Chapter 3: The effects of oil on morphological characteristics in Avicennia marina,
3.2 Materials and methods
3.2.1 Growth conditions
First rhizotron study
Propagules of A. marina, B. gymnorrhiza and R. mucronata were collected as described in Chapter 2.2.1. After collection, A. marina propagules were placed in water and pericarps allowed to shed naturally (24 hours). Root growth was monitored by growing plants in perspex rhizotrons with dimensions of 50 cm height x 31 cm length x 3.6 cm width. Rhizotrons were filled with a mixture of sand, potting soil and compost (1:2:1) and covered with black plastic to exclude light. Propagules of A. marina, B.
gymnorrhiza and R. mucronata were inserted into the soil to about 5 mm, 3 cm and 7 cm, respectively. The rhizotrons were tilted at an angle of 30° from the horizontal and watered daily with tap water and once monthly with 10% seawater. The rhizotrons were maintained in a glasshouse for 245 days. The temperature in the glasshouse during the experimental period was about 25 °C (day) and 18 °C (night).
Propagules of A.marina without pericarps were subjected to one of three treatments:
i. C – control propagules were planted in the sediment.
ii. ½O – 50% of the propagule was dipped in oil using a pair of forceps.
iii. O – propagules were completely dipped in oil and planted in the sediment.
Propagules of B. gymnorrhiza (about 15.5 ± 2 cm in height), and R. mucronata about 22 ± 0.9 cm in height) were subjected to one of two treatments:
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i. C – control propagules were planted in the sediment.
ii. ⅔O – 67% of the propagule from the base/radical end was dipped in oil using a pair of forceps.
The properties of the bunker fuel oil used in this study are indicated in Table 2.1, Chapter 2. There were four replications per treatment for A. marina and three for R.
mucronata. One oiled propagule of B. gymnorrhiza was not viable and excluded, so that there were four replications in the control and three in the oiled treatment.
Second rhizotron study
Propagules of the three species were collected from the Isipingo estuary (29° 59ʹ 59ʺ S, 30° 56ʹ 42ʺ E). Root growth was monitored by growing A. marina and B. gymnorrhiza in perspex rhizotrons with the same dimensions as in the first study. Rhizophora mucronata was grown in rhizotrons with dimensions of 50 cm height x 31.2 cm length x 7 cm width. The experimental set-up was identical to the first study. The rhizotrons were maintained in a glasshouse for 409 days. The second study concentrated on sediment oiling in all species. In A. marina, the completely oiled propagule treatment was replicated as there was 100% mortality in the first study.
Propagules of A. marina without pericarps were subjected to one of three treatments:
i. C – control propagules were planted in the sediment.
ii. SO – propagules were planted in sediment to which 200 ml of oil were carefully poured onto the soil surface.
iii. O – propagules were completely dipped in oil and planted in the sediment.
Propagules of B. gymnorrhiza (about 17 ± 2.5 cm in height), and R. mucronata (about 22 ± 1.2 cm in height) were subjected to one of two treatments:
i. C – control propagules were planted in the sediment.
ii. O – propagules were planted in sediment to which 200 ml of oil were carefully poured onto the soil surface.
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The sediment was oiled at the commencement of the experiment. There were three replications per treatment for A. marina and B. gymnorrhiza and two for R. mucronata.
3.2.2 Plant growth measurements
Measurements of shoot and internode length and number of leaves were determined after the experimental period.
3.2.3 Root growth and harvesting of plant parts
Root growth was monitored weekly by tracing new growth onto clear plastic transparencies attached to the outside of the rhizotron. At the end of the treatment (245 and 409 days, respectively), plants were carefully removed from rhizotrons and washed with water to remove soil. Plants were measured and separated into leaves, stems and roots. Plant parts were weighed and dried in an oven to constant mass at 70 °C for three days.
3.2.4 Leaf area and chlorophyll content
Leaf areas were determined by photocopying fresh leaves and scanning into a computer using image analysis software, SIS Pro Softward, version 3:1. Chlorophyll content was determined as described in Chapter 2.2.3.
3.2.5 Root/shoot ratio and relative root growth rate
Dry mass of roots and shoots were determined by weighing on a scale (Mettler Toledo AG 204, accuracy ± 0.1 mg, Mettler Toledo products, Switzerland). Root/shoot ratio was determined on a dry mass basis. Relative root growth rate (RRGR) expressed in
grams per day, was calculated according to Sánchez (2005) using the equation:
RRGR = DM / t
where DM = root dry mass, t = duration of the experiment in days, assuming that the initial root mass was zero.
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Root volume was determined using the Archimede’s principle (Harrington et al., 1994).
Plant roots were suspended in a known volume of water in a measuring cylinder. Root volume was approximated to that of the water displaced. Specific root volume (SRV) was calculated according to Merkl et al. (2005) and is defined as the ratio of volume (V) per unit dry mass (DM):
SRV = V / DM
where V = root volume, DM = root dry mass.
Roots were separated into coarse (>2 mm) and fine (<2 mm) diameter. Root diameter (RD) was measured from cross sections using an ocular graticule (Muthukumar et al., 2003). Root length was determined by scanning traced transparencies into a computer using image analysis software, SIS Pro Softward, version 3:1. Specific root length (SRL) was calculated according to Bouma et al. (2001) and is defined as the ratio of length (L) per unit dry mass (DM):
SRL = L / DM
where L = root length, DM = root dry mass.
3.2.7 Data analyses
Means and standard errors were calculated for all measurements. Resulting data were tested for normality using the Kolmogorov-Smirnov test and subjected to one-way ANOVA and Tukey-Kramer multiple comparisons test (P ≤ 0.05) using MINITAB version 16 (Minitab Statistical Software, MINITAB Inc., USA). Other data were subjected to two-way ANOVA and Tukey’s multiple comparisons test (P ≤ 0.05) using GraphPad Prism Version 6.05 (GraphPad Software, Inc., USA).
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