Synergistic toxic effects of zinc pyrithione and copper to three marine species: Implications on setting appropriate water quality criteria.

Abstract

Zinc pyrithione (ZnPT) is widely applied in conjunction with copper (Cu) in antifouling paints as a substitute for tributyltin. The combined effects of ZnPT and Cu on marine organisms, however, have not been fully investigated. This study examined the toxicities of ZnPT alone and in combination with Cu to the diatom Thalassiosira pseudonana, polychaete larvae Hydroides elegans and amphipod Elasmopus rapax. Importantly, ZnPT and Cu resulted in a strong synergistic effect with isobologram interaction parameter lambda>1 for all test species. The combined toxicity of ZnPT and Cu was successfully modelled using the non-parametric response surface and its contour. Such synergistic effects may be partly due to the formation of copper pyrithione. It is, therefore, inadequate to assess the ecological risk of ZnPT to marine organisms solely based on the toxicity data generated from the biocide alone. To better protect precious marine resources, it is advocated to develop appropriate water quality criteria for ZnPT with the consideration of its compelling synergistic effects with Cu at environmentally realistic concentrations.

Full text

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Synergistic toxic effects of zinc pyrithione and copper to three marine species:
Implications on setting appropriate water quality criteria
Vivien W.W. Baoa,*, Kenneth M.Y. Leunga,*, Kevin W.H. Kwoka, Amy Q. Zhanga, Gilbert C.S. Luib
aThe Swire Institute of Marine Science, Division of Ecology and Biodiversity, School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China
bDepartment of Statistics and Actuarial Science, The University of Hong Kong, Pokfulam, Hong Kong, China
a r t i c l e i n f o
Keywords:
Antifouling booster biocide
Combined toxicity
Diatom
Tube worm
Amphipod
a b s t r a c t
Zinc pyrithione (ZnPT) is widely applied in conjunction with copper (Cu) in antifouling paints as a sub-
stitute for tributyltin. The combined effects of ZnPT and Cu on marine organisms, however, have not been
fully investigated. This study examined the toxicities of ZnPT alone and in combination with Cu to the
diatom Thalassiosira pseudonana, polychaete larvae Hydroides elegans and amphipod Elasmopus rapax.
Importantly, ZnPT and Cu resulted in a strong synergistic effect with isobologram interaction parameter
k > 1 for all test species. The combined toxicity of ZnPT and Cu was successfully modelled using the non-
parametric response surface and its contour. Such synergistic effects may be partly due to the formation
of copper pyrithione. It is, therefore, inadequate to assess the ecological risk of ZnPT to marine organisms
solely based on the toxicity data generated from the biocide alone. To better protect precious marine
resources, it is advocated to develop appropriate water quality criteria for ZnPT with the consideration
of its compelling synergistic effects with Cu at environmentally realistic concentrations.
? 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Since the partial ban on the application of organotin biocides,
especially tributyltin (TBT) in antifouling paints for small marine
vessels (<25 m in length; Readman et al., 2002), a new generation
of surrogate antifouling biocides such as zinc pyrithione (ZnPT),
copper pyrithione (CuPT), Irgarol 1051, diuron and Sea-nine 211
have been increasingly used (Yebra et al., 2004). These surrogate
biocides are often applied in conjunction with copper (Cu) com-
pounds such as cuprous oxide, Cu thyocyanate or metallic Cu to
control Cu-resistant fouling organisms (Voulvoulis et al., 2002). To-
day, Cu contamination in coastal marine environments is univer-
sal; and one main source of Cu to the marine environment is Cu-
containing antifouling coatings on ship hulls (Srinivasan and
Swain, 2007). As a consequence of the restricted use of TBT, the
use of Cu-based antifouling biocides has been dramatically in-
creased leading to elevated levels of Cu in coastal waters and sed-
iments (Schiff et al., 2007). This problem is evident in many coastal
areas, particularly busy ports such as those in the United Kingdom
where the waterborne Cu level could be as high as 20 lg/L (Matthi-
essen et al., 1999) and marinas of the San Diego region where the
dissolved Cu concentrations in the surface water samples reached
an average of 8.5 lg/L (Schiff et al., 2007).
Different biocides often coexist with Cu in the marine environ-
ment, especially in coastal areas with heavy Cu contamination.
Understanding the interactions between different biocides (i.e.
additive, synergistic or antagonistic effect) is necessary in ecotoxi-
cology and ecological risk assessment (Hertzberg and Macdonell,
2002). Therefore, consideration solely based on the toxicity of the
surrogate biocide alone is insufficient to determine its ultimate
environmental impacts. The combined effect of Cu and the surro-
gate biocides should be of great concern, especially in the coastal
areas with high shipping activities (i.e. higher chances for being
contaminated with both Cu and other biocides).
Zinc pyrithione (zinc complex of 2-mercaptopyridine-1-oxide;
ZnPT), one of the most popular surrogate antifouling biocides,
has long been widely used as algaecides, bactericides and fungi-
cides and is well known for its application in antidandruff sham-
poos (Yebra et al., 2004). It was introduced to the market as a
substitute of organotin biocides by Arch Chemicals (Norwalk, CT,
USA) in 1991, and is currently viewed as the top prospect for
replacing TBT in antifouling paints (Doose et al., 2004). ZnPT-based
antifouling products are widely applied on yachts and large ocean-
going vessels in Europe, Japan and South Korea, and ZnPT is also
one of the most commonly used products in the United Kingdom
on small boats (<25 m in length; Thomas, 1999).
ZnPT was found to be highly toxic to aquatic plants and animals
(Turley et al., 2000), but it was assumed to be environmentally
0025-326X/$ - see front matter ? 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.marpolbul.2008.03.041
* Corresponding authors. Tel.: +852 2809 2179; fax: +852 2809 2197 (V.W.W.
Bao).
E-mail addresses: weiwei.bao@gmail.com (V.W.W. Bao), kmyleung@hkucc.h-
ku.hk (K.M.Y. Leung).
Marine Pollution Bulletin 57 (2008) 616–623
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Page 2

neutral because it could easily photo-degrade to less toxic
compounds (Turley et al., 2000, 2005). However, Bellas (2005)
demonstrated that the toxicity of ZnPT only decreased but did
not disappear after exposure to direct sunlight. ZnPT is also sug-
gested to persist in the marine environment where the influence
of the light is limited such as waters and sediments shaded under
parking vessels in marinas and harbours (Maraldo and Dahllöf,
2004), and it may accumulate in the sediment as manganese pyri-
thione and CuPT (Galvin et al., 1998).
Although ZnPT is largely used with Cu in antifouling paintings
worldwide, the combined effects of ZnPT and Cu to marine organ-
isms are still largely unknown, except those studies on the biolu-
minescent bacteria Vibrio fischeri (Zhou et al., 2006), the sea
bream Pagrus major and the toy shrimp Heptacarpus futilirostris
(Mochida et al., 2006), as well as the diatom Chaetoceros gracilis
(Koutsaftis and Aoyama, 2006). There is a paucity of data on the
toxicity of ZnPT alone or in combination with Cu to marine species.
The objective of this study was to investigate the acute toxici-
ties of ZnPT alone and in combination with Cu (CuSO4) to three
marine species including the diatom Thalassiosira pseudonana,
polychaete tubeworm Hydroides elegans and amphipod Elasmopus
rapax. The results are essential for evaluating the combined toxic-
ity of these two biocides to marine organisms. We also applied and
compared two different approaches, namely the isobologram and
the non-parametric response surface methods (Gessner, 1995;
Greco et al., 1995), for modelling the binary mixture toxicity of
ZnPT and Cu.
2. Materials and methods
Standard toxicity tests of ZnPT and Cu alone were first con-
ducted for each species, and the median lethal concentrations
(LC50, for the polychaete H. elegans and the amphipod E. rapax)
or median effect concentrations (EC50, for the diatom T. pseudo-
nana) of ZnPT and Cu alone were determined based on the test re-
sults. Suitable ZnPT and Cu concentrations were chosen according
to their LC50 or EC50 values for the binary mixture toxicity tests of
the two biocides. For better comparison of the toxicities between
the single biocide and binary mixture, the toxicities of ZnPT or
Cu alone to the three test organisms were assessed again at the
same time with the binary mixture toxicity tests. All concentra-
tions were nominal. The concentrations selected for the toxicity
tests of ZnPT and Cu alone, and the binary toxicity of ZnPT and
Cu, as well as the number of replicates for each of the treatments
are shown in Table 1. Filtered artificial seawater (FAS; sea salt: Tro-
pic Marine, Germany; salinity 33 ± 0.5‰, pH 8.1–8.4, filtered
through 0.45 lm membrane filter) was used for all the toxicity
tests. Tests were carried out at 25 ± 1 ?C while the test solutions
were kept at salinity 33 ± 0.5‰ and pH 8.1–8.4 throughout the test.
2.1. Chemical preparation
A stock solution of ZnPT (10 g/L) was made by dissolving ZnPT
(approx 95%; Sigma, USA) in dimethyl sulfoxide (DMSO; ACS re-
agent, P99.9%; Sigma, USA). A stock solution of Cu (1 g Cu/L)
was prepared by dissolving copper(II) sulphate pentahydrate (Cu-
SO4? 5H2O, formula weight 249.7; purity P99.5%; BDH Chemicals
Ltd. Pode, England) in distilled water. The stock solution was fur-
ther diluted in FAS in volumetric flasks to obtain working solutions
at designated nominal concentrations before dosing.
2.2. Test organisms
A pure T. pseudonana culture was obtained from the Depart-
ment of Biology and Chemistry, City University of Hong Kong, Hong
Kong and kept at controlled laboratory conditions with 16 h:8 h
light:dark photoperiod in autoclaved f/2-Si medium (Guillard’s
medium for diatoms, Guillard and Ryther, 1962). Adults of H. ele-
gans were collected from a fish farm at Sam Pui Chau, Sai Kung,
Hong Kong and acclimated in an aquarium tank with mild water
flow and aeration. They were fed with concentrated phytoplankton
(Phytoplex, Kent Marine, USA) and diatom Skeletonema costatum
on a daily basis. Juveniles of E. rapax were collected from the
aquarium of the Swire Institute of Marine Science, Cape d’Aguilar,
Hong Kong and acclimated in the laboratory for 24 h before the
acute toxicity test.
2.3. Algal growth inhibition test
Cell concentration of a fresh T. pseudonana culture at exponen-
tial growth phase (<1 week old) was determined using a haemocy-
tometer(NeubauerImproved,
According to the result, an appropriate amount of diatom culture
was added to each 10 mL autoclaved test vial (with autoclaved
plastic lids) with 5 mL autoclaved f/2-Si median to obtain an initial
algal concentration of 104cells/mL. Different levels of ZnPT or Cu
(or both) were then dosed into the vials, which were then ran-
domly positioned on a titer plate shaker (Lab-line, Melrose Park,
USA) at 40 rpm under 16 h:8 h light:dark photoperiod (light inten-
sity: ?920 lux; measured by Lux/Fc light meter, Tenmars, China)
for 96 h. Then 200 ll solutions from each test vial were pipetted
into a 96-well microplate (Nunc, Denmark) and fixed with 66.7 ll
HCl (1 M) for subsequent counting using the haemocytometer.
PrecicolorHGB,Germany).
2.4. Acute toxicity test for polychaete larvae
Trochophore larvae of H. elegans were used to assess toxicity of
ZnPT and Cu independently and combined, using 48 h static acute
toxicitytestswhichwereconductedin25compartmentsquarepetri
dish (Sterilin, UK; each compartment had a volume of ?6.5 mL).
Table 1
The nominal concentrations selected for the toxicity tests of ZnPT or Cu alone and the
binary toxicity tests of these two biocides, as well as the number (No.) of replicates for
each of the treatments, to Thalassiosira pseudonana, Hydroides elegans and Elasmopus
rapax
Test speciesZnPT (lg/L)Cu (lg/L)No. of replicates
T. pseudonana
0 (C)
0 (SC, 0.8 ppm)
2, 4, 8
0
0
0
0
10, 50, 200, 500, 800
1000
1300, 1800
10
50
200
3
3
3
3
5
2
3
3
3
2, 4, 8
2, 4, 8
2, 4, 8
H. elegans
0 (C)
0 (SC, 0.6 ppm)
2, 3, 4, 5, 6
0
1.3
2.6
0.2, 0.4, 1, 2, 3
0.2, 0.4, 1, 2, 3
0
0
0
60, 90, 125, 150,180
12.5, 62.5, 125
12.5, 62.5, 125
42
84
3
3
3
3
5
5
5
5
E. rapax
0 (C)
0 (SC, 10 ppm)
5, 10, 20, 50, 100
0
0
0
0
10, 20, 50, 200
100
10
20
3
5
3
3
6
3
3
5, 10, 15, 20
5, 10, 20
C, seawater control; SC, solvent control, with carrier solvent DMSO at ppm (v/v)
level.
V.W.W. Bao et al./Marine Pollution Bulletin 57 (2008) 616–623
617

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EstablishedproceduresbyQiuandQian(1997)werefollowedtoob-
tain the trochophore larvae. Each replicate consisted of 20 larvae.
Mortality of larvae was determined at the end of the 48 h test.
2.5. Acute toxicity test for the amphipod juveniles
To get a general idea about the size of the amphipod used for
the experiment, 30 acclimated juvenile E. rapax were randomly se-
lected for a linear measurement from the proximal end of the first
thoracic segment to the distal end of the fourth thoracic segment
using a stereo microscope fitted with an ocular reticle (Hacker
and Steneck, 1990). The measured length ranged from 0.36 to
0.85 mm with an average of 0.54 mm (95% C.I.: 0.50–0.58 mm).
Each replicate consisted of 10 individuals in 100 mL FAS. Test solu-
tions were renewed once at 48 h. Mortality was monitored every
24 h by observing reaction of amphipods to mild agitation with a
glass pipette under a stereo microscope.
2.6. Data analyses
LC50 (or EC50) values of ZnPT and Cu alone to each of the three
species were determined by the non-linear regression of the sig-
moidal dose response function (four-parameter logistic regression,
GraphPad Prism version 5.00, GraphPad Software, CA). The results
of combined toxicity tests were firstly analyzed using the Isobolo-
gram method (Prakash et al., 1996). ZnPT and Cu concentrations
that caused the combined toxicity effect equalled to 50% of the
control (e.g. 50% survivorship = 50% mortality for H. elegans and
E. rapax; 50% growth of the control for T. pseudnonana) were fitted
with the following regression function:
TUCu
i
¼ ½1 ? ðTUZnPT
Although the specification above may not look familiar, without
consideration of error term viabove will yield the following isobole:
i
Þ1=k?kþ vi:
ðTUCu
where TUCu
i.e., TUCu
EC50ZnPT), cCu
respectively, and LC50’s (or EC50’s) represent the LC50 (or EC50)
of corresponding substances. The similarity parameter k in the
regression function above was estimated by the non-linear least
squares approach using Matlab (The MathWorks Inc., USA). The k
of the isobole indicates the toxicity interaction of the two sub-
stances. When k = 1, the interaction is simple additive; when
k > 1, the interaction is synergistic, vice versa.
Secondly, the non-parametric response surface and its contour
were constructed using the average data of each treatment, and
computed using the PROC G3Grid with (for H. elegans and E. rapax)
and without spline (for T. pseudonana, as there was no observable
difference between the surfaces plotted with and without spline)
and PROC GCONTOUR procedures in SAS 9.1.3, SAS Institute Inc.,
Cary, NC. For the response surface with spline, the experimental
observations were fitted with a bivariate fifth-degree polynomial
spline while the smoothness of spline was maintained by a polyno-
mial up to order three, i.e. the function,
iÞ1=kþ ðTUZnPT
i
Þ1=k¼ 1;
i
¼ cCu
i
and TUZnPT
i=LC50Cu(or EC50Cu) and TUZnPT
and cZnPT
i
are the concentrations of Cu and ZnPT
i
are toxic units for Cu and ZnPT respectively,
ii
¼ cZnPT
i
=LC50ZnPT(or
pi¼ UðxCu
i;xZnPT
i
Þ ¼
X
5
j¼0
X
5?j
k¼0
qjkðxCu
iÞjðxZnPT
i
Þk
is estimated by the least squares approach subject to the constraints
of smoothness, where xCu
i
and xZnPT
i
ZnPT, respectively, piis the control rates for T. pseudonana or mor-
tality rates for H. elegans and E. rapax and qjk’s are unknown coeffi-
cients being estimated. In the cases where smoothing spline was
used, the penalized least squares approach was used for estimation
are concentrations of Cu and
instead. The details of spline fitting for the response surface are re-
ferred to Akima (1978) and Wahba (1979). The horizontal lines con-
necting the edges of the response surfaces at 20%, 40%, 50%, 60% and
80% of control (or mortality) levels were presented in contour plots.
A straight diagonal NW-SE isobol in a contour plot would be consis-
tent with the Loewe additivity (same as the additivity line in iso-
obologram), while a downward bowed isobol represents the
Loewe synergism, vice versa (Sühnel, 1990; Greco et al., 1995).
2.7. Confirmation for the transchelation of ZnPT and Cu
InordertoconfirmwhetherthetranschelationbetweenZnPTand
Cu occurred when they encountered each other in seawater, ZnPT
and Cu (as Cu2+in CuSO4? 5H2O) with different ratios of molar con-
centrations([ZnPT]:[Cu] = 0:0(seawatercontrol),1:0,0:1,1:0.5,1:1,
1:2 and 1:4, where 1 stands for a molar concentration of 315 nM;
n = 1 for the control, and n = 3 for the other treatments) were mixed
inFAStohave afinalvolumeof100 mLina darkcondition,andthen
extracted with 50 mL dichloromethane (DCM). After a clear separa-
tion,boththe waterphaseand theorganicphasewere collectedand
stored under 4 ?C and ?20 ?C, respectively until they were analyzed
forCuconcentrationsusinganatomic-absorptionspectroscopywith
afurnacesystem(AAnalyist800AAS,PerkinelmerInc.).AdilutedCu
stock used for this test with a nominal concentration of 50 lg Cu/L
wasalsoanalyzedatthesametimetochecktheactualstockconcen-
tration. The underlying reasoning was that Cu of inorganic form
(Cu2+) would stay in the water phase, while Cu of organic form (pre-
sumablyCuPT)fromthetranschelationofCuandZnPTwouldstayin
the organic phase after reaching equilibrium.
3. Results
3.1. Toxicities of ZnPT and Cu alone
ZnPT alone was found to be much more toxic than Cu alone to T.
pseudonana and H. elegans larvae (a significant difference was de-
fined as non-overlapping of 95% C.I.), while the 96 h-EC50 and
48 h-LC50 values of ZnPT to these two species were both at ppb le-
vel (Table 2, Figs. 1 and 2 when [Cu] or [ZnPT] = 0 lg/L). T. pseudo-
nana was quite tolerant to Cu, with a 96 h-EC50 value as high as
970 lg Cu/L (Table 2). Interestingly, Cu appeared to stimulate the
growth of T. pseudonana at lower concentrations (50–200 lg Cu/
L) and the highest growth rate was found at 200 lg Cu/L
(Fig. 1A). The 72 h-LC50 values of ZnPT and Cu were not signifi-
cantly different for E. rapax, but its 96 h-LC50 value of ZnPT was
obviously lower than its 96 h-LC50 value of Cu indicating that ZnPT
was more toxic to the amphipod than Cu (Table 2).
3.2. Combined toxicities of ZnPT and Cu
In general, ZnPT and Cu exhibited synergistic effects to all test
species (Figs. 1–3). For example, an average 96 h growth rate of
T. pseudonana was about 47% of the control when exposed to
2 lg/L ZnPT alone, but a binary mixture of 2 lg ZnPT/L and 10 lg
Table 2
LC50 or EC50 values (their 95% confidence intervals) of ZnPT and Cu alone to
Thalassiosira pseudonana, Hydroides elegans and Elasmopus rapax
Species End pointZnPT (95% C.I.), lg/L Cu (95% C.I.), lg/L
T. pseudonana
96 h-EC50 1.9 (1.6–2.3) 970 (870–1100)
H. elegans
48 h-LC504.4 (3.9–4.9) 120 (120–130)
E. rapax
24 h-LC50
48 h-LC50
72 h-LC50
96 h-LC50
>100
>100
70 (34–150)
29 (19–46)
180 (140–240)
110 (92–130)
84 (74–95)
78 (68–90)
618
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Cu/L dramatically decreased its average growth rate to 11% of the
control (Fig. 1B); and the higher the Cu level, the more obvious
the synergistic toxicity effect of ZnPT to T. pseudonana. Similar pat-
terns could also be observed for the other two test species (Figs. 2
and 3). For H. elegans, addition of 42 and 84 lg/L Cu considerably
reduced the 48 h-LC50 value of ZnPT from 4.4 (95% C.I.: 3.9–4.9;
[Cu] = 0) lg/L to 2.5 (95% C.I.: 2.3–2.7) lg/L and 0.5 (95% C.I.:
0.3–0.7) lg/L, respectively (Fig. 2A; values are also shown in
Fig. 4B as TU). Likewise, the binary mixtures of ZnPT and Cu
showed a very strong synergistic effect on E. rapax across all expo-
sure durations (Fig. 3). At 96 h of exposure, the average mortality of
E. rapax exposed to a mixture of 10 lg/L ZnPT and 10 lg/L Cu was
50%, which was almost 12 times of the mortality of those exposed
to 10 lg/L ZnPT alone (4% only). For E. rapax, addition of 10 and
20 lg/L Cu considerably reduced the 96 h-LC50 value of ZnPT from
29 (95% C.I.: 19–46; [Cu] = 0) lg/L to 11 (95% C.I.: 9.6–12) lg/L and
6.4 (95% C.I.: 5.9–7.0) lg/L, respectively (Fig. 3; values are also
shown in Fig. 4C as TU).
Such synergistic effects were further confirmed with the isobo-
lograms and their corresponding interaction parameter k of 2.3, 1.3
and 2.2 for T. pseudonana, H. elegans and E. rapax, respectively (i.e.
all k > 1; Fig. 4). Remarkably, all data points on the isobolograms
were distinctively below the Loewe additivity line, indicating a
strong synergistic effect (Fig. 4).
The non-parametric response surface was a powerful method to
model the binary mixture toxicity and could be applied to the re-
sults of all test species (Fig. 5). The isobols in the contour figures
for T. pseudonana (Fig. 5B) and H. elegans (Fig. 5D) were mostly
bowed downward, indicating a moderate Loewe synergism of ZnPT
and Cu. The isobols in the contour figure for T. pseudonana bowed
slightly to the right side at Cu levels from 50 to 200 lg/L, which
was due to the stimulation of algal growth at these low concentra-
tions (Fig. 5B). A stronger Loewe synergism of the mixture was ob-
served in E. rapax as the isobols in the contour figure were all
obviously curved downward (Fig. 5F).
3.3. Confirmation for the transchelation of ZnPT and Cu
The results clearly showed that when Cu was not in excess (the
ratio of molar concentrations for [ZnPT]:[Cu] P 1), all Cu added
Fig. 1. Growth of Thalassiosira pseudonana after 96 h exposure to (A) Cu alone at
differentconcentrationsand (B)combinations
concentrations.
of differentZnPT andCu
Fig. 2. Mortality of Hydroides elegans larvae after 48 h exposure to (A) ZnPT at
different Cu concentrations and (B) Cu at different ZnPT concentrations.
V.W.W. Bao et al./Marine Pollution Bulletin 57 (2008) 616–623
619

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was transchelated with ZnPT to form Cu-complexed organic com-
pounds (presumably CuPT), so that all Cu was only found in the or-
ganic phase (Table 3). When the ratio of molar concentrations for
[ZnPT]:[Cu] was <1, Cu was in excess, and the non-transchelated
portion of Cu stayed in the water phase (Table 3). The diluted Cu
stock with a nominal concentration at 50 lg Cu/L was estimated
to be 44.6 lg Cu/L. Based on the results, the recovery of the total
Cu added in the test solutions ranged from 69% to 93% with an
overall average of 78% (Table 3). Surprisingly, a low concentration
of Cu (29 nM) in the organic phase was also detected in the treat-
ment of [ZnPT]:[Cu] = 1:0 (Table 3), indicating that there was some
impurity of Cu-complexed organic compounds (probably CuPT) in
the chemical ZnPT purchased from Sigma (USA). If all Cu detected
were assumed to be CuPT, the proportion of the impurity would ac-
count for ?9.3% by weight.
4. Discussion
ZnPT is well known to be highly toxic to aquatic organisms with
a chronic NOEC (no observed effect concentration) 61.5 lg/L (Tur-
ley et al., 2000). Our results also support that ZnPT is highly toxic to
marine organisms as shown in the three test species with 96 h-
EC50 of 1.9 lg/L, 48 h-LC50 of 4.4 lg/L and 96 h-LC50 of 29 lg/L
for Thalassiosira pseudonana, H. elegans and E. rapax, respectively.
These acute toxicity endpoints of ZnPT are comparable to the re-
ported values for marine organisms in temperate regions. For in-
stance, 72 h-EC50 of ZnPT to the diatom C. gracilis was 3.2 lg/L
(Koutsaftis and Aoyama, 2006), while the 48 h-EC50 values of
ZnPT, based on inhibition of embryonic development, to the sea
urchin Paracentrotus lividus and the blue mussel Mytilus edulis were
2.4 lg/L and 2.5 lg/L, respectively (Bellas et al., 2005).
The current results of the combined toxicity tests clearly indi-
cate that there is a strong synergistic effect between ZnPT and Cu
to the three test species, especially T. pseudonana and E. rapax. It
is important to note that such synergism was consistently ob-
served in these two species even at low Cu concentration (i.e.
10 lg Cu/L that falls within the range of environmentally realistic
Cu concentrations in coastal waters). It is postulated that such syn-
ergistic effects are partially attributable to the formation of CuPT
by transchelation of ZnPT with Cu (Grunnett and Dahllöf, 2005),
since CuPT has been shown to be more toxic than ZnPT to marine
organisms. Mochida et al. (2006) observed that CuPT was more
toxic than ZnPT to the sea bream P. major and the toy shrimp H.
futilirostris (96 h-LC50 values of CuPT and ZnPT: 9.3 and 98.2 lg/L
for P. major, and 2.5 and 120 lg/L for H. futilirostris, respectively).
Indeed, CuPT was more toxic than ZnPT to E. rapax juveniles; the
96 h-LC50 of CuPT to E. rapax juveniles was determined to be
9.6 lg/L (95% C.I.: 8.1–11 lg/L; study by Stella Wong, unpublished
data) that was considerably lower than the corresponding LC50 for
ZnPT (29 lg/L). Also, CuPT was found to be more toxic to the cope-
pod Tigriopus japonicus (24 h-LC50 = 41 lg/L) than ZnPT (24 h-
LC50 > 500 lg/L) (Shipbuilding Research Association of Japan,
2002, cited in Yamada, 2006). Similarly, CuPT is more toxic than
ZnPT to suspension-cultured fish cells CHSE-sp and juvenile rain-
bow trout Oncorhynchus mykiss (Okamura et al., 2002). In concur-
rence with our results, synergistic effects of the binary mixtures
of ZnPT and Cu were consistently observed in the bacteria V. fisc-
heri (Zhou et al., 2006), P. major and H. futilirostris (Mochida
Fig. 3. Cumulative mortality of Elasmopus rapax exposed to different binary mixtures of ZnPT and Cu at 24 h, 48 h, 72 h and 96 h, respectively.
620
V.W.W. Bao et al./Marine Pollution Bulletin 57 (2008) 616–623