Download (Avicennia marina (Forsk.) Vierh.) seedlings to spills of crude oil

J. Exp. Mat’. Biol. Ecol., 171 (1993) 273-295
© 1993 Eisevier Science Publishers B.V. All rights reserved 0022-0981/93/$06.00
273
JEMBE 02015
The response of grey mangrove (Avicennia marina (Forsk.)
Vierh.) seedlings to spills of crude oil
D.L. Grant, P.J. Clarke and W. G. Allaway
School of Biological Sciences, The University of Sydney, N.S. W., Australia
(Received 3 December 1992; revision received 10 May 1993; accepted 26 May 1993)
Abstract: The effects of crude oil on grey mangrove seedlings were examined in glasshouse and field
experiments. In a glasshouse experiment, fresh oil was found to cause greater leaf loss than aged oil. Fresh
oil, which does not resemble oil washed into mangroves, should, therefore, not be used as a treatment in
oil spill studies since it may exagerate effects. A field experiment examined the effects of weathered B ass Strait
crude oil (11/m2) on seedling survival. The results were unequivocal: 96.4~o of seedlings treated with
weathered Bass Strait oil died within 14 days, while all untreated seedlings survived. A further field experiment also examined the effects of light, canopy gaps and Bass Strait crude oil in the sediment on propagule
establishment and survival. Establishment and survival were not enhanced by light and canopy effects but
crude oil in the sediment inhibited establishment and decreased the number of seedling surviving for several years. We predict the population response of a stand of Avicem~ia marina (Walp.) Moldenke to a series of oil spills frpria previous studies of population dynamics and the results of our experiments.
Key words: Avicennh7 marina; Botany Bay; Field experiment; Mangrove; Oil spill
INTRODUCTION
Mangroves are trees and shrubs that grow in sheltered intertidal areas (Macnae,
1968). Mangrove propagules are usually buoyant and their dispersal will depend on the
prevailing winds, currents and tides (Chapman, 1976; Saenger, 1982; Clarke, 1993a).
When petroleum hydrocarbons are discharged in the marine environment their fate will
be determined by the same factors. Consequently, spilt oil often ends up stranded in
the intertidal mangrove zone (Lewis, 1983). It is not surprising then that following an
oil spill mangrove propagules may end up stranded on oil-contaminated sediments.
Avicetmia marina var. australasica (Walp.) Moldenke is the dominant mangrove
growing on the shores of Botany Bay, a busy industrial port 12 km south of Sydney,
Australia. Transport of oil associated with two large oil refineries adjacent to the bay
has resulted in oil spills. Between 1955 and 1987, 31 spills ~vere recorded in Botany
Bay with an average size of 49 000 1 (McGuinness, 1988). Several of these spills have
contaminated the mangroves on the southern shore of the bay (Allaway et al., 1985;
Anink et al., 1985). The dispersal of oil following a spill may be quite slow: the adCorrespondence address: W. G. Allaway, Schooi of Biological Sciences, The University of Sydney, N.S.W.
2006, Australia.
274
D.L. GRANT ET AL.
vection of an oil slick on the sea surface is estimated to be between 1.3 and 3.5~o of
the wind velocity (Nelson-Smith, 1972). In previous spills in Botany Bay, it has taken
from 3 to 6 h for the oil to travel from the point of discharge to the mangrove shore
(SPCC, 1984). During this time the oil would have undergone physical, chemical and
biological alteration (Harrison et al., 1975; McAuliffe, 1976; Atlas, 1981). However,
fresh oil is used as a treatment in the majority of experimental investigations of the
effects of oil spills on the biota (e.g. Baker, 1971; Mathias, 1977; Lai et al., 1984;
Allaway et al., 1985; Getter et al., 1985).
Studies of the effects of oil spills on mangroves in Botany Bay have reported a variety of responses ranging from no observed effect (Allaway et al., 1985; McGuinness,
1990) to widespread seedling mortality and dieback of mature trees (Allaway, 1982;
Anink et al., 1985). This is consistent with reports in the international literature (for
a review see Wardrop, 1987). A suite of sublethal effects including anomalous aerial
pneumatophores (Snedaker et al., 1981; Allaway et al., 1985), expanded lenticels, periderm fissuring and reduction in leaf size (Getter et al., 1985) has also been associated
with oil contamination. The bulk of the literature purporting to show effects of oil spills
on mangroves is based upon post-spill observations (e.g. Diaz-Piferrer, 1964; Rutzler
& Sterrer, 1970; Spooner, 1970; Jernalov & Linden, 1981; Jackson et al., 1988). While
effects appear to be obvious, inferences drawn from such observations are usually
weak. Authors appear to confuse replication which requires the repetition of a treatment with repeated observations. Large scale field experiments have also been susceptible to pseudoreplication where subsamples of a single treatment such as a single
oiling are regarded as replicates (see e.g. Getter et al., 1985). Despite the methodological fla~vs, observed effects are often so gross that stochastic processes can be ruled out
as a likely explanation for their occurrence. Useful conclusions can be drawn from
post-even studies if the effects can be correlated with the presence of the contaminant
and if causal links have already been established (Underwood & Peterson, 1988). A
causal relationship between oil contamination in the field and the mortality of seedlings
of Avicennia marina has yet to be experimentally established.
The longer term effects of oil persisting in the sediment have received considerably
less attention than the immediate effects of oil contamination on man~roves. Areas of
oiled mangroves in Botany Bay, which were reported to have suffered extensive mangrove dieback (Allaway et al., 1985), now show signs of recolonisation by seedlings
(McGuinness, 1990; Clarke & Allaway, 1993). Other areas which suffered a high incidence of seedling mortality following spills in 1979, 1981 and 1985 (Allaway et al.,
1985; Anink et al., 1985) but in which the canopy remained intact have also been
recolonised by seedlings. Observations like these have been used to suggest that mangroves in Botany Bay display no long-term effects of oil spills. McGuinness (1990)
sampled five sites in Botany Bay to measure abundances of macro-invertebrates in oiled
and unaffected mangrove areas. He found significantly lower densities of Avicennia
seedlings in two oil-contaminated sites than in two unaffected sites, but the lowest
density of seedlings was recorded for a fifth site that he had designated as unoiled.
MANGROVE RESPONSE TO OIL SPILL
275
Although the site was not contaminated by spills in 1981 and 1985, it was oiled in the
1979 World Encouragement spill (Allaway et al., 1985) and sediment samples from the
site were found to contain high levels of hydrocarbons (Anink et al., 1985).
Re-examination of the data suggest that there is a pattern of difference in seedling
density between previously oiled and unoiled sites in Botany Bay. Data collected after
oil spills have occurred are difficult to interpret in the absence of baseline data. Spatial heterogeneity within the mangroves (see Clarke & Myerscough, 1993), also makes
it difficult to obtain suitable control sites for comparison. At best such data can be used
to infer an association between the event of an oil spill and the pattern of difference.
Synergisms and other types of interactions between factors cannot easily be explored.
The conditions influencing establishment of mangrove propagules in areas where
dieback has occurred and in oil-contaminated areas where the canopy has remained
intact will be different. While the effects of oil-contaminated sediments may be a feature common to both areas, other conditions associated with dieback may enhance
seedling establishment. Several models could be put forward to account for observed
differences in seedling density. These models must take into account the following
factors: (1) oil in the sediment may inhibit seedling establishment; (2) seedlings establishing in dieback areas will be exposed to full sunlight whereas seedlings establishing
under the canopy will experience low light conditions. Light has been implicated as an
important factor in seedling establishment and survival (Saenger, 1982; Smith, 1987;
Clarke & Allaway, 1993); (3) the absence of trees may result in a reduction in competition for nutrient or root space. Manipulative field experiments can be designed in
such a way that competing models can be eliminated and interactions between factors
can be identified.
The aims of this study were to examine effects of crude oil on seedlings of Avicennia
marina in Botany Bay and to predict the response of a population to repeated crude
oil contamination. A preliminary glasshouse experiment compared the effects of fresh
and aged oils on seedlings. Two oils, Bass Strait crude and Light Arabian crude, which
are commonly spilt in Botany Bay, were used as treatments. The effects of aged Bass
Strait crude oil on seedlings were then examined in a field experiment. A second manipulative field experiment examined the effects of light, the absence of trees, and
sediments contaminated with Bass Strait crude oil, on establishment and survival of
Avicennia marina seedlings.
METHODS
STUDY SITES
The study sites for the field experiments were in Weeney Bay, a small enclosed bay
on the southern shore of Botany Bay, NSW, Australia (Fig. 1). Two species of mangrove are found in this area: Avicennia marina var. australasica (Walp.) Moldenke,
which is the most abundant species, and Aegiceras corniculatmn (L.) Blanco (Clarke
&Hannon, 1969). The latter is common but less abundant and in Botany Bay it usually
276
D.L. GRANT ET AL.
Botany Bay
Fig. 1. Map of Botany Bay in south-eastern Australia showing the field study sites.
grows at the landward edge of the mangroves. Mature trees of Avicennia marina grow
to heights of 5 to 10 m and occur in a band parallel to the shore 150 m wide at Site
1 and 75 m wide at Site 2. The algal flora within the mangrove zone is depauperate
in species, although some species are abundant. A conspicuous feature of the lower end
of the study site is a dense carpet of the brown alga Hormosira banksii (Turner) Dec.
The pneumatophores of Avicennia marina are often covered with epiphytic algae of the
Bostrychia-Caloglossa association (King & Wheeler, 1984). The epifauna found commonly within the study sites, includes several species of gastropods - Bembicium
auratum (Quoy and Gaimard), Salinator solida (Von Martens), Ophocardelis ornatus
(Ferussac), Melosidula zonata (H. and A. Adams), Austrocochlea constricta (Lamarck),
Assiminea tasmanica (Tenison-Woods) and Pyrazus ebeninus (Bruguiere). The crab
Heloecius eordiformis (H. Milne Edwards) is also abundant and its activities have a
significant effect on the microtopography (Warren & Underwood, 1986). Avieennia
trunks and pneumatophores may be encrusted with barnacles and the Sydney rock
oyster Saccostrea commercialis (Iredale and Roughley).
EFFECTS OF AGED OILS; PRELIMINARY GLASSHOUSE EXPERIMENT
Fruits ofA vicennia matYna collected from many trees in Weeney Bay were propagated
in plastic pots on a medium of washed sand saturated with a solution of 100~o seawater and nutrients. After 5 months, 30 healthy seedlings ~ 24 cm in height were selected. Samples of two fresh oils, Bass Strait crude and light Arabian crude, were aged
on seawater in plastic bins at ambient temperature (16 ° C). Aged oil was scooped from
the water surface after 24 h separating it from any of the lighter fraction that may have
MANGROVE RESPONSE TO OIL SPILL
277
dissolved in the water. Oil aged in this manner is similar to oil that has been floating
on the open water for 6-8 h (McAuliffe, 1976). Plants were randomly assigned to one
of five treatments: fresh Bass Strait crude oil (BF); aged Bass Strait crude oil (BA);
fresh light Arabian crude oil (AF); aged light Arabian crude oil (AA); and no oil (NO).
Each treatment was replicated six times (n = 6). To avoid the problem of a gross effect obscuring any differences between the oils, a low dose was selected, based upon
A!laway et al. (1985) finding that 200 ml.m-a did not kill all the glasshouse grown
seedlings.
Individual plants still in their pots were placed in plastic bins, 80 cm deep. Sea water
was gravity fed into the bins at a rate approximating the rising tide. When the water
had risen to a height covering the plants it was released at a rate approximately the
falling tide. A measured dose of oil equivalent to 200 ml.m-2 was added to the bins
when the water level had risen to a height of 8 cm. Seedlings were coated with oil as
the artificial tide rose and fell. After treatment the plants were taken to a low light
greenhouse. The soil was kept waterlogged and the evaporated soil water was regularly
replaced with fresh water to maintain a constant salinity. At intervals throughout the
experiment, the soil solution was completely exchanged by flushing ~vith 100~o sea
water. Individual leaves were followed on each plant throughout the course of the
experiment. Over the next 96 days, the plants were monitored for leaf initiation, defoliation and mortality.
FIELD EXPERIMENTS:
EFFECTS OF AGED BASS STRAIT CRUDE OIL ON AVICENNIA
MARINA SEEDLINGS
Various methods have been employed to apply oil in field experiments including
directly spraying oil on to plants (e.g. Wardrop et al., 1987) and floating oil on the water
and letting the tide apply the oil. The latter method requires some containment of the
spill but resembles more closely the way plants are contaminated in a real oil spill.
Booms have been used to great effect (e.g. Teas et al., 1987; Jackson et al., 1988) but
the deployment of booms is very costly and more suited to larger scale investigations
of the effect of oil on mature trees. In seedling experiments zinc boxes (Lai et al., 1984)
and plastic sheeting (McGuinness, 1990) have been used to enclose the oil. These
enclosures rely on water seepage to be filled or drained and cannot be relied on to
prevent oil seepage into the environment. McGuinness (1990) partially dealt with this
problem by sucking up residual oil after the treatment. In a real oil spill the slick usually
remains stranded in the intertidal area for several days (Wardrop, 1987) and seedlings
may be exposed to the oil for more than one tidal cycle. Taking these factors into
consideration, an apparatus was developed for oiling seedlings in the field. It consisted
of a large perspex box open at two ends and reinforced on all four vertical corners with
aluminium. The dimensions of the fully assembled box were 90 x 90 x 120 cm deep.
When the box was placed on mangrove sediments it sank into the mud effectively
sealing it. Drains were placed in the sediment to allow water flow: each drain consisted
278
D.L. GRANT ET AL.
of two 50 cm lengths of PVC piping joined by a "U" bend. The pipes were buried in
the sediment with the two open ends flush to the ground. In addition to acting as an
inlet and outlet for tide driven water, the drain acted as a reservoir for residual oil. Oil
was unable to pass through the pipe because it remained full of water. Four identical
boxes were made. Two sites in Weeney Bay were selected for the experiment (Sites 1
and 2, Fig. 1). The experiment was repeated at three different times (and locations) in
Site 1 and two different times (and locations) in Site 2. The five separate experiments
were performed sequentially, starting at Site 1 in January 1987 and finishing at Site 2
in March 1989.
In each experiment, four plots were randomly selected in the mid-tidal region. (The
landward edge of the mangroves was not used, to make sure that the seedlings were
completely inundated by the high tide and thus uniformly covered with oil.) Seedlings
were tagged with surveyors tape so that they could be individually identified. Drains
were dug into each plot and the boxes were fixed into position with wooden stakes.
Two of the boxes were treated with oil. The remaining pair of boxes were not oiled.
Oil was aged for 24 h in the manner described in the glasshouse experiment. A measured dose of aged Bass Strait Crude oil (1 1.m-2) was applied to the surface of the
water on a rising tide in the two oil treatment boxes. This dose had caused seedling
mortality in previous glasshouse experiments (Allaway et al., 1985) and is not unrealistic for a possible oil spill in Botany Bay. Boxes were removed after the surface slick
had disappeared but drains were left in place until the end of each experiment. Each
box was monitored for seedling mortality.
FIELD EXPERIMENT: EFFECTS OF OIL IN THE SEDIMENT ON ESTABLISHMENT AND
SURVIVAL OF AVICENNIA MARINA SEEDLINGS
A field experiment with five factors was chosen as the basic experimental design.
There were three orthogonal factors: canopy (fixed, with two levels - canopy gap and
canopy present); light (fixed, with two levels - full sunlight and shade); and oil (fixed,
with two levels - oil and no oil). Two nested factors, areas (random and nested in
canopy with two levels) and plots (random and nested in light and areas with two levels)
provided a measure of spatial heterogeneity. Figure 2 shows a schematic diagram of
the experimental design.
Canopy
The canopy factor was incorporated to test for fight independent effects due to the
presence or absence of trees. This required the large scale mechanical removal of
Avicennia trees from two areas to simulate dieback. An additional criterion was that
the sediment in these areas should be free from oil contamination. In the interest of
conserving mangroves, areas were sought where the vegetation had been recently removed. Several areas were available at Site 1 in Weeney Bay (Fig. 1) where swathes
MANGROVE RESPONSE TO OIL SPILL
CANOPY
AREA A
279
CANOPY GAP
AREA B
Ught * F.~b
AREA A
~
AREA B
a
Light
Light
Shade
[
~
a
Light *
F
Shade
~b
~b Shade*
Light *
Light
Shade
Light
Shade *
Detail of plot a
Fig. 2. Schematic diagram sho~ving the arrangement of the five actors in the substrate experiment. The
treatments were: canopy, light, oil, areas, and plots. Light * indicates removal of branches above the plot
to create conditions of full sunlight. Shade * indicates that an artificial canopy was constructed above the
plot to produce shade conditions.
of vegetation from the high water mark down had been cleared to provide access for
oyster farmers. Two of these areas ~ 15 x 150 m were selected as canopy gap areas A
and B. Two randomly selected vegetated areas were used as canopy areas A and B.
Light
To test for effects due to light, two plots exposed to full sunlight and two shaded plots
were required in each of the four "areas". In each area four random plots 2 x 2 m were
pegged out. In the two "areas" where the trees had been removed all plots were initially exposed to full sunlight. In each of these areas wooden frames were constructed
over two plots and covered with shadecloth to approximate shade conditions under the
canopy. In the "areas" with intact canopy all plots were initially shaded. Branches
responsible for the shading were removed from two plots in each area to create conditions of full sunlight.
Crude oil
In each of the 16 plots, four squares 0.5 x 0.5 m were randomly selected and pegged
out. A measured dose equivalent to 1 l’m-a Bass Strait Crude oil was applied to the
280
D.L. GRANT ET AL.
substrate in two of the squares. The remaining two squares were left unoiled. Plastic
covers were placed over all of the squares for 24 h to prevent contamination of the water
column and surrounding environment with oil. Six hundred-forty ripe fruits with pericarps intact were collected from Avicennia trees around Weeney Bay. Ten fruits or
propagules were placed on each of the squares and a polythene mesh cage measuring
0.5 x 0.5 x 0.15 m was fixed in position over each square. The 0.01 m mesh prevented
the seedlings from drifting away but allowed water, sunlight and small animals to pass
through. These cages were removed after the establishment of seedlings. The establishment and survival of seedlings was monitored for a 2-year period.
RESULTS
EFFECTS OF AGED OILS~ PRELIMINARY GLASSHOUSE EXPERIMENT
Physical differences between the oils, particularly in the way in which the oils spread,
~vere immediately apparent when the oils were poured on to the water. The differences
~vere consistent with the phenomenon described by McAuliffe (1976) of viscous oils
breaking up at cooler temperatures. Fresh Bass Strait crude oil which was more viscous than the fresh light Arabian oil broke up into large patches. During the aging
process, the aged Bass Strait crude oil formed discrete waxy globules that floated on
a thin opaque oil film. When the oil was applied to the plants, the manner in which
they were coated was also different. The fresh oils and the aged light Arabian crude
covered the above ground parts of the plant more uniformly than the aged Bass Strait
crude oil where distinct globs of oil adhered to the leaves and stem. Over the experimental period (96 days), five of the six seedlings treated with fresh light Arabian oil died.
+ NO,BA,AA
~ BF
"--O--- AF
20
40
60
80
Days since treatment
100
Fig. 3. Mortality ofAvicennia marhta seedlings over a 96 days in the glasshouse experiment. Plants have been
exposed to five treatments: no oil (NO); aged Bass Strait crude oil (BA); fresh Bass Strait crude oil (BF);
aged light Arabian crude oil (AA); fresh fight Arabian crude oil (AF).
MANGROVE RESPONSE TO OIL SPILL
281
2018
14
6
4
0
0
10 20 30 40 50 60 70
80 90 100
Days following treatment
Fig. 4. Mean number of leaves on Avicennia marina seedlings over 96 days in the glasshouse experiment.
Plants have been exposed to five treatments: no oil (NO); aged Bass Strait crude oil (BA); fresh Bass Strait
crude oil (BF); aged light Arabian crude oil (AA); fresh light Arabian crude oil (AF) (n = 6).
The only other seedling to die during the experiment ~vas one that had been treated with
fresh Bass Strait crude oil (Fig. 3).
Sub-lethal effects
The number of leaves on all seedlings was fairly constant for the first 38 days. The
plants that had not been treated with oil displayed a steady rate of leaf initiation and
development so that the number of leaves had doubled by 96 days. The number of
leaves on plants treated ~vith aged oils increased slightly over the 96-day period, but
TABLE I
One-factor analyses of variance of the effects of oil on the proportion of new 1eaves and the proportion of
leaves lost 96 days after treatment. In this and following tables: degrees of freedom (dO; sums of squares
(SS); mean squares (MS); ***p<0.001. (Cochrans’s tests on both sets of data were not significant).
Source
df
Proportion of new leaves
Proportion of leaves lost
SS
MS
F-ratio
SS
MS
F-ratio
2.28
0.09
24.02***
4.31
2.41
1.08
0.10
11.16"**
Betxveen treatments
Within treatments
4
25
9.13
2.38
Total
29
11.51
6.73
282
D.L. GRANT ET AL.
TABLE II
Results of multiple comparisons test (SNK tests) on the proportion of new leaves and the proportion of leaves
lost 96 days after treatments. The treatments were: fresh Bass Strait crude oil (BF); aged Bass Strait crude
oil (BA); fresh light Arabian crude oil (AF); aged light Arabian crude oil (AA); no oil (NO). Level of significance p < 0.05.
Proportion of new leaves
Ran order
Treatments
Mean
1
2
3
4
BF = AF = BA = AA<
0.10 0.11
0.34
0.45
Proportion of leaves lost
5
NO
1.59
1
2
3
NO = AA = BA<
0
0.02
0.03
4
5
BF = AF
0.55 0.94
there was a steady decline in leaf number of those plants treated with fresh oils (Fig. 4).
The first signs of leaf damage were apparent within 3-4 days when necrotic blotches
appeared on the leaves in areas closely associated with visible patches of oil. In plants
treated with fresh light Arabian oil, defoliation was concomitant with the death of the
stems. Analyses of the data 96 days after treatment show that there were significant
effects of oil on defoliation and leaf initiation (Table I). Ptants treated with fresh oils
lost significantly more leaves than plants treated with aged oils but there was no significant difference between the plants treated with aged oils and the controls with respect to leaf loss (Table II). There was no difference between any of the oil treatments
~vith respect to leaf initiation, but there were more new leaves on the control plants than
the oil-treated plants (Table II).
FIELD EXPERIMENT~
EFFECTS
OF AGED
BASS
STRAIT CRUDE
OIL ON AVICENNIA
MARINA SEEDLINGS
Aged Bass Strait oil had rapid and almost immediate effects on the seedlings in the
field. The leaves of treated seedlings immediately changed colour from bright leaf green
TABLE III
Results of field oiling experiments. The experiment was repeated sequentially in three areas at Site 1 and
two areas at Site 2. The table shows the number of surviving seedlings96 days after treatment. The two
treaments were: no oil and aged Bass Strait crude oil. The column "Days" refers to the number of days before
which 100~o mortality was observed. The superscript * indicates that this number is based upon the days
before all seedlings other than the persistent survivors had died.
Exp.
1
2
3
Treatment
No oil
Oil +
No oil
Oil +
No oil
Oil +
Site 1
No. survivors
Mortality ~o
16
0
14
1
14
0
0
100
0
95
0
100
Site 2
Days
No. survivors
Mortality ~o
Days
21
11
2
12
0
0
87
0
100
20*
14"
18
14
283
MANGROVE RESPONSE TO OIL SPILL
to a dark greyish green. Seedlings that had not been oiled remained healthy and
maintained the original colour of their leaves. Within 3 days some oiled seedlings began
to droop displaying a lack of turgor. A diagnosis of death was made when all leaves
on the seedlings had fallen off or curled up and ~vhen the stem had begun to ~vither.
The results from each of the experiments were consistent (Table III). With the exception
of three very hardy seedlings that displayed no signs of stress, all those treated with
oil were dead within 21 days. None of the seedlings in the control boxes died. The
results of each experiment show that contamination with aged Bass Strait crude oil
results in the death of 96.47o of treated seedlings, while no untreated seedlings died.
The result is clear cut and inferential statistics are not required.
FIELD EXPERIMENT; EFFECTS OF OIL IN THE SEDIMENT ON ESTABLISHMENT AND
SURVIVAL OF AVICENNIA MARINA SEEDLINGS
Within 2 days of placing propagules on the substratum, the majority of the pericarps
had split and become detached from the embryo. Embryo development was epigeal so
the events were easy to follow. By day 10, hypocotyl extension had begun and lateral
root primordia were visible. The hypocotyl and root displayed a positive geotropic
response, arching downward towards the sediment. By the 18th day some of the
seedlings had become anchored to the sediment. The hypocotyls began to straighten
TABLE IV
Results of 5 factor analyses of variance of the number of surviving seedlings ofA ~,icelmia marina 4 and 12 wk
after treatment. The factors were as follows: canopy (C); light (L); areas (A); plots (P). The data were
transformed with (x + 1)I/2.
Source
Canopy
Light
Oil
CxL
LxO
CxO
CxLxO
Areas
LxA
OXA
L×OxA
Plots
OxP
Residual
df
1
1
1
1
1
1
1
2
2
2
2
8
8
32
4 wk
12 wk
SS
MS
F-ratio
SS
MS
F-ratio
0.28
1.11
4.30
0.03
0.01
0.25
0.24
3.27
0.70
0,53
0.63
3.13
3.25
3,72
0.28
1.11
4.30
0.03
0.01
0.25
0.24
1.63
0.35
0.26
0.31
0,39
0,40
0,12
0,17 NS
3.18 NS
16.35a
0,10 Ns
0.35 NS
0.95 NS
0.76 NS
4,17 NS
0,89 NS
0,65 NS
0,78 NS
3,36**
3.44**
0.11
0.42
1,10
1.01
0.001
0.34
0,95
6,89
0.92
0.87
0.15
2.8
2,25
3.64
0.11
0.42
1.10
1.01
0.001
0.34
0,95
3.44
0.46
0.43
0.07
0.35
0.29
0.114
0.41 NS
3.10 NS
25.10"
2.20 NS
0.01 NS
0.77 NS
12.83 NS
9.87**
1.31 NS
1.50 NS
0.25 NS
3.07**
2.54**
There was no significant effect of the oil × area interaction at 4 wk (p> 0.50). A new F-ratio can be determined for testing for effects due to oil where F= MSoil/MS(pooled o x A and O x P) ; this F= 11.376 (p < 0.005).
284
D.L. GRANT ET AL.
10 9"
10
9
(a)
(b)
7
6
10
0
20
40
60
80
100
0
120
20
40
60
80
100
120
d)
.
76-
0
10
9
8
765=
4-
20
40
60
80
100
120
0
10-
20
40
60
80
100
120
40 60
80
100 120
9-
(e)
86-
5=
4=
20 40 60 80 100 120
0 20
10
(h)
(g) 9
8
7
6
5
4
3
~_~
,T, . ,T, ,
0 20 40 60 80 100 120
0
0
20 40 60 80 100 120
Weeks
Fig. 5. (a) Mean number of seedlings surviving oil (1~) and no oil (V1) treatments in canopy gaps over 106
weeks. Each graph is of a different plot with (a)A1-SI-P1; (b)A1-S1-P2; (c)A1-S2-P1; (d)A1-S2-P2;
(e)A2-S1-P1; (f)A2-Sl-P2; (g)A2-S2-P1; (h)A2-S2-P2. Al=area 1, A2=area 2, Sl=shade added,
$2 = open area, P1 = plot 1, P2 = plot 2. Standard errors shown. (b) Mean number of seedlings surviving oil
MANGROVE RESPONSE TO OIL SPILL
10
9
8
7
6
5
4
3
2
1
0
0
10
9
8
7
6
54321O=
0
10
9
8
7
6
5
4
3
2
1
0
~n 0
c~10
.~ 9
m8
m7
6
109876543
2
1
0
20 40 60 80 100 120
0
10
(k) 9
8
7
6
5
4
3
2
1
0
20 40 60 80 100 120
0
10(m) 9876543
2
1
0
20 40 60 80 100 120
0
10
9
8
7
6
5
4
3
285
(i)
(J)
20
40 60 80 100 120
(I)
20 40
60
80
100 120
(n)
20 40 60 80 100 120
(o)
(P)
Ot
20
40 60 80 100 120 0 20 40 60 80 100 120
Weeks
and no oil treatments under full canopy over 106 weeks. Each graph is of a different plot with (i)A1-S1Pl; (j) A1-S 1-P2; (k) A1-S2-P1; (1) AI-S2-P2; (m) A2-S l-P1; (n) A2-S l-P2; (o) A2-S2-P1; (p) A2-S2-P2.
A1 = area 1, A2 = area 2, S 1 = shade, $2 = cut open, P1 = plot 1, P2 = plot 2. Standard errors shown.
286
D.L. GRANT ET AL.
immediately and by day 21 ~ 25 ~o of the seedlings were anchored and standing erect.
On subsequent visits, it became apparent that some seedlings were becoming disengaged from the sediment and that these seedlings were capable of re-anchoring. A shoot
bearing the first pair of plumular leaves emerged above the cotyledons after approximately 30 days. The first internode was very long compared to the hypocotyl. A second pair of leaves were well developed in some plants by day 42. The cotyledons
shrivelled up or dropped off between 8 and 12 weeks. The number of seedlings anchored to the substrate in each cage increased until approximately 12 wk after the
commencement of the experiment.
SEEDLING MORTALITY
Several categories of seedling mortality were observed. (1) For some seedlings death
occurred when the hypocotyl failed to extend and the cotyledons developed necrotic
lesions and rotted. (2) On oiled sediments only, some seedlings extended their hypocotyl
down to the substratum but the developing roots were apparently unable to penetrate
the substratum. The root tips became stublike and necrotic. These seedlings eventually died. (3) Other seedlings anchored on the oiled substrate initially but subsequently
became uprooted. The roots looked very similar to the stublike roots described above.
These seedlings lived for many days but were unable to re-anchor. (4) Seedlings developed to the two or four leaf stage but did not thrive and eventually died.
Cochrans test for heterogeneity of variances (Underwood, 1981), was done before
analysis of 4, 12, 63 and 106 wk survival data. The Cvalues at 4 wk (C= 0.163), 12 wk
(C= 0.129), 63 wk (0.189) and 106 wk (0.076) were less than the critical value (0.280)
for 32 treatments with 1 degree of freedom (p< 0.05). This test, however, may not be
powerful enough to detect heterogeneity when ~< 5 (Day & Quinn, 1989). To guard
against Type 1 error, data from 4, 12 and 63 wk were transformed using (1 + x)~/2,
which is appropriate for data containing many zeros (Underwood, 1981). The 106 wk
data were not transformed because the very low C value suggested that this was not
warranted.
No significant effect of the light or canopy factors on seedling establishment was
detected after analysis of variance on the data at 4 and 12 wk (Table IV). This prompted
the removal of the artificial canopies as they were prone to storm damage and proved
increasingly difficult to maintain. Branches removed to expose shaded plots to sunlight
were also starting to regenerate. In subsequent analysis of the data there were only four
factors; canopy, oil, areas and plots and therefore light independent effects of the
canopy could not be measured. A significant oil x plot interaction at both 4 and 12 wk
makes it difficult to interpret main effects ofoil and plots at these times. Data have been
graphed for oil and no-oil treatments for each plot across time (Fig. 5). In both oiled
and unoiled plots, a distinct 12 wk establishment period in which the number of
seedlings established in each square increased was followed by a gradual decline in
seedling numbers that continued throughout the course of the experiment. In most plots
MANGROVE RESPONSE TO OIL SPILL
287
[] No Oil
10
5
4
3
2
1
0
[] oil+
ab
Light
Shade
AREA A
Light Shade
Light Shade
AREA B
AREA A
a b Plot
Light Shade
AREA B
CANOPY
CANOPY GAP
Fig. 6. Mean number of seedlings established 12 wk after treatment. Columns are arranged in pairs that
represent oil and no oil treatments in each plot. Initial number of propagules = 10. Standard errors shown.
Significant differences between pairs of oil and no oil treatments (SNK tests,/)<0.05) are shown by *.
more seedlings survived in unoiled plots than the oiled plots but the error bars suggest
that this difference was not always significant at the 95 7o confidence limits. Figure 6
shows the number of seedlings surviving oil and no-oil treatments in each plot at the
TABLE V
Results of 4-factor analyses of variance of the number of surviving seeedlings of A~,icennia marh~a 63 and
104 weeks after treatment. The factors were as follows: canopy (C); oil (O); areas (A); plots (P). The data
from 63 wk were transformed with (x + 1)1/2. The 106-wk data were not transformed.
Source
Canopy
Oil
Areas
CxO
OXA
Plots
OXP
Residual
df
1
1
2
1
2
12
12
32
106 wk
63 wk
SS
MS
F-ratio
SS
MS
F-ratio
0.25
105.62
32.56
9.00
7.31
13.13
13.12
95.00
0.25
105.62
16.28
9.00
3.66
1.09
1.09
1.09
0.01 NS
28.73*
14.70"*
2.46 NS
3.34 NS
0.55 NS
0.55 NS
0.62
45.65
30.12
3.06
5.62
19.94
20.75
31.00
0.62
45.56
15.06
3.06
2.81
1.66
1.73
0.97
0.04 NS
16.35~
9.04**
1.08 NS
1.63 NS
1.71 NS
1.78 NS
a There was no significant effect of the oil x area interaction at 106 wk (p>0.25). A new F-ratio can be
determined for testing for effects due to oil where F = MSoil/M S(pooled o x a and o x p), this F = 25.54 (p < 0.01).
288
D.L. GRANT ET AL.
d
r- 0
Oil+
No Oil
Oil treatments
Fig. 7. Mean number of seedlings present on oiled and unoiled substrates after 106 weeks. Initial number
of propagules = 10, standard errors shown.
end of the 12 wk establishment phase. A postetJo~J pairwise comparisons of the means
using Student-Newmans-Keuls tests (Underwood, 1981) show that while there were
significant oil effects in some plots, the difference was not detectable in others. The
magnitude of the oil effect varied from plot to plot and this appears to explain the
interaction. At 63 and 106 wk there was no oil x plot interaction (Table V), but there
was a significant main effect of oil on seedling survival (Fig. 7).
At 4, 12, 63 and 106 wk there was a significant effect of areas (Tables IV and V).
After 106 wk the mean number of seedlings surviving in canopy area A was greater than
the number surviving in canopy area B but no greater than canopy gap area A and
canopy gap area B (SNK, p<0.05).
DISCUSSION
Effects of aged oil
The glasshouse experiment showed that even at a low dose (200 ml.m-a), oiling of
Avicennia seedlings in a glasshouse can produce lethal and sublethal effects. While
mortality only occurred when plants were treated with fresh oils, defoliation and the
suppression of leaf initiation occurred when plants were treated with aged oils. Fresh
oils were more damaging than aged oils. Differences between the effects of Bass Strait
oil and light Arabian oil were not apparent once the oils had been aged. This has
important implications for the interpretation of studies in which oil dose responses and
comparative effects of oils have been determined using fresh oil treatments (see Mathias,
1977; Jagtup & Untawale, 1980; Lai et al., 1984). It is not surprising that the aged oil
treatments had different effects from those of fresh oils. The high rate of evaporation
MANGROVE RESPONSE TO OIL SPILL
289
of low molecular weight hydrocarbons has been extensively reported (McAuliffe, 1976).
Harrison et al. (1975) found that all n-C9 hydrocarbons were lost fi’om an oil slick in
40-90 min (n-C12 in 3-8 h). The light Arabian oil initially had a higher proportion of
aromatics than the Bass Strait oil which probably accounts for its greater toxicity. After
aging, much of the aromatic fraction would have evaporated so the two oils would have
become more alike in content and in effect. The conclusions of the glasshouse experiment cannot be extended beyond the specific set of conditions under which this experiment was done and these conditions only superficially resemble field conditions,
e.g. the sand potting mix does not resemble the more organic sediments found in
mangrove swamps. However, the results suggest that the conclusions of field experiments in which fresh oil is used as a treatment must also be regarded with caution given
that spilt oil usually ages before contact with mangroves.
Effects of aged oil on seedling survival
In the glasshouse and field experiments the doses used as experimental treatments
fall between the range of a small to large scale oil spill (200-1000 ml.m-2). The
thickness of the slick can be made to approximate a real oil spill but the size of the
spill will be limited by the dimensions of the container. Allaway et al. (1985) noted that
the amount of oil available in a small container may not be sufficient for the wetting
properties of the oil on the leaves. In a real spill, oil is stripped from the slick by seedling
stems and leaves because the slick reconverges on rise or fall of the water level. The
amount of oil available for reconvergence and recoating in an experimental enclosure
is limited. This suggests that experimentally treated plants may not be subjected to the
same degree of oiling as plants contaminated in a real spill. Also, the dose used in our
experiment cannot be extrapolated to assess the effects of spills of known volume and
area because of the adherence of oil to the sides of the experimental enclosures.
In the field oiling experiments, seedlings treated with a dose of 1 l’m-2 of Bass Strait
crude oil consistently displayed the same gross effect of widespread mortality that has
been reported in the post-spill literature (e.g. Allaway, 1982). The variation within
treatments was very low compared to the variation between treatments. Three of the
seedlings survived the oil treatments without showing any signs of the characteristic
sublethal effects like defoliation; there are several possible explanations including uneven oiling of individual plants or genetic variation. McGuinness (1990) reported no
effect of oiling on Avicennia seedlings with a dose of 1 1.m-2 weathered Dubai light
crude, although he qualified these findings by stating that his sampling design was not
intended to test for effects of oil on mangrove seedlings.
Effects of light on establishment and survival
There were no significant effects of light or canopy on seedling establishment or
survival and there was no mortality of the control plants that were gro~vn under low
290
D.L. GRANT ET AL.
fight conditions in the glasshouse. Propagules that had not anchored in the sediment
remained viable for up to 12 wk coinciding with the period cotyledons persisted on
seedlings. Seedlings transfer mass from the cotyledons to the plant body providing a
reservoir of energy for seedling development and this may account for lack of differences during establishment. Following establishment the number of seedlings in all
treatments decreased at rates similar to the new world Avicennia bicolor and Avieennia
ge~7ninans (Rabinowitz, 1978). Prolonged seedling survival in the understorey appears
to relate to maximisation of photosynthesis during exposure to high intensity sun flecks
(Ball & Critchley, 1982).
The data from our experiment are not consistent with the hypothesis that light is the
only limiting factor and suggest that co-factors are required for seedling recruitment.
The lack of difference, of course, might reflect the ability of the design to resolve small
differences. Clarke & Allaway (1993) found that canopy effects alone were not significant, but both light and sediment disturbance were required for seedling recruitment.
Smith (1987) has shown that predators can also influence recruitment in canopy gaps
in tropical mangroves, but at the latitude of our experiments levels of predation are low
(Clarke & Myerscough, 1993).
The significant difference between the number of seedlings surviving among canopy
areas, might be explained in terms of sediment disturbance, although sediments appeared to be similar. Nutrient levels have also been identified as a key factor in seedling growth (Clarke & Alla~vay, 1993). One possible explanation is that a greater input
of terrigenous nutrients into one area increased seedling survival. Clearly, whatever the
cause, field experiments in mangroves must take into account spatial heterogeneity.
Effects of crude oil on establishment and survival
The magnitude of the oil effect was greater in some plots than in others and seedlings in general grew better in some plots. The oil x plot interactions that were significant in the analysis at 4 and 12 wk also demonstrate the importance of including
factors that measure spatial heterogeneity. An important factor not taken into account
in the experiment was the size of sediment particles and the degree of waterlogging of
the sediment. Each of the areas had a range of sediment types from fine silty mud to
sand so it was inevitable that sediment sizes in plots would vary. It was noted that those
plots experiencing the greatest mortality were plots with fine sediments that never
seemed to drain. Dicks (1986) observed a similar interaction between sediment size and
the effects of oil though this was not experimentally tested. Little (1987) also found that
sediment grain size and moisture content influenced the residence time of crude oil in
the sediment and that mud particles tended to retain surface-bound contaminants more
strongly than sands. In the longer term, the plot effect proved not be significant in our
experiment.
By the end of the establishment phase, it was clear that oil was having a significant
effect on the survival of seedlings in many but not all of the plots. However, after 2
MANGROVE RESPONSE TO OIL SPILL
291
years the significant main effect of oil made it cleat" that oil in the substrate can inhibit
the survival of seedlings. An important qualification to the interpretation of this result
is that the propagules were placed on the sediments only a short time after these
sediments had been oiled. The observed effects may not occur if the propagules land
on the substratum after the oil has weathered for a longer period and this may account
for the observed colonisation of previously oiled areas with well aerated soils (Clarke
& Allaway, 1993). Oil retained in waterlogged anoxic sediment will break down more
slowly than that retained in aerobic sediments (Atlas, 1981), and oil has been shown
to persist in anoxic sediments for up to 30 years (Corredor et al., 1990). It has been
shown that Rhizophora mal~gle cannot re-establish immediately after an oil spill (Jackson et al., 1988) and attempts at regenerating experimentally oiled areas with Somwratia
caseolaris were said to have failed because of oil in the sediment (Dutrieux et al., 1990).
Predicted population mspo~se to repeated oiliJ~g
Underwood & Peterson (1988) suggested that the first step after rejecting a null
hypothesis that there is no effect of a pollutant is to determine whether the observed
effect really matters. The results of the field oiling experiment on seedlings are unequivocal but is this biologically significant? Clarke & Allaway (1993) have shown that
seedlings are unlil¢ely to recruit to the sapling and adult populations unless they establish in large canopy gaps with sediment disturbance or newly deposited sediments.
If these places are oiled then recruitment to the adult population will be impaired until
a new cohort is able to colonise the regeneration niche. The results of the establishment experiment show that about four times as many seedlings survived on unoiled
substrates than on experimentally oiled substrates. A single oil spill may cause widespread seedling mortality and oil in the sediment may inhibit the establishment of
propagules but if nearby adults continue to be fecund the long-term consequences may
not be serious.
It is evident from the literature, however, that mangroves contaminated with oil are
often subjected to further oil spills (Dicks, 1986; Snowden & Ekweozor, 1987; Jackson et al., 1988; Corredor et al., 1990). This constitutes a repeated pulse disturbance
that may lead to longer term press effects (se~su Underwood, 1989). In Botany Bay,
the same area of mangroves has been oiled in three successive spills over a 6-year period
(Allaway, 1987), but neither the pulse or press effects of multiple oilings have been
experimentally investigated. In particular, the interactions of differing components of
the population to repeated disturbances are poorly understood.
It is logistically difficult to conduct experiments in which the full range of frequencies, magnitudes, oil types and age classes can be manipulated to assess the impact of
perturbations on natural populations. Whilst further experiments are essential for accurately predicting impact, a working hypothesis can be developed based on our results, field correlations, and population experiments. The latter includes reproductive
biology (Clarke & Myerscough, 1991; Clarke, 1992), dispersal (Clarke, 1993a), estab-
292
D.L. GRANT ET AL.
lishment (Clarke & Myerscough, 1993), seedling and sapling survival (Clarke & Aliaway, 1993; Clarke & Myerscough, 1993) and population structure for south-eastern
Australia (Clarke, 1993b). Figure 8 describes a possible population response of three
age classes of Avicemda to three oil spills spaced by ~ 5 years, each of ~vhich causes
massive mortality in seedlings, and lesser mortality to saplings and adults. An initial
oiling decreases the seedling population (A) and recovery is inhibited by residual oil
effects as demonstrated by our experiments. The magnitude of recovery is difficult to
predict as it will depend upon duration of residual effects. A subsequent oiling has the
same effect (B) but the magnitude of recovery might also be reduced because the
number of adults has decreased (Allaway, 1987). Finally, a third spill some 10 years
after the first reduces all populations to a very low level (C). Seedling recovery is initially very slow because propagules have to be dispersed from longer distances into the
disturbed area (Clarke, 1993a). After the initial colonists have recruited to the sapling
stage seedling numbers increase rapidly due to increased population fecundity (D).
Sapling populations rise to densities higher than pre-impact levels because of the increased levels of light and sediment resources (Clarke & Allaway, 1993). These densities then decrease to pre-impact proportions because of self-thinning (E) (Clarke,
1993b). Following this phase the simulation suggests that pre-impact population structure would not be restored until at least 30 years after the last perturbation.
We have established that fresh oils damaged plants more than aged oils, that aged
Bass Strait crude oil applied on the water surface ldlled most seedlings, and that aged
Bass Strait crude oil on the sediment inhibited seedling establishment and survival.
These conclusions could not have been drawn from post-spill observations because
1000
oE 10
III
30
35
40
45
Fig. 8. A working hypothesis of the population response of Avicelmia marimba to three major crude oil spills.
Letters indicate phases in population change (see text).
MANGROVE RESPONSE TO OIL SPILL
293
there are no controls for environmental heterogeneity and no replication. Observations
and correlations between oil spills and mortality of seedlings can, however, be used to
develop useful hypotheses that can be tested in well designed field experiments.
ACKNOWLEDGEMENTS
We thank L. Wilson, K. Pavlovic, C. Grant, B. Gladstone and M. Hovenden for
assistance with field work, M. Cole for advice on growth and assistance in glasshouse
experiments, A.J. Underwood for advice on experimental design, K. McGuiness for
comments on the paper, and Caltex (Australia) for providing the hydrocarbons.
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