were found up to a maximum distance of
200 m from a dual carriageway with 35 000 vehicles per 12 h
(Angold, 1997). Increased growth was observed in vascular
plants, especially Calluna vulgaris (heather) and grasses such
as Molinia. Cladonia species decreased in cover, abundance,
and number close to the road, with effects on size up to 80 m
away. These effects were consistent with those from experimental studies of enhanced nitrogen deposition on dry heathlands,
and the author attributed them to NOx. Bernhardt-Romermann
et al. (2006) also related vegetation composition to distance
from the road, but in coniferous forests in southern Germany.
The edgeeeffect of two motorways (with 64 000 and
103 300 vehicles/day) extended 230 m downwind of the road,
and 80 m upwind. They observed an increased abundance in nitrogen-tolerant species near to the motorway. As estimated nitrogen deposition, and ammonium and nitrate levels in the soil
were elevated up to 230 m from the motorway, the authors concluded that the effects on the vegetation were driven by changes
in nitrogen loading of the system. From the soil chemistry, basic
compounds from winter road salt applications were also assumed to have an influence.
Attribution of the observed effects in our study to specific
pollutants is difficult, as only NO2 was measured. However,
as effects were observed up to 50e100 m, a significant contribution from certain pollutants is unlikely. Particulates (including metals and particle-bound PAHs) and gases with a high
deposition velocity (e.g. HONO and NH3), decrease rapidly
in concentration (Truscott et al., 2005) and are unlikely to be elevated beyond 15 m. Effects of salt and other spray are also unlikely. Soil salinity decreased rapidly with distance from
motorways sampled in England, including the M62; beyond
2 m soil salinity was very low, except in a severe winter
(Thompson et al., 1986). Gases with a lower deposition velocity
(e.g. NO and NO2) will decrease in concentration less rapidly.
The consistency between the edgeeeffect distance for the majority of the measured parameters and the NO2 pollution profile,
suggests that the effects were mainly caused by NOx or by other
air pollutants with a similar deposition velocity. Additionally,
the effects observed are broadly consistent with the known
mode of action of nitrogenous pollutants, particularly NOx. Increased growth, chlorophyll concentrations and nitrogen concentrations can all be attributed to a greater nitrogen input
near to the motorway, whilst NOx can also disrupt cell membranes, leading to leakage of electrolytes.
Background pollution levels and climatic variables at other
sites may exacerbate or alleviate the effects of motor vehicle
pollution, thereby changing the edgeeeffect distance. However, the evidence from this study, and from others in the

339

literature, suggests that effects on vegetation are likely up to

a maximum distance of 100e200 m from busy roads. Therefore, based on our data and that in the literature, new road
building and road expansion should avoid a buffer zone of
up to 100e200 m from sensitive sites, particularly those
containing a key bryophyte component.
Acknowledgements
Funding was provided by NERC and the University of
Bradford.
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