Ecotoxicology (2007) 16:393402
DOI 10.1007/s10646-007-0134-4
Comparative toxicity of hydrophobic contaminants to microalgae
and higher plants
M. K. Chung R. Hu M. H. Wong K. C. Cheung
Accepted: 12 January 2007 / Published online: 14 April 2007

Springer Science+Business Media, LLC 2007
Abstract To enable rapid and sensitive screening of
phytotoxic compounds in terrestrial system, a 4 day solid-
phase microalgal bioassay was developed. Three species of
microalgae (Selenastrum capricornutum, Chlorococcum
hypnosporum and Chlorococcum meneghini) were chosen
to investigate their responses to DDTs (DDT, DDD and
DDE) and PAHs (naphthalene, phenanthrene and pyrene)
spiked sands. The bioassay results showed that PAHs and
DDTs were toxic to microalgae in a 4-day exposure tests
but not to seed germination of ryegrass (Lolium perenne).
Phenanthrene was the most phytotoxic. Among three
investigated endpoints, fluorescence emissions by micro-
algae were less sensitive than cell density (optical density
OD650) and chlorophyll a concentration as endpoints. In
general, S. capricornutum was the most sensitive species
for PAHs (EC50 for phenanthrene = 9.4 mg kg1), while C.
meneghini for DDTs (EC50 for DDE = 20.0 mg kg1).
Comparison of the microalgal tests with US EPA standard
seed germination/root elongation test (using Lolium per-
enne) demonstrated the superior screening potential of
phytotoxic hydrophobic compounds using the proposed
bioassay. Using OD650 as the endpoint, EC10 of selected
microalgae for PAHs and DDTs were 0.4364.3 mg kg1
and 0.67117 mg kg1 respectively, which were much
lower than the EC10 of L. perenne for both PAHs (94
187 mg kg1) and DDTs (113483 mg kg1). The results
M. K. Chung
M. H. Wong
K. C. Cheung (&)
Croucher Institute for Environmental Sciences and Department
of Biology, Hong Kong Baptist University, Kowloon Tong,
Kowloon, Hong Kong SAR, PR China
e-mail: biochung@hkbu.edu.hk
R. Hu
Institute of Hydrobiology and Aquatic Ecoscience, Jinan
University, Guangzhou, P.R. China
encourage further studies involving wider types of vascular
plants and more comparison with standard phytotoxicity
tests from different authorities using contaminated soils to
verify the effectiveness of the microalgal bioassay.
Keywords DDTs
EC50
PAHs
Persistent Organic
Pollutants (POPs)
Inhibition
Toxic effects
Soil

Ecotoxicology
Introduction
Plants are particularly important components in ecosys-
tems since they are the primary food producers. Though
microalgae can be barely seen by naked eye, they form an
important component of the soil ecosystem as they
comprise up to 27% of the total microbial biomass in the
soil (McCann and Cullimore 1979). Since 1980s, phyto-
toxicity tests have been required by environmental legis-
lations and included as parts of the guidelines developed
by different authorities for environmental monitoring and
assessment (European Chemicals Bureau 1992; US
Environmental Protection Agency 1996b; Organization
for Economic Cooperation and Development 2002).
Among the test species, microalgae are used as the sur-
rogates for screening phytotoxic chemicals in aqueous
toxicity assays because they are generally more sensitive
than vascular plants (Benenati 1990; Juneau et al. 2003).
For example, sensitive species of microalgae were elim-
inated in medium (27 mg kg1 soil) and highly DDT
contaminated soils (34 mg kg1 soil), suggesting that
these organisms could be used as bioindicators of
pollution (Megharaj et al. 2000). Apart from sensitivity,
microalgae are generally easier to handle and less time
and labor intensive than testing with vascular plants.
123
394
Furthermore, algal toxicity study is a short-term test and
also a chronic sublethal test as it studies the effects of
toxicants with several generations of microalgae in a large
number. As a result, damages on cell division/reproduc-
tion could be assessed without a lengthy assay. In
terrestrial system, seed germination/root elongation and
early seedling growth tests are commonly used to assess
phytotoxicities of chemicals or contaminated substrates.
However, seed germination/root elongation test could be
insensitive to hydrophobic chemicals such as PAHs
(Mitchell et al. 1988; Sverdrup et al. 2003) while pro-
longed period of testing is required for early seedling
growth test (14 days to even months).
To overcome the inherent limitations of these tests,
the present study aimed at developing a fast (4 days) and
sensitive solid-phase screening method. Microalgae were
used as the screening organisms towards hydrophobic
persistent toxic substances (PTS) including naphthalene,
phenanthrene, pyrene, DDT, DDD and DDE spiked
sands. PAHs and DDTs were selected because of their
abundance in the environment and their notorious char-
acteristics such as high persistency and bioaccumulative
potential in food chains. Two commercially available
microalgal species, Selenastrum capricornutum and
Chlorococcum hypnosporum, along with one locally
isolated soil microalga Chlorococcum meneghini were
chosen as test species for the study. Comparison with
seed germination/root elongation test using Lolium per-
enne was also made to determine the feasibility of the
proposed microalgal solid-phase test as a screening
surrogate for higher plants.
Materials and methods
Preparation of BBM
Modified Bolds Basic Medium (BBM) (Metting 1994)
with addition of vitamins B1 and B12 was used for cul-
turing the microalgae. The medium was adjusted to pH 6.5
before sterilization in an autoclave (Tomy SS-325) at
121C for 15 min.
Preparation of chemical stocks
Naphthalene (99%), pyrene (98%), DDT (98%), DDD
(97%) and DDE (99%) were purchased from Aldich
Chemical Co. (Germany), and phenanthrene (96%) was
obtained from Sigma Chemical Co. (Germany). Stocks of
PAHs and DDTs dissolved in reagent grade acetone (Tedia,
Co., USA) were wrapped with aluminium foil and stored
at 4C to minimize photodegradation and loss from
evaporation.
123
M.K. Chung et al.
Substrate and vessel preparation
Quartz sand with 0.180.5 mm in diameter as recom-
mended by Organization for Economic Cooperation and
Development (OECD) (2003) was supplied by Fantasy
Quartz Sand Supply Co. (Hong Kong) .The sand was wa-
shed with 10% HCl acid and Decon 90 detergent (Decon
Laboratories Ltd., USA) and sterilized in an autoclave
(Tomy, SS-325). Glass Petri dishes with 9 cm in diameter
were also cleaned with the standard procedures mentioned
above.
Spiking of toxicants
Spiking procedures were followed those described in Kim
and Osako (2003). Sterilized sand was weighed to Erlen-
meyer flasks, mixed with chemical stocks to the designated
concentration (mg kg1 dry mass) and made up to a ratio of
1:1.5 with reagent grade acetone (volume of acetone:
weight of sand). The flasks were wrapped with aluminium
foil and the mouths were wrapped with Kimwipes paper to
minimize contamination. They were allowed to evaporate
at 25C until dry on an orbital shaker (Lab-Line Instrument
Inc., India) at 150 rpm. Four random spiked sand samples
were analyzed to verify the spiking efficiency using soxhlet
extraction followed by quantification with GC-MS (Kong
et al. 2005).
Mass culture of microalgae
Axenic culture of freshwater unicellular green algae
S. capricornutum and C. hypnosporum were purchased
from Carolina Biological Supply Co. (USA) on a bacteria-
free Bristol agar slant. C. meneghini was isolated from a
local lateritic red soil (near Plover Cove reservoir, Hong
Kong). One mL of the stock culture was transferred asep-
tically to a 1000 mL Erlenmeyer flask with 500 mL ster-
iled modified BBM medium. Aeration and mixing were
provided with a 0.2 lm syringe filtered (Nalgene) air at
1520 mL min1 air flow rate. The flask was incubated
under illumination with cool white fluorescent lamps at a
light intensity of 47.3 lmol m2 s1 in a temperature con-
trolled room (22 1C). The light/dark cycle was set to
16/8 h. Cultures at the middle of the exponentially growth
phase were harvested by centrifugating at 3,300 rpm
(Beckman Allegra 6R) at 20C for 5 min in sterile 50 mL
centrifuge tubes. The filtrate was decanted and the cell
residues were washed twice with fresh modified BBM
medium and then resuspended and counted using a haem-
ocytometer (Neybauer, Germany). All the cell counting
was based on the average of two separate counts of each
sample at 400 under a light microscope (Olympus,
CH30).
Comparative toxicity of hydrophobic contaminants
Experimental setup and incubation conditions for solid-
phase algal test
The experimental design for the solid-phase microalgal
toxicity test was based on Nyholm (1989) and a number of
standard guidelines (American Society for Testing and
Materials 1991; European Chemicals Bureau 1992; US
Environmental Protection Agency 1996b). Moreno-Garrido
et al. (2000) demonstrated that the initial concentration of
algal species has strong effect on the toxicity endpoints
under the concept of cellular toxic quote (amount of toxic
per cell). Therefore, extensive preliminary trials were made
and an initial concentration of 5 105 cells g1 dry sand
with incubation for 4 days were found to be optimum for
the purpose of rapid screening.
Twenty g of dry sand was added to the glass Petri dishes
with a total volume of 7 mL modified BBM medium
(100% Water Holding Capacity (WHC) (Swift and Bignell,
2002) of dry sand plus 1 mL of medium). The sand was
mixed carefully and smoothed with a sterilized metal
spoon. There were four replicates for each concentration of
toxicant. The dishes were arranged under a randomized
block design in a temperature controlled room (25 1C)
and illuminated with cool white fluorescent lamps at light
intensity of 47.3 lmol m2 s1 for 4 days. The light/dark
cycle was set to 16/8 h.
Organic solvents such as acetone and ethanol had del-
eterious effects on the organisms (ElJay 1996). Therefore
solvent controls were setup and compared with control. In
addition, controls without microalgae were prepared for
each concentration to provide reference values for signal
correction for any potential interferences during measure-
ment.
Endpoints measurements in solid-phase microalgal test
To study the effects of PAHs and DDTs on different
microalgal species, three different endpoints (fluorescence
values, algal optical density (OD650) and chlorophyll a
concentration) were measured to reflect the physiological
and biochemical responses.
Fluorescence values
At the end of the 4th day, sand in Petri dishes was rinsed
into 50 mL centrifuge tubes with 15 mL of deionized water
and mixed briefly. Supernatants with algal suspension were
transferred to another 15 mL centrifuge tube and subjected
to centrifugation at 3300 rpm (Beckman Allegra 6R) for
5 min. The clear supernatants were discarded and another
15 mL of deionized water was added to the parent 50 mL
centrifuge tubes containing sand and algal residue. The
above extraction procedures were repeated twice. Extracted
395
microalgae were concentrated to 0.5 mL in 1.5 mL ep-
pendrof tube and then pipetted to the algae cuvette (Opti-
Science, FL-AC) and dark-adapt for 15 min at 25C, which
is the time needed to reduce the fluorescence level (mini-
mum fluorescence yield, Fo) to constant. Fo, Fm (maximal
fluorescence), and Fv/Fm (variable fluorescence to maximal
fluorescence ratio) were measured using a Pulse-amplitude-
modulated (PAM) chlorophyll fluorometer (Opti-Science,
OS1-FL) with a saturating pulse of ~15,000 lE for 0.8 s.
Reduced pastoquinone pool (Fv/2) was calculated using the
formula Fv = Fm Fo.
Cell density
The absorbance value has been employed as a surrogate of
direct cell counting (Schrader et al. 1998; Hyenstrand et al.
2000). Graphical plot of algal cell density against wave-
length of 650 nm (A650) with a UV/VIS spectrophotometer
(PerkinElmer, Lambda 35) for the investigated species was
shown to be highly correlated (Fig. 1). Thus the algal
suspensions were pipetted to 1.5 mL plastic cuvettes
(Plastibran), and the cell density was measured by spec-
trophotometry. Deionized water and control extracts at
corresponding spiked concentrations were used as blank
and signal correction respectively.
Chlorophyll a concentration
Algal suspensions were transferred to eppendrofs and
centrifuged at 9,000 rpm (Eppendorf 5417C) for 5 min.
Supernatant was discarded without disturbing the algal
pellet, and washed sand was added carefully together
with reagent grade acetone (Tedia Co.). The samples
were drilled in dim light, and stored in dark at 4C
for 2 days. Extracts were measured for chlorophyll a
Algal cell density vs A 650
6000
Selenastrum capricornutum
Chlorococcum hypnosporum
5000
Cell density (10 cells/ml)
Chlorococcum meneghini
4000
4
3000
2000
1000
0
-1000
0.0 0.5 1.0 1.5 2.0 2.5
A 650
Fig. 1 Relationship between cell density ( 104 cells mL1) and
absorbance at 650 nm (A650) of three different microalgae. R2 for
S. capricornutum, C. hypnosporum and C. meneghini were at 0.996,
0.992 and 0.995 respectively
123
396
concentration according to US EPA method 446 (Eliza-
beth 1997). The final concentration was expressed as
mg kg1 of dry sand.
Experimental setup and growth conditions for seed
germination/root elongation test
Untreated seed of Lolium perenne purchased from Herbi-
seed Co., England were surface sterilized by shaking with
1% active HOCl (Chlorox) for 30 s and rinsed thoroughly
with deionized water before use. The experimental design
for seed germination/root elongation test was based on US
EPA guideline (US Environmental Protection Agency
1996a) with some modifications as described by Folsom
and Price (1991). For fast screening purpose, 5 days were
chosen for the test.
About 20 g of dry quartz sand was added to the glass
Petri dishes with a total volume of 6.5 mL deionized water
(100% WHC of dry sand plus 0.5 mL of deinoized water).
For each dish, 10 seeds of similar size with no observable
damage were pushed gently into the sand with a depth not
exceeding 2 times of the seed diameter and were subse-
quently smoothed with a metal spoon. There were four
replicates for each concentration. The dishes were covered,
wrapped with aluminium foil and arranged under a ran-
domized block design in a temperature-controlled green-
house (25 2C) for 5 days. Controls and solvent controls
were also prepared and the germination rates in both con-
trols were greater than 80%.
Endpoints measurement for seed germination/root
elongation test
Seed germination rate and root length were selected as
endpoints to reflect the growth and health status of the
seed. The definition of germination varies among different
studies (3 to 4 mm) (US Food and Drugs Administration
1987; US Environmental Protection Agency 1996a). Nev-
ertheless, seed was only regarded as germinated when
having root length greater or equal than 3 mm, as a balance
between sensitivity and fault counting. Only seed defined
as germinated was measured for root length. When
Table 1 Toxicant concentration ranges (mg kg1 dry mass) for each corresponding microalgae used in definite test
S. capricornutum
Naphthalene 1, 10, 100, 500, 700, 1000
Phenanthrene 1, 5, 10, 50, 100, 500
Pyrene 1, 5, 10, 100, 300, 500
DDT 1, 10, 50, 100, 250, 500
DDD 1, 10, 50, 100, 250, 500
DDE 1, 10, 100, 500, 700, 1000
123
M.K. Chung et al.
multiple roots were observed, only the longest one was
measured. Measurement was done with an electronic dig-
ital caliper (Aerospace Co., China) by first carefully
straighten the root.
Range finding test
Concentration ranges used in definitive tests were in
accordance with the results of the range finding tests as
presented in Table 1 for microalgal bioassay. Maximum
concentration was set to 1,000 mg kg1 of spiked toxicants
even less than 50% of inhibition to the endpoints was
found.
Calculation of effective concentrations and statistical
analyses
Effective concentration (ECx) represents the concentration
of toxicant that results in a X% reduction of endpoint rel-
ative to control at a given duration of time. EC50 and EC10
were calculated to study the concentration of each toxicant
for a 50% and 10% inhibition of corresponding endpoints.
Calculations of EC50 and EC10 were done by using Linear
Interpolation (ICp) Method (Norberg King 1993) with the
software provided by US EPA (ICp method program,
version 2.0). Probit analysis was used to calculate the EC
values for seed germination test (EPA probit analysis
program, version 1.5). Dose-response curves were plotted
using SigmaPlot graphic software (version 8.0, Systat
Software Inc.).
Results and discussion
For all tests, solvent controls were found to have no sig-
nificant difference when compared with controls
(P < 0.01). The recoveries of spiked toxicants were >90%
on average. The general trends observed in dose-response
curves from three selected microalgae were similar and
thus only the curves for S. capricornutum are presented.
EC50 and EC10 of all three species are presented in the
Table 2, 3, 4.
C. hypnosporum C. meneghini
50, 100, 300, 500, 700, 1000 50, 100, 300, 500, 700, 1000
10, 50, 100, 250, 300, 500 1, 5, 10, 50, 100, 500
50, 100, 300, 500, 700, 1000 50, 100, 300, 500, 700, 1000
50, 100, 300, 500, 700, 1000 10, 50, 100, 300, 500, 700
50, 100, 300, 500, 700, 1000 50, 100, 300, 500, 700, 1000
50, 100, 300, 500, 700, 1000 0.1, 1, 5, 10, 100, 300
Comparative toxicity of hydrophobic contaminants 397

Table 2 EC50 and EC10 values with 95% confidence interval (mg kg1) of six different toxicants for three different algal species using
fluorescence parameter Fv/Fm as the measurement endpoint after incubation for 4 days (N = 4)
S. capricornutum C. hypnosporum C. meneghini

Naphthalene EC50 >1000 >1000 >1000

EC10 41.80 (0.71556.1) 196 (89.17360.00) >1000
Phenanthrene EC50 >500 >500 >500
EC10 46.95 (2.49437.3) 9.27 (8.3515.71) 7.52 (3.8320.93)
Pyrene EC50 >500 >1000 >1000
EC10 89.00 (0.96413.8) 66.92 (49.86150) >1000
DDT EC50 >500 >1000 >700
EC10 30.70 (4.9756.95) >1000 77.5 (33.75157.22)
DDD EC50 >500 >1000 >700
EC10 6.48 (0.5257.47) >1000 35.35 (28.0149.49)
DDE EC50 >1000 >1000 >300
EC10 7.62 (1.9578.33) 47.28 (44.3254.17) 0.21 (0.071.01)

Table 3 EC50 and EC10 values with 95% confidence interval (mg kg1) of 6 different toxicants for three different algal species using OD650 as
the measurement endpoint after incubation for 4 days (N = 4)
S. capricornutum C. hypnosporum C. meneghini

Naphthalene EC50 419.27 (366.58470.47) 473.88 (460.72488.84) 529.78 (494.91556.94)

EC10 7.26 (3.3556.88) 64.23 (42.9783.48) 25.79 (20.8935.09)
Phenanthrene EC50 9.39 (7.3632.32) 147.92 (127.59253.71) 29.28 (20.6538.01)
EC10 0.43 (0.330.63) 2.98 (2.833.14) 1.42 (0.673.81)
Pyrene EC50 376.91 (331.46430.7) 349.96 (284.17400.22) 364.48 (252.75595.29)
EC10 0.63 (0.540.76) 35.62 (31.6140.86) 18.82 (14.3828.91)
DDT EC50 236.71 (215.06276.43) 172.44 (135.41196.98) 66.80 (45.9179.80)
EC10 0.79 (0.523.83) 19.03 (14.9626.24) 2.91 (2.363.97)
DDD EC50 327.22 (292.23366.40) 456.52 (444.77466.48) 178.60 (138.01256.45)
EC10 0.77 (0.621.10) 117.93 (109.82125.62) 12.98 (11.4715.23)
DDE EC50 521.90 (379.15575.74) 256.83 (246.87246.06) 19.99 (9.9834.75)
EC10 0.67 (0.491.47) 48.13 (41.6654.72) 1.14 (0.801.73)

Table 4 EC50 and EC10 values with 95% confidence interval (mg kg1) of 6 different toxicants for three different algal species using chlorophyll
a content as the measurement endpoint after incubation for 4 days (N = 4)
S. capricornutum C. hypnosporum C. meneghini

Naphthalene EC50 724.77 (672.78760.69) 422.96 (403.39439.07) 464.22 (454.43474.62)

EC10 70.00 (17.0398.11) 84.00 (84.4688.74) 141.63 (112.55165.32)
Phenanthrene EC50 6.54 (4.918.02) 282.97 (85.59290.01) 8.90 (8.579.27)
EC10 0.59 (0.381.44) 3.07 (2.933.81) 2.35 (2.142.53)
Pyrene EC50 >500 626.08 (178.91711.67) 944.52 (900.86999.77)
EC10 0.82 (0.512.76) 26.2 (27.9130.62) 34.87 (28.3645.60)
DDT EC50 202.56 (127.79379.45) 659.52 (553.57823.08) 54.34 (48.5160.79)
EC10 3.01 (0.825.87) 3.86 (26.8539.21) 2.63 (2.562.70)
DDD EC50 246.01 (200.69492.00) 511.11 (440.65607.79) 149.71 (109.84179.02)
EC10 5.32 (0.7922.57) 56.90 (56.0257.76) 14.27 (13.3815.32)
DDE EC50 >1000 704.06 (688.89724.19) 8.83 (8.429.29)
EC10 26.68 (0.84523.03) 74.82 (71.6778.24) 2.08 (1.922.23)

123
398 M.K. Chung et al.

140 120
130 Naphthalene Phenanthrene
110
120
100
110
100 90
90
80
80
70
70
60 60
0 200 400 600 800 1000 1200 0 100 200 300 400 500 600
Fluorescence parameters (% of control)

130 140
Pyrene
120 120 DDT

110
100
100
80
90

80 60

70 40
0 100 200 300 400 500 600 0 100 200 300 400 500 600

160 160
DDD DDE
140 140

120 120

100 100

80 80

60 60

40 40
0 100 200 300 400 500 600 0 200 400 600 800 1000 1200

Toxicant concentrations (mg/kg)

Fig. 2 Dose response relationship showing the effects of six different toxicants (naphthalene, phenanthrene, pyrene, DDT, DDD and DDE) on
fluorescence parameters [Fo (), Fm (.), Fv/Fm (r) and Fv/2 ( d)] of S. capricornutum after incubation for 4 days

Influences on photosynthetic potential trends in Fig. 2. S. capricornutum and C. meneghini were

more sensitive to DDTs in a broad sense (EC10: 7.62
Fo is an re-emission from the excited chlorophylls in PS II 30.7 mg kg1 and 0.2177.5 mg kg1 respectively) while C.
antenna (Maxwell and Johnson 2000) and a general hypnosporum to PAHs (EC10: 9.27196 mg kg1) (Table 2).
increasing trend was observed for Fo as a function of toxicant Treatments with PAHs and DDTs had negative im-
concentrations (Fig. 2). There were sharp increases initially pacts on Fm, Fv/Fm and Fv with gradual reduction trends
and then plateaus were observed for Fo. The above results (Fig. 2). Fv/Fm represents the quantum efficiency of open
clearly demonstrated that the efficiency of energy transfer PS II centre, which is a proxy measurement of photo-
from light harvesting protein to PS II were reduced under the synthesis efficiency and describes the potential yield of
influence of PAHs and DDTs, but such stress was rather the photochemical reaction (Maxwell and Johnson 2000).
constant along the concentrations, which is also true for Fv/ It has been observed that DDT interacted with photo-
Fm. It has been reported that the inhibition of electron synthetic electron transport chain in vascular plants at 2
transport system by DDT can be attributed to their interac- sitesin the oxidizing side of PS II and in the interme-
tion to the proteinaceous component of the thylakoid mem- diate of electron transport chain (Akbar and Rogers
brane (Moreland and Novitzky 1984). The binding of DDT 1985), and it could also be the cause of reduction in
was estimated to be 900 molecules per photosynthetic unit fluorescence parameters in microalgae. An increase on Fo
(490 chlorophyll molecules). Thus the slow changes of and a decline on Fv/Fm were commonly used to indicate
fluorescent parameters observed after 200 mg kg1 of spiked the incident of inhibitory effects on vascular plants in
DDT could be caused by the saturation of binding sites on the response to environmental stresses (Groom and Baker
thylakoid membrane, and higher concentration of DDT 1992; Epron et al. 1995) which could be applied on
could only further increase the turn over rate of the saturated microalgae. The results presented were in line with other
sites. The above mechanism is also suspected to be appli- similar studies, which demonstrated the inhibition of
cable to the rest of the toxicants as they exhibited similar photosynthetic system by DDTs in microalgae (Powers

123
Comparative toxicity of hydrophobic contaminants 399

et al. 1979; Samson and Popovic 1988). The use of For S. capricornutum, the inhibitory trends for cell density
fluorescence parameters to detect PAHs toxicities has were similar to those of chlorophyll a concentration except
also been reported in vascular plant (Gensemer et al. for pyrene and DDE. This general finding was also ob-
1999). served for C. hypnosporum and C. meneghini. The trends
In general, the sensitivity of these parameters was in the for pyrene and DDE were characterized by a strong de-
hierarchy of Fm > Fv/2 > Fv/Fm > Fo. Of the four fluo- crease of cell density but not the chlorophyll a content to
rescence parameters, Fv/Fm was used for comparison only an increase of toxicant concentrations. It is assumed that
owning to its relationship with photosynthetic potential. chlorophyll synthesis is a major objective in photoauto-
The EC50 and EC10 values of Fv/Fm are presented in trophic organisms in absence of cell division (Hagen et al.
Table 2. The order of chemical toxicities for S. capricor- 2001) and a decrease of chlorophyll content per unit of
nutum, C. hypnosporum and C. meneghini was: plant material does not necessarily mean inhibition of
growth (Verdisson et al. 2001). DiEguez-Rojo and Gon-
DDD > DDE > DDT > naphthalene > phenan-
zAlez (2003) observed that chlorophyll content increased
threne > pyrene
per cell while the cell density decreased for the unicellular
Phenanthrene > DDE > pyrene > naphthalene > DDT,
green alga Tetraselmis suecica under the effects of BOA
DDD
(2-benzoxazolinone) when compared with control. Souku-
DDE > phenanthrene > DDD > DDT > naphthalene,
pova et al. (1999) also demonstrated that microalgae could
pyrene
acclimate the sub-lethal concentration of toxicant such as
respectively. DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). Micro-
algae can alleviate the inhibitory effects on photosynthetic
Effects on cell growth and chlorophyll a concentration system by increasing the amount of photosynthetic antenna
that drives more excitation to the remaining functional
The inhibitory effects of PAHs and DDTs on cell growth reaction centers of PS II. Though in our study, chlorophyll
and chlorophyll a concentrations are illustrated in Fig. 3. a per algal cell was not measured, the rather constant total

Fig. 3 Dose response 120 120

Naphthalene Phenanthrene
relationship showing the effects 100
of 6 different toxicants 100

(naphthalene, phenanthrene, 80
80
pyrene, DDT, DDD and DDE) 60
on cell density (OD650) (m) and 60
chlorophyll a content (d) of S. 40
Cell Density (OD650) and chlorophyll a content (mg/kg) (% of control)

capricornutum after incubation 20

40
for 4 days
0 20
0 200 400 600 800 1000 1200 0 100 200 300 400 500 600

110 110
100 Pyrene 100 DDT

90 90
80 80
70 70
60 60
50 50
40 40
30 30
0 100 200 300 400 500 600 0 100 200 300 400 500 600

110 120
100 DDD DDE
100
90
80
80
70 60
60
40
50
20
40
30 0
0 100 200 300 400 500 600 0 200 400 600 800 1000 1200

Toxicant concentrations (mg/kg)

123
400
chlorophyll a concentration recorded (chlorophyll a con-
tent per cell cell density) for pyrene and DDE (Fig. 3)
revealed the algal acclimation occurred for these two
compounds. In general, inhibition of cell density and
chlorophyll a content ranged from 70 to 90% within the
tested ranges (Fig. 3). For endpoint OD650 (Table 3), the
toxicity of phenanthrene was more prominent than the rest
of the toxicants to S. capricornutum (EC50: 9.39 mg kg1)
and C. hypnosporum (EC50: 147.92 mg kg1), but this did
not hold true for C. meneghini, as it showed lower EC50
towards DDE (20 mg kg1). Similar results can also be
found in Table 4.
The assessment of phytotoxic nature of DDTs in terms
of cell density was documented (Megharaj et al. 2000).
Zbigniew (1987) found that treatment of various con-
centrations of PAHs to Scenedesmus quadricauda de-
creased the cell density and chlorophyll a content
significantly. Gaur (1988) demonstrated that naphthalene
caused a reduction in cell density of S. capricornutum.
Inhibitory effects of pesticides (atrazine and nicosulfuron,
lindane, pentachlorophenol, isoproturon and methyl para-
thion) to algae were also revealed (Seguin et al. 2001;
Mostafa and Helling 2002). In addition, metabolite of
DDT (DDE) also inhibited algal growth (Powers et al.
1979). The results presented were in agreement with these
studies.
EC50 and EC10 values calculated as a reduction on cell
density and chlorophyll a content are listed in Table 3 and
4. In accordance with EC50, the toxicity orders of toxic
chemicals for S. capricornutum, C. hypnosporum and
C. meneghini were:
Cell density (OD650):
Phenanthrene > DDT > DDD > pyrene > naphtha-
lene > DDE
Phenanthrene > DDT > DDE > pyrene > DDD > naph-
thalene
DDE > phenanthrene > DDT > DDD > pyrene > naph-
thalene
respectively.
Chlorophyll a level:
Phenanthrene > DDT > DDD > naphthalene > DDE,
Pyrene (Pyrene was out of maximum tested concentra-
tion range but was smaller than the EC50 for DDE )
Phenanthrene > naphthalene > DDD > py-
rene > DDT > DDE
DDE phenanthrene > DDT > DDD > naphtha-
lene > pyrene
respectively. The responses of C. meneghini to DDE
differed significantly when compared with S. capricornu-
tum and C. hypnosporum, which was very sensitive to DDE
contamination.
123
M.K. Chung et al.
Sensitivity of solid-phase microalgae test with different
endpoints
Inhibitory effects of PAHs and DDTs on cell density and
chlorophyll a concentration were more prominent than
fluorescence parameters. In general, EC50 values of fluo-
rescence were greater than the rest of the parameters for all
algal species (Tables 24). EC50 values of cell density and
chlorophyll a content ranged from 9.59 to 530 mg kg1 and
6.54 to >1000 mg kg1 respectively (Table 3 and 4). Al-
though the responses of microalgae varied among param-
eters and species, cell density (OD650) and chlorophyll a
concentration were the more sensitive parameters (EC50
c.a. 500 mg kg1). It might be due to the different sus-
ceptibility of biochemical processes in microalgae, where
the sites of actions were possibly not chiefly on the pho-
tosynthetic system but on other biochemical reactions
(Djomo et al. 2004).
Preliminary study of the screening potential by solid-
phase microalgal test
The first step of developing a screening method by mic-
roalgae is to simulate certain mechanism of actions of
toxicants in terrestrial system without severe interferences
from unwanted substances which may mask the responses
of microalgae. The microalgal bioassay presented in this
paper is aimed to mimic the situations in soils by using
quartz sand as the substrate and has considered certain
unique modes of actions in terrestrial system. It is
hypothesized that the toxicants coated on the sand exerted
their actions to microalgae via both indirect and direct
pathways. For the indirect pathway, the coated toxicants
are desorbed from the sand to the ambient solution, and
become freely available to the testing species though their
solubility are low (Log Kow > 3). They can be absorbed or
adsorbed by microalgae and subsequently enter the cell by
diffusion or other means. For the direct pathway, the
microalgae are in direct contact with the coated toxicants
and mass transfer of toxicants to cell surface occurs. To
assess the screening potential of phytotoxic hydrophobic
compounds by the 4-day solid-phase microalgal assay, the
results from the microalgal assays (Table 24) were com-
pared with those obtained in 5 day seed germination/root
elongation test (Table 5). Most of the EC50 values were
greater than 1,000 mg kg1 for L. perenne for different
endpoints (seed germination rate and root length). Con-
cerning the EC10, it ranged from 94 to 483 mg kg1 of
toxicants for using root length as an endpoint, which was
still far less sensitive than the EC10 values calculated
from microalgal bioassays. The data suggested that a ger-
mination study alone of the grass would not be enough to
screen phytotoxic chemicals, which is also in line with the
Comparative toxicity of hydrophobic contaminants 401

Table 5 EC50 and EC10 values with 95% confidence interval commonly used in phytoremediation, and with different
(mg kg1) of six different toxicants for L. perenne using germination types of contaminated soils, should be conducted in the
rate and root length as the measurement endpoint after growing for
future in order to verify the screening potential of the
5 days (N = 4)
bioassay presented here.
Seed germination Root length
Acknowledgments The authors acknowledge the financial support
Naphthalene EC50 >1000 >1000 from the Strategic Research Fund from the Science Faculty, HKBU
EC10 >1000 154.93a and technical assistance from Mr. W.C. Li. This study was supported
Phenanthrene EC50 >1000 >1000 by the Area of Excellence (AoE) Scheme under the University
EC10 >1000 94.68a Grants Committee of the Hong Kong Special Administrative Region
(CITYU/AoE/0304/02).
Pyrene EC50 >1000 >1000
EC10 >1000 187.69a
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