Ecotoxicology and Environmental Safety 142 (2017) 538–543

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Toxicity of diesel water accommodated fraction toward microalgae, MARK

Pseudokirchneriella subcapitata and Chlorella sp. MM3
⁎
Kavitha Ramadassa, Mallavarapu Megharajb, , Kadiyala Venkateswarlua,c, Ravi Naidub
a
Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, SA 5095, Australia
b
Global Centre for Environmental Remediation (GCER) and Cooperative Research Centre for Contamination Assessment and Remediation of the Environment
(CRCCARE), Faculty of Science, University of Newcastle, ATC Building, Callaghan, NSW 2308, Australia
c
Formerly Department of Microbiology, Sri Krishnadevaraya University, Anantapur 515055, India

A R T I C L E I N F O A B S T R A C T

Keywords: Diesel is a commonly used fuel and a key pollutant on water surface through leaks and accidental spills, thus
Diesel creating risk directly to planktons as well as other aquatic organisms. We assessed the toxicty of diesel and its
Water accommodated fraction water accommodated fraction (WAF) towards two microalgal species, Pseudokirchneriella subcapitata and
Microalgal toxicity Chlorella sp. MM3. The toxicity criteria included were: chlorophyll a content as a growth parameter and
Oxidative stress
induction of enzyme activities linked to oxidative stress. Increase in concentrations of diesel or its WAF
Superoxide dismutase
significantly increased toxicity towards growth, measured in terms of chlorophyll a content in both the algae.
Peroxidase
Activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) in
response to addition of diesel or diesel WAF to the microalgal cultures were dose-dependent. Diesel WAF was
more toxic than diesel itself, suggesting that use of WAF may be more relevant for environmental risk assessment
of diesel. The overall response of the antioxidant enzymes to toxicants’ stress followed the order:
POX≥SOD > CAT. The present study clearly demonstrated the use of SOD, POX and CAT as suitable biomarkers
for assessing diesel pollution in aquatic ecosystem.

1. Introduction tocopherols, carotenoids and phycocyanin (Suzuki and Mittler, 2006).

Diesel, a refined product from crude oil, is one of the most
extensively used petroleum hydrocarbons. Diesel oil consists of a
complex mixture of hundreds of aliphatic and aromatic hydrocarbons.
Global consumption of diesel increased substantially by 23% during
2000–2008, whereas the increase for gasoline demand was only 7% in
the corresponding period (IEA, 2008). The expected demands for diesel
and gasoline between 2012 and 2035 are > 5 and 2 million barrels/d,
respectively (IEA, 2013). This increase in global consumption of diesel
has a direct impact on environmental contamination when leaked from
storage mostly into the aquatic ecosystems. If released into the water
bodies, hydrocarbons and other chemical compounds from petroleum
products including diesel threaten important aquatic organisms such as
microalgae which are the primary producers.
Increased generation of reactive oxygen species (ROS) like free
radicals, hydrogen peroxide, and singlet oxygen results in oxidative
stress (Regoli et al., 2002). Superoxide dismutase (SOD), catalase
(CAT), glutathione reductase (GSH) and peroxidase (POX) are the key
antioxidant enzymes, whereas the non-enzymatic mechanism includes
the response of such mediator compounds as ascorbic acid, glutathione,
When nontarget organisms are exposed to toxic xenobiotic compounds,
antioxidants like SOD, CAT, GSH, etc. can serve as excellent biomarkers
that reflect contaminant-mediated oxidative stress (Geret et al., 2003).
Microalgae are very sensitive to a variety of organic and inorganic
toxicants through their rapid physiological response (McCormick and
Stevenson 1998; Megharaj et al., 2000; Ramakrishnan et al., 2010). The
effects of diesel alone towards several toxicity criteria like chlorophyll
content and other accessory pigments, growth rate, lipid peroxidation,
etc. in microalgae have been reviewed (Lewis et al., 2013). Although
microalgae have the ability to encounter ROS by stimulation of their
antioxidants defense system, their response to organic pollution has not
been fully understood (Choo et al., 2004; Subashchandrabose et al.,
2012).
Petroleum hydrocarbons are affected by a series of physical,
chemical and biological processes in water bodies, and the aquatic
toxicity is only due to their water accommodated fraction (WAF)
(Mackay et al., 1980), possibly increasing the durability of hydrocarbon
pollution. There have been no studies on the effects of diesel WAF on
nontarget microorganisms such as microalgae. Hence, the objective of
the present study was to evaluate growth and biochemical responses of

⁎
Corresponding author.
E-mail address: megh.mallavarapu@newcastle.edu.au (M. Megharaj).

http://dx.doi.org/10.1016/j.ecoenv.2017.04.052
Received 28 October 2016; Received in revised form 20 April 2017; Accepted 24 April 2017
Available online 04 May 2017
0147-6513/ © 2017 Elsevier Inc. All rights reserved.
K. Ramadass et al.
two microalgae, Pseudokirchneriella subcapitata and Chlorella sp. MM3,
upon their exposure to diesel WAF in relation to diesel itself. These two
microalgae were selected because P. subcapitata is a standard OECD
(Organization for Economic Co-operation and Development) test spe-
cies and Chlorella sp. is distributed most widely in aquatic environ-
ments, and both are often used in toxicity tests due to their sensitivity to
different contaminants (Muñoz et al., 1996; Ramadass et al., 2015,
2016).
2. Materials and methods
2.1. Microalgal cultures
P. subcapitata (non-motile, unicellular, crescent-shaped
(40–60 µm3), commonly found in most freshwaters) was obtained from
CSIRO Collection of Living Microalgae (Hobart, Australia), while
Chlorella sp. MM3 was from the Microalgal Culture Collection at the
Centre for Environmental Risk assessment and Remediation (CERAR),
University of South Australia. Axenic cultures of the algal species were
maintained in Bold's basal medium (BBM) at 25 ± 2 °C in a growth
chamber under continuous illumination of 200 µE m−2 s−1 PPFD
(Subashchandrabose et al., 2012; Ramadass et al., 2016).
2.2. Preparation of WAF and analysis
Commercial diesel oil used in the study was purchased in Adelaide,
South Australia. WAFs were prepared according to Bejarano et al.
(2006) with a slight modification. Diesel oil (80 mL) was layered on top
of 720 mL membrane-filtered (0.22 µm filter) water contained in a
1000 mL glass bottle by means of a syringe. The bottle was sealed tight,
headspace air purged through the Teflon septa with a stainless-steel
needle attached to a gas-tight syringe, and the 200-mL headspace filled
with nitrogen (> 99% purity) to prevent oil degradation/oxidation.
The bottle was placed in a refrigerated incubator (20 ± 1.5 °C) on a
magnetic stirrer plate. The water extract portion was drained out after
24 h of continuous stirring, and was considered as 100% WAF.
Benzene, Toluene, Ethylene and Xylene (BTEX), and other volatile
compounds in WAF were directly estimated following EPA methods
5030 and 8260B (GC-MS with purge-and-trap extraction for volatile
organics). Standard volatile organic compound mix (VOC Mix 502-
Alltech VOC-2JM-A) was used to quantify BTEX and other organic
compounds. 4-Bromofluorobenzene, at a concentration of 20 µg mL-1,
was used as a surrogate. Total petroleum hydrocarbons (TPHs) in WAF
samples were extracted with methylene chloride, concentrated to
1.0 mL under N2 stream, and analyzed by GC fitted with a flame
ionization detector (GC-FID Agilent model 6890). o-Terphenyl was used
as a surrogate at 20 μg mL-1 in each sample extraction. Chromatography
was performed on a fused-silica capillary column BPX-5 from SGE
(15 m×0.32 mm internal dia) coated with HP-5 (0.10 µm film thick-
ness). Helium was used as the carrier gas at 2.5 mL min-1, and the FID
detector temperature was kept at 300 °C. Splitless injection with a
sample volume of 1.0 µl was applied. The oven temperature was
increased from 50 to 300 °C at a gradient of 25 °C min-1, and held at
this temperature for 5 min. The total run time was 19.6 min.
Hydrocarbons were quantified using Agilent Chemstation Software by
integration and calibration of peaks of known concentrations (Risdon
et al., 2008). The recovery, following continuing calibration verifica-
tion (CCV) at the start and end of every 10 samples, was 95–110% of
true value. Hexane blank was run after every 10 samples to verify
cleanliness of the system. The measured concentration of TPHs in 100%
WAF was 53.14 mg L-1, and TPH concentration in each dilution of WAF
was predicted based on nominal dilution of the initial 100% stock
solution. Thus, the predicted nominal concentrations of TPHs in the test
medium were 0.53, 1.33, 1.99, 2.66, 3.32, 3.98, 5.31, 6.64 and
7.97 mg L-1 corresponding to 1.0%, 2.5%, 3.75%, 5.0%, 6.25%, 7.5%,
10.0%, 12.5% and 15% of WAF, respectively. Polycyclic aromatic
539
Ecotoxicology and Environmental Safety 142 (2017) 538–543
hydrocarbons (PAHs) from WAFs were extracted using methylene
chloride. The surrogate chemicals used for analysis were 2-Fluorobi-
phenyl and p-terphenyl-d14. PAHs in WAFs were analyzed following
EPA SW-846 Method 3510 C.
2.3. Algal toxicity assay
Algal growth inhibition test was conducted by exposing each
microalga to different concentrations of diesel oil (0–12.5 mg L-1) and
dilutions of WAF (0–15%) for 2 weeks. Whole oils were added into BBM
inoculated with algal cells from a 7-d-old exponentially-growing
culture. The initial cell density of a culture was maintained at 5×105
cells mL−1, and the total volume of the test medium was 100 mL. WAFs
were also tested for their toxicity and compared with the whole oil
toxicity. Aliquots of freshly extracted WAF were combined with
appropriate volumes of sterilized milliQ water and BBM in clean 250-
mL conical flasks to provide 0–15% WAF in 100 mL. Algal cells were
inoculated into the flasks. Cultures with no diesel and 0% WAF which
contained only sterilized milliQ and BBM served as controls. The test
cultures were maintained in a temperature-controlled (25 °C) orbital
shaker set at 120 rpm under cool white fluorescent illumination of
about 200 µE m−2 s−1 PPFD. At the end of 4, 8 and 12 days, algal
growth was measured in terms of chlorophyll a, an indicator of algal
biomass (Megharaj et al., 1986; Deasi et al., 2010; Ramadass et al.,
2016). Median lethal concentration (EC50) values for 96 h exposure (US
EPA 1993) were calculated from the values of per cent inhibition in
growth relative to those of untreated controls by Probit analysis using
Minitab 16 statistical software (Palma et al., 2008). The acute toxicity
experiment was performed twice under reasonably constant test con-
ditions, and the data showed that the precision (CV) of EC50 values in
four replicates of each sample was 11.2%.
2.4. Antioxidant enzyme assays
After 96 h of incubation, cells were harvested by spinning algal
cultures at 4600×g for 15 min, and the cell pellet was washed twice
with sterile ultrapure water. The cells were resuspended in the
respective enzyme assay buffer and lysed using an ultrasonicator
(Branson digital sonifier). Cell lysates were then centrifuged at
9167×g for 5 min, and the supernatant was transferred to fresh
microfuge tubes and stored on ice or at 4 °C until the enzymes were
assayed.
To determine total soluble protein, washed cells from the cultures
were suspended in 50 mM Tris (pH 7.50) containing 0.5 mM phenyl-
methylsulfonyl fluoride and lysed in an ultrasonicator. After centrifuga-
tion, the clear supernatant was used for analysis of soluble protein, and
assay of antioxidant enzymes like SOD, CAT and POX to determine the
oxidative stress induced in the selected microalgae upon exposure to
diesel as described previously (Ramadass et al., 2016). In brief, the
ability of superoxide radical to inhibit the reduction of cytochrome C
was used for measuring SOD activity. The reaction mixture contained
50 mM of potassium phosphate buffer (pH 7.8), 0.1 mM EDTA,
0.01 mM cytochrome C, 0.05 mM xanthine, 0.005 U of xanthine
oxidase and 100 µl cell extract. The increase in absorbance at 550 nm
was recorded for 5 min. To determine POX activity, an aliquot (100 µl)
of cell extract was added to 3 mL of assay mixture containing 14 mM
potassium phosphate buffer (pH 7.0), 0.027% H2O2, and 0.5% pyr-
ogallol. Change in absorbance at 420 nm was monitored in a spectro-
photometer every 20 s for 5 min. The activity of CAT was measured in
the reaction mixture that consisted of 50 mM potassium phosphate
buffer (pH 7.0), 0.035% H2O2, and 100 µl cell extract. The time
required for a decrease in absorbance from 0.45 to 0.40 was read
at 240 nm. The activities of all the three enzymes were expressed
as U mg-1 protein.
K. Ramadass et al. Ecotoxicology and Environmental Safety 142 (2017) 538–543

Table 1
Volatile and semi-volatile hydrocarbons present in diesel WAF.

Aromatics Diesel Safe limit for Safe Safe limit

WAF used drinking limit for for marine
in this water fresh water
study water

Volatile aromatics (µg L−1) 532 1 950 500

Benzene
Toluene 1448 800 ID ID
Ethyl benzene 253 300 ID ID
m/p-Xylene 628 ID 350 ID
o-Xylene 514 ID 200 ID
Naphthalene 158 NA 16 70
1,3,5-Trimethylbenzene 100 NA NA NA
1,2,4-Trimethylbenzene 464 NA NA NA
Semi volatile hydrocarbons
(mg L−1)
C8-C14 12.13 NA 50 NA
C15-C28 40.23 NA 100 NA
C29-C36 0.78 NA 100 NA
Total (C8-C14) 53.14

Safe limits are the 95% species protection values as per the Australian and New Zealand
Guidelines for Fresh and Marine Water Quality (ANZECC & ARMCANZ 2000). ID =
insufficient data to derive an investigation level. NA=not available.

2.5. Statistical analysis

The experimental data were analyzed for significance (P≤0.05)

using statistical software tool SPSS version 18.0.0 (SPSS Inc., Chicago,
USA). Antioxidant enzyme activities were compared to determine
whether mean values differed significantly following Tukey's Test using
SPSS software.

3. Results and discussion

Fig. 1. Effect of (a) diesel, and (b) diesel WAF on chlorophyll a in P. subcapitata. Error
3.1. Hydrocarbons in diesel WAF bars represent standard deviation (SD) (n=4).

Analysis of WAF prepared from diesel for volatile organic com-
pounds revealed the presence of major toxic compounds such as BTEX,
alkylated benzenes and naphthalene, and most of these compounds
were above the levels of safe limits (environmental relevant concentra-
tions) for an aquatic ecosystem (Table 1). The semi-volatile TPHs in
WAF were about 53 mg L-1. Analysis for PAHs showed the predomi-
nence of napthalenes and 2.7 µg L-1 of phenanthrene. Other PAH
compounds like fluorene, acenaphthene and benzopyrene were present
below detectable limits.
3.2. Microalgal toxicity of diesel and diesel WAF
Chlorophyll a, a photosynthetic pigment which serves as a biomass
indicator of aquatic microalgae that support food webs, is the most
frequently measured biochemical parameter to study the effect of
environmental pollution (Ramakrishnan et al., 2010;
Subashchandrabose et al., 2012; Ramadass et al., 2016). In fact,
chlorophyll a content in combination with other variables was used
as a good substitute for microalgal biomass (Ramakrishnan et al.,
2010). When exposed to diesel, there was a significant dose-related
response in growth, measured in terms of chlorophyll a, of the two
microalgae used in the present study (Figs. 1a, 2a). However, P.
subcapitata exhibited an initial stimulation at 0.5–2 mg L-1 of diesel
followed by inhbition at higher doses of 3.5–11 mg L-1 (i.e., hormetic
effect), while exposure of Chlorella sp. MM3 to diesel was inhibitory.
Diesel was completely toxic to growth of P. subcapitata at the highest
concentration of 12.5 mg L-1 used in the present study. On the other
hand, growth of Chlorella sp. MM3 was totally inhibited at 8.75 mg L-1
of diesel. From the data on growth, measured in terms of chlorophyll a,
it is clear that Chlorella sp. MM3 was more sensitive to diesel than P.
subcapitata.
Most of the work on aquatic toxicity with petroleum hydrocarbons
is usually expressed in terms of per cent water-soluble fraction (WSF) or
%WAF because it is a significant indicator for the expected toxicity
level when petroleum oil is released into the aquatic environment
(Nayar et al., 2005). In assessing toxicity, the solubility of hydrocarbon
components in petroleum products is an important property and water
solubility of a substance determines the possible routes of exposure.
Low molecular weight (LMW) compounds are more soluble than high
molecular weight (HMW) compounds, and C4-C8 compounds have a
solubility level of approximately 2000 ppm. Thus, solubility is inversely
proportional to the molecular weight of compounds, and generally, the
most soluble components are the most toxic. Hence, WAF was prepared
from diesel to investigate its potential for causing toxicity to the test
microalgae. Solubility of aromatic hydrocarbons in water is greater
when compared to the alkanes of similar molecular weight and
disappear more slowly from solution compared to alkanes (Anderson
et al., 1974). Furthermore, aromatic compounds tend to accumulate in
organisms to a greater extent and are retained longer than alkanes (Neff
et al., 1976). These are the critical factors that determine the high
toxicity of diesel oil. In the present study, volatile hydrocarbon analysis
in WAF prepared from diesel revealed the presence of higher amounts
of toxic monoaromatic compounds (Table 1). The soluble aromatics of
diesel (such as benzene, alkylated benzenes, toluene, alkylated to-
luenes, ethylbenzene, xylenes, and naphthalenes) are mainly respon-
sible for the diesel toxicity.
The data presented in Figs. 1b and 2b depict the effect of WAF on
growth, in terms of chlorophyll a, of two microalgae. WAF, at 2.5% was
either innocuous or slightly stimulatory to growth of P. subcapitata,
while other concentrations were inhibitory. Increase in WAF concen-

540
K. Ramadass et al.
Fig. 2. Effect of (a) diesel, and (b) diesel WAF on chlorophyll a in Chlorella sp. MM3. Error
bars represent SD (n =4).
tration resulted in inhibition in growth of Chlorella sp. MM3. Toxicity,
in terms of EC50 to the test microalgae, of diesel alone differed greatly
from diesel WAF. In case of P. subcapitata, the estimated 96 h EC50
values for diesel and diesel WAF were 6.03 ± 0.22 mg L-1 and
4.52 mg L-1 (8.24%), respectively. Similarly, 96 h EC50 values for diesel
and diesel WAF toward Chlorella sp. MM3 were 3.90 ± 0.06 mg L-1 and
2.6 mg L-1 (4.8%), respectively. These results clearly suggest that diesel
WAF is more toxic to algal growth than diesel itself.
Growth stimulation in P. subcapitata may be attributed to its ability
to utilize compounds from diesel as sources of organic carbon for
growth. Uptake and metabolization of oil constituents have been
suggested as probable mechanisms for growth stimulation in algae
(O'Brien and Dixon, 1976). Stimulation of algal growth at low oil
concentration of diesel and gasoline was reported earlier (Gordon and
Prouse, 1973; Dunstan et al., 1975; Megharaj et al., 2000). Inhibition in
growth by high concentrations of diesel could be due to its interference
with cell membrane permeability and pigment production. However,
the relative concentration of LMW and HMW hydrocarbons is the key
factor that influences the toxicity. Diesel contains 13–27 times more
concentration of LMW hydrocarbons than HMW hydrocarbons (Qian
et al., 2001). The LMW hydrocarbons with low-boiling points, unsatu-
rated compounds, aromatics and acids present in diesel cause mem-
brane damage (Kauss and Hutchinson, 1975), and also increase
membrane permeability and reduce proton motive force in cells. Also,
it is evident that some LMW hydrocarbons are highly toxic to marine
biota (Anderson et al., 1974). When the proton motive force declines,
the electrochemical gradient across the thylakoid membrane will be
compromised, resulting in a decrease in photosynthetic yield by the two
photosystems (I and II) (Piehler et al., 2003). Oil pollution, including
diesel and gasoline, inhibited the growth of Chlorella pyrenoidosa
541
Ecotoxicology and Environmental Safety 142 (2017) 538–543
(Coffey et al., 1977), and two species of Chlorella, C. homospora and
C. vulgaris (El-Sheekh et al., 2000). When applied to algae, diesel
disrupted the optimal physical state of cytoplasmic membranes, and
disturbed the osmotic balance in algal cells (Mohammady et al., 2005).
Increase in cell permeability stimulates the influx of a pollutant and
probably results in accumulation of a high quantity of hydrocarbons,
causing cell swelling. As a result, the pollutant may have a positive
significant effect on cellular production of chlorophyll a as evidenced in
the present study.
3.3. Effect of diesel and diesel WAF on antioxidant enzymes in microalgae
Antioxidant enzymes are considered to be the signals of distress at
molecular level, and hence useful as toxicity biomarkers. Critical
antioxidant enzymes such as SOD, CAT, ascorbate peroxidase, and
glutathione S-transferase help an organism during oxidative stress
(Torres et al., 2008). Antioxidant enzymes may be activated by a slight
oxidative stress; however, this activity may be suppressed severely
when oxidative stress becomes more, resulting in a loss of the
compensatory mechanism. Wojcik et al. (2006) and Maity et al.
(2008) applied these biomarkers in toxicity studies on vertebrates,
invertebrates, and plants. Toxic contaminants like trace metals, poly-
cyclic aromatic hydrocarbons, and polychlorobiphenyls have the ability
to enhance the formation of ROS in cells (Liu et al., 2009), leading to
oxidative stress and several alterations in cell metabolism such as
protein degradation or lipid peroxidation of membranes. Normally,
many ROS generating processes are slow in algal cells when exposed to
toxic metals and xenobiotics. In order to assess any oxidative damage
occurring due to diesel exposure, antioxidant enzymes such as SOD,
POX and CAT in the test microalgae were assayed after 96 h of exposure
to diesel itself and diesel WAF.
In P. subcapitata, the SOD activity remained unchanged up to
2 mg L-1 diesel, increased significantly up to 6.5 mg L-1, and was
completely inhibited at 8 mg L-1 (Fig. 3a). Diesel, up to 1.25 mg L-1,
had no effect on SOD activity in Chlorella sp. MM3 (Fig. 4a); however,
2.5 and 3.75 mg L-1 concentrations caused an increase in enzyme
activity, and the activity was completely inhibited at 5 mg L-1 level.
Diesel exposure increased the POX activity in P. subcapitata significantly
at the concentrations of 2–6.5 mg L-1 (67–187% increase over control),
and the activity was completely inhibited at concentrations from 8 to
12.5 mg L-1. Exposure of Chlorella sp. MM3 to diesel up to 0.5 mg L-1
had no effect on POX activity; however, an increase in diesel concen-
tration from 1.25 mg L-1 to 3.75 mg L-1 increased the enzyme activity,
and was completely inhibited at 5 mg L-1. Diesel up to 5 mg L-1 had no
effect on the activity of CAT in P. subcapitata, but 6.5 mg L-1 concentra-
tion enhanced the enzyme activity by 46% over control. As with the
other two enzymes (SOD and POX), higher concentrations of diesel
(8–12.5 mg L-1) resulted in complete inhibition of CAT activity.
Chlorella sp. MM3 showed an increase in the activity of CAT up to
3.75 mg L-1 of diesel, but 5 mg L-1 concentration resulted in complete
loss of activity. In general, SOD, POX and CAT were more sensitive to
diesel exposure in Chlorella sp. MM3 than in P. subcapitata.
Exposure of both the microalgal species to WAF also influenced the
antioxidant enzymes. SOD activity increased when P. subcapitata and
Chlorella sp. MM3 were exposed to WAF at 7.5% and 1%, respectively
(Figs. 3b and 4b). An increase in POX activity was observed with 5%
diesel WAF in P. subcapitata and 2.5% in Chlorella sp. MM3. Activity of
CAT was stimulated in P. subcapitata when exposed to 5% WAF. All the
three tested antioxidant enzymes were completely inhibited with 10%
and 6.35% diesel WAF in P. subcapitata and Chlorella sp. MM3,
respectively. However, addition of diesel WAF to the culture medium
was innocuous to CAT in Chlorella sp. MM3 up to 3.75%, while 5%
stimulated the activity. These results on antioxidant enzymes also
clearly suggest that Chlorella sp. MM3 was more sensitive to WAF
obtained from diesel than P. subcapitata.
Increase in activity of antioxidant enzymes such as SOD, POX and
K. Ramadass et al.
Fig. 3. SOD, POX and CAT activities (bars in the order) in P. subcapitata exposed to (a)
diesel, and (b) diesel WAF. For an enzyme, mean values related to different concentra-
tions of diesel or its WAF sharing the same letter on top of the bars are not significantly
different at P≤0.05 (Tukey's test).
CAT with increasing diesel concentration indicates a clear correlation
between free radical generation and antioxidant production which
reflects the oxidative stress caused in the microalgal cells. However,
when the toxicity of diesel was more, the enzyme activities were
completely inhibited or impaired. The production of ROS by microalgae
may be due to the toxic components present in diesel, and over-
production of ROS would explain the damage occurred to the antiox-
idant defense system as evident from suppression of the enzyme
activities. Cheung et al. (2001) reported similar results with algae upon
their exposure to crude oil. Elevated levels of ROS production can cause
oxidative stress, resulting in mutagenesis, carcinogenesis, protein
oxidation and degradation, carbohydrate damage, or lipid peroxidation.
Overall, the present data indicate an inverse relationship between
chlorophyll content and antioxidant production in the algal species
under stress conditions mediated by diesel.
Available information on the response of algal antioxidant systems
to environmental stresses seems to be little and broadly diffused. To our
knowledge, no information is available on the microalgal antioxidants’
response to diesel exposure. However, significant changes in SOD and
POX were observed in Euglena, isolated from lake water contaminated
with organic compounds including PAHs and BTEX (Li et al., 2014).
Also, Lei et al. (2006) found stimulation of SOD activity in P. subcapitata
and inhibition in Chlorella sp. by pyrene. Therefore, more intensive
studies, involving large number of microalgae implicated in ecosystem
health, are required to establish the toxic nature of diesel pollution.
4. Conclusion
The results of this study clearly demonstrate that diesel exposure
542
Ecotoxicology and Environmental Safety 142 (2017) 538–543
Fig. 4. SOD, POX and CAT activities (bars in the order) in Cholrella sp. MM3 exposed to
(a) diesel, and (b) diesel WAF. For an enzyme, mean values related to different
concentrations of diesel or its WAF sharing the same letter on top of the bars are not
significantly different at P≤0.05 (Tukey's test).
cause significant damage to the growth of microalgae. Furthermore,
WAF obtained from diesel is more toxic than diesel itself. Hence, diesel
WAF may be more relevant for environmental risk assessment of diesel.
For the first time, the activities of antioxidant enzymes such as SOD,
POX and CAT were used as toxicity criteria for understanding oxidative
stress in microalgae exposed to diesel pollution. Thus, the present data
may provide a better insight into the toxicity and oxidative stress
caused by diesel exposure, and suggests the suitability of antioxidant
enzymes as biomarkers for assessing the oxidative stress in microalgae.
In all, our investigation suggests that contamination of water bodies
with diesel could potentially result in serious consequences to ecosys-
tem health by disrupting primary producers such as microalgae that are
located at the base of the food chain. This laboratory study allowed for
greater control of variables so that actual effect of diesel toxicity on
microalgae could be evaluated. It provides a consistent and reproduci-
ble approach for evaluating the acute and sub-lethal toxicity of diesel-
contaminated water ecosystem that allows for extension to filed-scale
studies. Both laboratory and field evaluations are mandatory for a
weight-of-evidence assessment to determine the driving stressor-expo-
sure responses.
Acknowledgment
This research was supported by the Australian Government,
University of South Australia through an IPRS Scholarship, and CRC
for Contamination Assessment and Remediation of the Environment.
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