the impact of petroleum hydrocarbons (diesel) on periphyton in an impacted tropical estuary based on...

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Journal of Experimental Marine Biology and Ecology

302 (2004) 213–232

The impact of petroleum hydrocarbons (diesel)

on periphyton in an impacted tropical estuary

based on in situ microcosms

S. Nayara,*, B.P.L. Gohb, L.M. Choua

aMarine Biology Laboratory, Department of Biological Sciences, National University of Singapore,

Block S1, 14 Science Drive 4, Singapore 117 543, SingaporebNatural Sciences Academic Group, National Institute of Education, Nanyang Technological University,

1 Nanyang Walk, Singapore 637 616, Singapore

Received 7 October 2002; received in revised form 19 September 2003; accepted 22 October 2003

Abstract

The distribution of petroleum hydrocarbons and their effects on the periphytic algal biomass

using in situ microcosms were investigated in Ponggol estuary located on the northeastern coast of

Singapore. Dissolved or dispersed petroleum hydrocarbon (DDPH) concentrations in the surface and

bottom waters and absorbed or adsorbed petroleum hydrocarbon (AAPH) concentrations in

sediments were monitored from July 1999 to June 2000. Results showed concentrations ranging

from 4.42 to 248.94 Ag l� 1, from 0.35 to 1099.65 Ag l� 1, and from 20.55 to 541.01 mg kg� 1 for

DDPH in surface and bottom waters and AAPH in sediments, respectively. Accidental spillages of

fuel from dredgers operating in the estuary, fuel and engine oil from recreational boats, shipping

operations in the adjacent strait, and runoff monsoon drains in the vicinity were some of the possible

sources of petroleum hydrocarbons in the estuary. An assessment of environmentally realistic

concentrations of petroleum hydrocarbons on periphytic algal biomass using in situ microcosms

revealed signs of acute toxicity. A reduction in periphytic algal biomass (with respect to controls) of

68–93% was observed for various treatments exposed to diesel.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Petroleum hydrocarbons; Diesel contamination; Periphyton; Microcosms; Ecotoxicology; Chlorophyll

a; Tropical estuary

0022-0981/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.jembe.2003.10.016

* Corresponding author. Present address: Tropical Marine Science Institute, National University of

Singapore, 14 Kent Ridge Road, Singapore 119 223, Singapore. Tel.: +65-9134-9818; fax: +65-6779-2486.

E-mail address: [email protected] (S. Nayar).

S. Nayar et al. / J. Exp. Mar. Biol. Ecol. 302 (2004) 213–232214

1. Introduction

Most surveys on dissolved or dispersed petroleum hydrocarbon (DDPH) and

absorbed or adsorbed petroleum hydrocarbon (AAPH) contamination in coastal seas

and estuaries have centered on North America and Europe (Gordan et al., 1978;

Marchand et al., 1988; Bidleman et al., 1990; Zhang et al., 1993; Rajkumar and

Persad 1994; Law et al., 1997; Sharma et al., 1997; Woodhead et al., 1999; Soclo et

al., 2000; Notar et al., 2001). Little information is available from Southeast Asia,

where industrialization and urbanization have proceeded rapidly during the past

several decades. As one of the busiest ports in the world and a regional hub for

oil refining, Singapore faces a high risk of oil pollution (Tang et al., 1997). Marine

pollution in Singapore has been attributed to exhaust emissions from boats, increased

shipping activities, release of antifouling paints from boats, industrial sources, and

dredging (Sin et al., 1991; Goh and Chou 1997; Orlic et al., 1997; Tang et al.,

1997).

In the coastal waters of Singapore, pollutants such as petroleum hydrocarbons have

been found to be in concentrations detrimental to marine life (Tang et al., 1997). One

of the approaches to test the toxicity of these pollutants to marine biota is by the use

of ecotoxicological tests (Giddings et al., 1994). In most situations, ecotoxicological

studies conducted in laboratories are poor simulations of natural field conditions,

resulting in inaccurate extrapolation errors (Cairns and Pratt, 1989). The highest level

of validation in toxicity testing is provided by manipulative studies in natural

ecosystems (Effler et al., 1980). In situ mesocosms or microcosms are an alternative

in this context, as methods that involve introducing pollutants directly into the

ecosystem are environmentally unacceptable due to the large quantities of the

chemicals required (Geckler et al., 1976). Although a few studies have examined

the response of bacteria, microphytobenthos, and phytoplankton to petroleum hydro-

carbons in microcosms and mesocosms (e.g., Farke et al., 1985; Cretney 1987; Lee

and Levy 1987; Yanshun et al., 1987; Yu et al., 1987; Plante-Cuny et al., 1993;

Carman et al., 1995, 1996), there are few studies involving periphytic algae (but see

Riquelme and Garcia 1986; Singh et al., 1987; Pudo and Fubara 1988; Belanger et al.,

1993, 1996).

The purpose of this ecotoxicological investigation was to obtain information upon

which to predict the possible impact of environmental levels of diesel, one of the major

components of the petroleum hydrocarbon pollution in this estuary, on periphytic algae

using in situ microcosms. Periphytic algae are ubiquitous and a dominant primary

producer forming the base of aquatic food webs (Kairesalo, 1980; Robert et al., 1995).

They are an important ecological component of aquatic ecosystems (Boston et al., 1991;

McCormick and Stevenson, 1998) and have been reported to be a good indicator of

aquatic pollution (Fjerdingstad, 1964) and an ideal candidate group for ecotoxicological

tests. The present study was divided into three parts encompassing field monitoring of

environmental concentrations of DDPH and AAPH in the water column and sediments of

the estuary, periphyton settlement studies, and short-term exposure of periphyton to

environmental concentrations of DDPH and AAPH made up using diesel in in situ

microcosms.

S. Nayar et al. / J. Exp. Mar. Biol. Ecol. 302 (2004) 213–232 215

2. Materials and methods

2.1. Site description

Ponggol estuary (latitude: 01j25V27UN–01j25V45UN; longitude: 103j53V20UE–103j55V10UE) is a mangrove-fringed estuary located on the northeastern coast of

Singapore (Fig. 1). The mouth of the estuary opens into the East Johor Strait. The estuary

Fig. 1. Location of the sampling stations, the mooring pontoon, and the unimpacted site along Ponggol estuary,

Singapore.


is exposed to anthropogenic activities such as dredging, reclamation, mangrove defores-

tation, dumping of dredge spoils, recreational boating from an adjacent marina, and

shipping activities along the East Johor Strait (Nayar et al., 2003).

2.2. Field monitoring

Surface and subsurface water and sediment samples were collected from three stations

along the estuary at high tide, on a fortnightly basis, from July 1999 to June 2000 (Fig. 1).

The 1-year sampling spanned a period during which the estuary was affected by

developmental activities mentioned above. In situ measurements of temperature, pH,

dissolved oxygen (DO), and salinity were also taken. Temperature and DO were measured

with a YSIR 55 DO meter pH with a WTWR pH330 pH meter and salinity with a WTWRLF330 salinity meter. Water samples were collected at each station using 1 l acid-washed

polyethylene (PE) bottles with a pump-based sampler at the surface (30 cm below the

surface) and subsurface (30 cm above the bottom) depths. The average depths at the three

stations were 5.03, 2.20, and 2.0 m at stations 1, 2, and 3, respectively. Similarly, sediment

samples were collected using an Ekman grab at the three stations. To avoid contamination

from the grab, grabbed sediments were subsampled from the center of the grab using an

acid-washed plastic spatula. Sediment samples were transferred into 250 ml acid-washed

PE bottles. Both water and sediment samples were chilled and transported (30 min) to the

laboratory at + 4 jC.At the laboratory, DDPH in the water samples were immediately analysed, in

duplicates, according to the spectrofluorometric method of Parsons et al. (1984). For

sediments, about 10 g of the accurately weighed sample was placed into acid-washed

and baked porcelain crucibles and oven-dried at 100 jC until constant weights were

obtained. The moisture content of the sediments was determined based on the difference

between the wet and dry weights. AAPH measurements were performed in duplicates

using weighed portions (about 10 g) of the wet sample, mixing with anhydrous sodium

sulphate and extraction with spectrophotometric grade dichloromethane (MerckR). Thespectrofluorometric protocol for petroleum hydrocarbons of Parsons et al. (1984) was

used. Sample fluorescence was corrected using procedural blanks subjected to a similar

extraction method using equivalent amounts of anhydrous sodium sulphate. Results were

expressed as milligrams of Total petroleum hydrocarbons (TPH) per kilogram of dry

sediments.

All spectrofluorometric analysis were carried out using a Shimadzu RF 1501 spectro-

fluorometer with an excitation wavelength set at 310 nm and an emission wavelength of

374 nm. The instrument was calibrated using Chrysene standards (MerckR).Turbidity in the water samples was measured with a HatchR 2100P turbidimeter.

Samples for DOC were filtered through a MilliporeR 0.2-Am pore size cartridge filter and

analysed with a Skalar FormacsR carbon analyser.

2.3. Periphyton settlement studies

The purpose of the settlement study was to establish the suitable settlement depth

and duration of settlement to yield a measurable quantity of periphytic algae for the


diesel exposure studies described later. Another objective of the study was to obtain

preliminary information on the community structure of periphytic algae in the

estuary.

Based on previous research (Thielcke and Ratte 1994; Sreekumar 1995; Vinyard

1996; Nystroem et al., 2000), the choice of the settlement substrate was glass. Rafts of

polyvinyl chloride (PVC) were constructed into frames on which glass slides could be

placed for measurements of settlement at the surface with 1, 2, and 3 m depths. Glass

slides of 13.75 cm2 were cleaned with absolute alcohol prior to attachment onto the

rafts. The rafts and glass slides were submerged and anchored at the unimpacted site

(Fig. 1). The settlement studies were carried out in May 2001. The unimpacted site

was located on a branch of the estuary, away from the zone of boat traffic, and was

constantly flushed by the waters of the East Johor Strait. Preliminary investigations on

the petroleum hydrocarbons at this site revealed undetectable concentrations of DDPH

and AAPH (Nayar, 2003). Glass slides were retrieved each day from each depth for

the duration of the experiment (5 days). For determination of periphyton chlorophyll a

and cell counts, the retrieved glass slides were transported to the laboratory in 250-ml

glass bottles containing 200 ml of filtered estuarine water maintained at 4 jC in the

dark.

Dissolved oxygen, pH, and salinity were measured in situ. Water samples were also

collected from the four depths using an acid-rinsed Van Dorn water sampler. About 150 ml

of the sample was filtered through a 0.2-Am membrane filter and stored in the dark at 4 jCfor the analysis of nutrients. Nutrients in the water samples were estimated following the

standard colorimetric protocol of Parsons et al. (1984). A ShimadzuR RF 1601 was used

for all spectrophotometric measurements.

For measurements of periphyton productivity, a set of glass slides retrieved from

each depth was transferred into paired 250-ml light- and amber-coloured bottles

containing 200 ml of filtered estuarine water taken from the same depth. Each bottle

was spiked with 5 ACi of NaH14CO3 (ICNR Radiochemicals) and incubated in situ

for 30 min at the corresponding depth at which the glass slides were collected. All

incubations were carried out on a multilevel floating raft anchored to the mooring

pontoon of the marina (Fig. 1). After incubation, the bottles were retrieved and

transported immediately to the laboratory at + 4 jC (transit time approximately 30

min) in the dark. In the laboratory, periphyton on the glass slides was scraped off

using a flat blade scalpel and resuspended in the filtered seawater it had been

incubated in. The slurry was further filtered onto a WhatmanR 0.2-Am pore size

membrane filters under vacuum with repeated rinsing using filtered estuarine water to

rinse off traces of unfixed radiotracer on the filter paper. Filters were then placed

into 20-ml glass scintillation vials after which 1 ml of 0.5 N hydrochloric acid was

added to each vial, to remove inorganic carbon. Vials were left uncovered in a clean

fume hood for 24 h, after which 10 ml of scintillation cocktail Universol (ICNRRadiochemicals) was dispensed into each vial, and capped tightly. A WallacR 1414

liquid scintillation counter, calibrated using WallacR 14C unquenched standards, was

used to assay the radioactivity of the filters using the protocol of Parsons et al.

(1984). Periphyton productivity was measured in duplicates for each day and each

depth.


Periphyton on another set of glass slides was scraped using a clean flat blade scalpel

and resuspended in filtered seawater to be used for periphyton chlorophyll a and cell

counts.

Resuspended periphyton was filtered onto WhatmanR 0.2-Am pore size membrane

filters under vacuum for chlorophyll a measurements. The acetone extraction fluorometric

protocol of Parsons et al. (1984) was followed, and fluorescence was read using a

Shimadzu RF 1501 spectrofluorometer. Periphyton chlorophyll a concentrations were

measured in duplicates for each day and each depth.

Resuspended periphyton was fixed with formalin. The larger cells (>20 Am) were

directly enumerated under an OlympusR BX50 binocular light microscope, while smaller

cells ( < 20 Am) were enumerated using the DAPI epifluorescence technique on the same

microscope (Porter and Feig, 1980).

2.4. Short-term exposure of periphyton to diesel in in situ microcosms

This experiment was designed based on the results from the monitoring of petroleum

hydrocarbon concentrations and investigation on the settlement of periphyton. The

primary objective of the short-term exposure studies was to assess the possible toxicity

to periphytic algae of environmentally realistic concentrations of petroleum hydrocarbons

(diesel) measured during the monitoring studies.

Based on the findings of Basheer et al. (2003a,b) and Nayar (2003), diesel was

found to be one of the major sources of petroleum hydrocarbon contamination in and

around Ponggol estuary. Therefore, diesel was used as the contaminant in treatments

simulating environmentally realistic concentrations of petroleum hydrocarbons in the in

situ microcosms. Treatment concentrations used in the experiments were the mean and

highest concentrations of petroleum hydrocarbons, measured from the dissolved fraction

(DDPH) and sediments (AAPH) as shown in Table 1. Surface waters from an

unimpacted site in the estuary (Fig. 1) were collected using a pump and filtered

through a WhatmanR 0.2-Am filter paper to remove all particulates. This water was

used as the diluent water for the microcosms. Background levels of DDPH in the

diluent water were below detectable levels and hence did not require any adjustment

while making up the final treatment concentrations. For the two DDPH concentrations,

diesel fuel was used for the treatments in clear 250-ml glass microcosm bottles (Schott

Duran). Control microcosms comprised 200 ml of diluent water (without diesel)

contained in 250-ml bottles.

For the two treatments of AAPH, 40 g of sediment slurry collected from the

unimpacted site in the estuary was spiked with the appropriate concentrations of

diesel corresponding to environmentally measured concentrations of AAPH. Diluent

Table 1

Concentrations of petroleum hydrocarbons recorded from Ponggol estuary during the monitoring study

Fraction Minimum Mean Highest

DDPH (Ag l� 1) 0.35 41.01 1099.65

AAPH (mg kg� 1 dry weight) 20.55 148.23 541.04


water was added to the microcosm bottles containing the spiked sediments to make

the volume to 200 ml. Controls contained 40 g of sediment slurry collected from the

unimpacted site in the estuary made up to a volume of 200 ml with diluent water in

250-ml microcosm bottles. All treatments and controls were conducted in duplicates.

Based on the results from the preliminary settlement studies, periphyton for the

microcosm experiments was allowed to settle on glass slides left submerged on rafts for

3 days at 1 m depth at the unimpacted site. At the end of 3 days, the glass slides were

carefully transferred to each of the microcosm bottles. All microcosms were incubated at 1

m depth from the surface, on a floating raft at the mooring pontoon in Ponggol Marina

between June and July 2001. One duplicate set of slides from treatments and controls was

retrieved daily over 3 days and transported (30 min) to the laboratory at 4 jC in the dark

for analysis. In the laboratory, the samples were immediately processed for measurement

of periphyton chlorophyll a following the acetone extraction fluorometric protocol of

Parsons et al. (1984). The fluorescence of the samples was read using a ShimadzuR RF

1501 spectrofluorometer.

Temperature, pH, DO, salinity, dissolved organic carbon (DOC), and turbidity were

measured from the unimpacted site and the microcosms following the protocol described

in Section 2.2.

2.5. Statistical analysis

Results obtained for the monitoring studies, settlement studies, and microcosms were

analysed using the statistical package Minitab ver. 13.23 and Statistica 98 (release 5.1).

One-way analysis of variance (ANOVA) and t test comparisons were used to determine

significant differences between the days and depths of settlement. One-way ANOVA

and Dunnett’s test were used to determine if there were statistically significant differ-

ences in responses between controls and treatments in the in situ microcosm studies.

Assumptions of normality and homogeneity of variance were tested with Kolmogorov–

Smirnov and Levene’s tests. The threshold level of statistical significance for this study

was a = 0.05.

3. Results

3.1. Field monitoring

DDPHs in the surface and subsurface waters of Ponggol estuary during the field

monitoring ranged from 4.42 to 248.94 Ag l� 1 and from 0.35 to 1099.65 Ag l� 1,

respectively (Fig. 2). AAPH concentrations in sediments during the period of study ranged

from 20.55 to 541.01 mg kg� 1. Distinct peaks in DDPH and AAPH were observed during

the study, coinciding with major oil spill events in the estuary (Fig. 2).

Factorial ANOVA on the results for DDPH registered a significant difference

between sampling dates ( p < 0.05). DDPH concentrations on November 12, 1999,

February 10, 2000, and February 21, 2000 were significantly higher than rest of the

sampling days. No significant differences were observed between the three stations

Fig. 2. Distribution of DDPHs in surface and subsurface waters and AAPHs in sediments of Ponggol estuary,

Singapore. All graphs are representations of meanF S.E. The three triangular heads on the figure represent the oil

spill events.


and depths, nor were the interaction effects significant (Table 2). A two-way ANOVA

on the data for AAPH showed significant differences between the sampling dates

( p < 0.05; Table 2). AAPH concentrations on February 10, 2000, March 7, 2000, April

Table 2

Results of factorial and two-way ANOVA for DDPH in the water column and AAPH in sediments, respectively,

in Ponggol estuary, Singapore

Sources of variance df effect F p

(1) For water column

Between sampling dates 23 2.13 0.015

Between stations 2 0.97 0.386

Between depths 1 0.70 0.409

Samplings� stations 46 1.28 0.206

Samplings� depths 23 0.57 0.928

Stations� depths 2 0.28 0.759

(2) For sediments

Between sampling dates 23 3.24 0.001

Between stations 2 0.99 0.379


18, 2000, and June 30, 2000 were significantly higher than the rest of the sampling

days.

Results of the water quality parameters measured during the monitoring study in

the estuary and at the unimpacted site registered negligible variations (Table 3). DOC

concentrations in the estuary and at the unimpacted site were observed to be high,

with mean concentrations of 128 and 120 ppm, respectively.

3.2. Settlement studies

Results of periphyton settlement, measured in terms of productivity, chlorophyll a,

and cell counts, showed an increase in periphyton production and biomass at all depths

on all days (Fig. 3a–c). Among the four depths, settlement was greatest on slides

placed at 1 m depth. Among the days, increased settlement was observed from day 3

onwards. One-way ANOVA performed on the three settlement parameters showed

statistically significant differences between the days of settlement (Table 4). Settlement

for days 3–5 were significantly different from days 1 and 2. In addition, paired

Student’s t tests showed no significant differences in settlement between days 3–5. No

significant differences were observed in settlement at different depths ( p>0.05).

Table 3

Table summarising water quality parameters measured during the monitoring study, at the unimpacted site and in

in situ microcosms

Parameters Monitoring Unimpacted site Diesel microcosms

Temperature (jC) 30.58F 0.54 30.33F 0.52 30.17F 0.35

pH 7.96F 0.09 8.13F 0.06 8.14F 0.03

DO (mg l� 1) 8.47F 0.72 8.53F 0.61 6.14F 0.48

Salinity (ppt) 25.23F 0.67 26.17F 0.32 27.2F 0.10

Turbidity (NTU) 8.51F 0.53 3.77F 0.10 5.67F 0.23

Values represent meanF S.E.M.

Fig. 3. Settlement determined in terms of (a) periphyton productivity, (b) periphyton chlorophyll a, and (c)

periphyton cell counts in Ponggol estuary.


Table 4

One-way ANOVA for periphyton settlement studies in Ponggol estuary

Parameter Effect df effect F p value t test comparison

Periphyton productivity Depths 3 2.43 0.081

Days 4 10.11 0.000 5 > 4 > 3 > 2 > 1

Periphyton chlorophyll a Depths 3 1.10 0.362

Days 4 28.6 0.000 5 > 4 > 3 > 2 > 1

Periphyton cell counts Depths 3 1.75 0.197

Days 4 6.49 0.003 5 > 4 > 3 > 2 > 1

Results of the t test comparison are arranged in the ascending order of their means and lines are drawn over ‘‘days

of settlement’’ that are not significantly different from each other ( p>0.05).


3.3. In situ periphyton microcosms

Changes in periphyton chlorophyll a were taken as the response variable used to

assess the effect of petroleum hydrocarbons on periphyton settlement. Over the 3 days

of the study, periphyton chlorophyll a in the control microcosms registered a

significant increase with time (Fig. 4). Treatments, simulating the highest and mean

concentrations of petroleum hydrocarbons in the dissolved and sediment fractions,

showed a reduction in periphyton biomass over the 3 days of exposure.

One-way ANOVA revealed significant differences between various treatments in this

study ( p< 0.01). Dunnett’s test revealed significant differences between the treatments

simulating the mean and highest concentrations of diesel with respect to the controls

( p < 0.01; Table 5).

Fig. 4. Changes in periphyton chlorophyll a when exposed to background levels of petroleum hydrocarbons in in

situ microcosms.

Table 5

Results of one-way ANOVA and Dunnett’s test for different treatments on each day of exposure to petroleum

hydrocarbons in in situ periphyton microcosms

Parameters Day 1 Day 2 Day 3

df 4 4 4

F(4,9) 30.24 51.68 115.54

p 0.001 0.000 0.000

Dunnett’s test C>MD>HD>MS>HS C>MD>HD>MS>HS C>MD>HD>MS>HS

The abbreviations C, MD, HD, MS, and HS stands for control, mean concentrations in dissolved fraction, highest

concentrations in dissolved fraction, mean concentrations in sediments fraction, and highest concentrations in

sediment fraction, respectively.


Results of the water quality parameters measured from the in situ microcosms were

comparable to that in the environment (Table 3). DOC concentrations in the microcosms

were high, with a mean of 133 ppm.

4. Discussion

Results from the monitoring study on the distribution of DDPH and AAPH in Ponggol

estuary revealed concentrations that were comparable to other highly impacted coastal

ecosystems worldwide (Table 6). In Ponggol estuary, high concentrations of DDPH and

AAPH have been attributed to historical and current anthropogenic impacts occurring in

and around this estuary. Basheer et al. (2003a,b) attributed higher levels of petroleum

hydrocarbon contamination in the coastal waters of Singapore to shipping, petrochemical

industries, and marine oil spillages.

Herrmann and Hubner (1982) stated river runoff to be an important source of petroleum

hydrocarbons, particularly polycyclic aromatic hydrocarbons (PAHs), in coastal marine

environments. Rivers and estuaries are low-energy ecosystems and are prone to oil spills

and accumulation of contaminants (Lee and Levy 1987; Little, 1987). Studies generally

reveal that petroleum hydrocarbons in rivers and estuaries are relatively higher than that in

coastal seas (Fernandes et al., 1997; Hutagalung et al., 1997; McCready et al., 2000). The

sources of petroleum hydrocarbons in Ponggol estuary were accidental spillages of diesel

from storage tanks on dredgers operating in the estuary, fuel and engine oil leaks from

recreational boats in the Marina, oil tanker traffic and shipping operations in the adjacent

Johor Strait, and land runoff from the numerous monsoon drains that empty into the river

(Nayar, 2003). In studies on the speciation of PAHs in the coastal waters (Basheer et al.,

2003a) and sediments (Basheer et al., 2003b) of Singapore, diesel was reported as one of

the principal petroleum hydrocarbon contaminants around Ponggol. The study on the

speciation of PAH by Basheer et al. (2003a,b) was carried out at around the same time

when the monitoring component of TPH in the present study was undertaken.

The present study revealed generally higher concentrations of DDPH in subsurface

waters near the bottom than in surface waters. Elevated concentrations of DDPH in the

subsurface/bottom waters, as observed in this study, may be attributed to resuspension of

sediment particles from the superficial sediment layer, to which petroleum hydrocarbons

Table 6

Comparison of DDPH in the water column and AAPH in the sediments of Ponggol estuary with those reported

from impacted coastal ecosystems in other parts of the world

Sample

number

Sampling

locality

Concentration

range

Technique

used

References

DDPH

1 Ponggol estuary, Singapore 0.35–1099.7 UVF This study

2 Southern Baltic sea 2.0–130.0 UVF Law and Andrulewicz

(1983)

3 Qatar 1.2–428.0 UVF El-Samara et al. (1986)

4 Saudi Arabia 4.3–546.0 UVF El-Samara et al. (1986)

5 Winyah, USA 0.23–25.0 UVF Bidleman et al. (1990)

6 Estuaries in UK 9.3–48.0 UVF MAFF (1993)

7 Coastal waters of Malaysia 320.0–2280.0 UVF Abdullah (1995)

8 Gulf of Thailand 0.01–76.2 UVF Wattayakorn et al. (1997)

9 Arabian Gulf < 0.10–59.00 UVF

and GCMS

Douabul and Al-Shiwafi

(1998)

AAPH

1 Ponggol estuary, Singapore 20.55–541.01 UVF This study

2 Dulang oil field, Malaysia 718.0–974.0 GCMS Mahadi et al. (1992)

3 Near shore Gulf 62.0–1400.0 GCMS Fowler et al. (1993)

4 Abu Ali, Persian Gulf < 1–6800 HPLC

and GCMS

Krahn et al. (1993)

5 Coastal Malaysia 18.2–847.4 GCMS Abdullah (1997)

6 Coastal Lagoon, Guadeloupe 25.4–4104.4 GCMS Bernard et al. (1996)

7 Inshore Mombasa, Kenya Up to 3600 GCMS Williams et al. (1997)

8 Upper Laguna Madre, TX, USA 2.60–692.0 GC Sharma et al. (1997)

9 Pacific Colombian Coast ND–400.0 UVF

and GC

Gonzalez et al. (1999)

10 Coastal England and Wales ND–43470.0 HPLC-FID Woodhead et al. (1999)

11 Coastal Patagonia, Argentina ND–737.6 UVF Commendatore et al.

(2000)

12 Sydney Harbour, Australia < 0.1–380.0 GCMS McCready et al. (2000)

13 Coastal Singapore 13.63–84.92 GCMS Basheer et al. (2003b)

ND—below detectable limits; UVF—UV fluorescence; GC—gas chromatography; GCMS—gas chromatography

and mass spectroscopy; HPLC—high-performance liquid chromatography; HPLC-FID—high-performance liquid

chromatography with flame ionisation detection.

All DDPH and AAPH concentration ranges are expressed as micrograms per liter and milligrams per kilogram,

respectively.


may have been bound (Witt, 1995). High molecular weight (HMW) fractions of petroleum

hydrocarbons preferentially adsorb to particulate material and may be incorporated into

sediments (Connell and Miller, 1984; Witt, 1995). Due to their lipophilic character and

high persistence, petroleum hydrocarbons tend to accumulate in high concentrations in the

sediments (Neff, 1979). Petroleum hydrocarbons and other oils also show a tendency to

adhere to phytoplankton and other suspended particulates, causing them to sink to the

bottom. This process may result in a reduction of particulate oil concentrations in the water

column (Lee et al., 1985; Yu et al., 1987), while increasing the proportion of petroleum

hydrocarbon concentrations in the sediments. Diesel, one of the principal sources of


petroleum hydrocarbon pollution in the study area, does not readily evaporate unlike

lighter fuels such as gasoline (Clark, 1989), rendering it persistent in the environment. This

may explain the relatively elevated concentrations of AAPH measured from the sediments

of Ponggol estuary, as compared with DDPH in the overlying water column.

Such high concentrations of DDPH and AAPH recorded from the estuary may exert

adverse effects on the biotic components, particularly those organisms that are sessile and

that may not leave the impacted site in the event of a severe oil spill (Dickman, 1973). This is

of particular concern, since studies report increased bioavailability of sediment-bound

petroleum hydrocarbons compared with pyrogenic sources that are ultimately transported to

the benthos and other organisms living in the water column (Connell and Miller, 1984; Qian

et al., 2001). Periphytic algae form the base of food webs and have been reported to respond

immediately to alterations in the water quality (Robert et al., 1995; Lewis et al., 2002).

Periphytic algal community in Ponggol estuary from this study showed that out of 32

microalgal species recorded, 88% comprised diatoms and about 9% comprised cyanobac-

teria (Nayar et al., 2003). Dominance of diatoms in the periphyton community has been

reported by researchers elsewhere (Genter et al., 1987; Pudo and Fubara, 1988; Bobkova and

Smirnova, 1994; Sreekumar and Joseph, 1995; Greenwood et al., 1999; Havens et al., 1999;

Kostel et al., 1999; Muller, 1999; Baffico, 2001; Brandini et al., 2001). Light intensity and

currents have also been reported to play an important role in the settlement of periphyton

(Robert et al., 1995; Strueder, 1999; Ledger and Hildrew, 1998; Wellnitz and Rinne, 1999;

Abe et al., 2000). Photoinhibition of periphyton has been reported to occur at the surface due

to high irradiance (Dodds et al., 1999). In studies conducted by other researchers, production

and biomass maxima were observed at a 1-m depth, followed by a decline with depth,

supporting the findings of this study (Strueder, 1999; Brandini et al., 2001).

Periphyton chlorophyll a was taken as the response parameter in the short-term in situ

microcosms to study the effect of environmentally measured concentrations of petroleum

hydrocarbons to periphyton. A few published works have similarly used periphyton

chlorophyll a as a biomarker of pollution on periphyton in manipulated experiments

(Grzenda and Brehmer, 1960; Crossey and La-Point, 1988; French and Evans, 1988; Singh

and Gaur, 1989; Dahl and Blanck, 1996; Arnegard et al., 1998; McCormick and

Stevenson, 1998; Paulsson, 2000).

Oil spills in the estuary consisted primarily of diesel, which originated from the storage

tanks of the dredgers operating in the estuary, as well as recreational boats at the marina.

The short-term in situ microcosms simulating environmental concentrations (of petroleum

contaminants) also utilised diesel as the pollutant. Results from the exposure of periphyton

to the mean and highest concentrations of DDPH and AAPH showed inhibition of all

periphyton growth, as seen from the decrease in periphytic chlorophyll a. In a marine

mesocosm study, Yu et al., (1987) reported suppression of phytoplankton at petroleum

hydrocarbon concentrations exceeding 1.5 mg l� 1. Although they did not observe any

changes in species composition, they observed a significant decline in cell counts,

chlorophyll a, and primary production.

Oil spills have been suggested to result in the eventual dominance of microflagellates

because of the sensitivity of diatoms to oil (Hsiao, 1978; Yu et al., 1987). These findings

would be of interest in an ecosystem like Ponggol estuary where the periphytic algal

community is dominated by diatoms. Similarly, Riquelme and Garcia (1986) observed that


marine periphytic flora colonizing introduced glass substrates were adversely affected

when exposed to oil, resulting in a significant decline in the number of bacteria,

protozoans, and primary producers. Singh et al. (1987) observed that a slight decline in

oil and phenol concentrations from 5.1 to 1.2 mg l� 1 and from 1.56 to 0.20 mg l� 1,

respectively, resulted in an increase in species diversity of periphyton, which was

dominated by cyanobacteria. Singh and Gaur (1989) also reported a significant decline

in chlorophyll a and cell numbers of an epilithic community exposed to oil refinery

effluents. The toxicity of petroleum hydrocarbons generally depends on the relative

concentrations of the HMW and the low molecular weight (LMW) fraction of hydro-

carbons. It has been demonstrated that some of the LMW hydrocarbons are highly toxic to

marine biota (Anderson et al., 1974; Calder and Lader, 1976). Petroleum products such as

diesel, one of the principal sources of petroleum hydrocarbon polluting Ponggol estuary,

contains 13–27 times more concentration of LMW hydrocarbons than HMW hydro-

carbons (Qian et al., 2001). This explains why exposure of periphytic algae in the in situ

microcosms to diesel resulted in a significant decline in periphyton biomass measured in

chlorophyll a over the 3 days of exposure.

5. Conclusions

This study provides comprehensive information on the distribution of DDPH and

AAPH in Ponggol estuary, Singapore, during a period when anthropogenic impacts were

taking place. This is the first study from Singapore integrating results obtained from

monitoring studies and ecotoxicological assays carried out on one of the biotic compo-

nents of an estuary. Ecotoxicological assessment of environmentally realistic concen-

trations of petroleum hydrocarbons on periphytic algal biomass in in situ microcosms

revealed signs of acute toxicity. The use of periphyton has potential in ecotoxicological

testing, since it is a reliable and sensitive measure requiring modest investments.

Acknowledgements

This research was partly supported by the research grants MBBP/MB1/BG1 made

available to the Tropical Marine Science Institute by the National Science and Technology

Board, Singapore, and the Singapore Institute of Biology research grant RTF 30/2001.

Thanks are due to Mr. Abdul Latiff for his help during the field work, and Ponggol Marina

for boat berthing facilities and the use of a pontoon for the microcosm experiments. We

also thank the anonymous reviewers for their valuable comments. [RW]

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