Environmental Pollution 158 (2010) 1825 1833
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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol
Influence of environmental factors on the response of a natural population
of Daphnia magna (Crustacea: Cladocera) to spinosad and
Bacillus thuringiensis israelensis in Mediterranean coastal wetlands
a,b b c a b,*
C. Duchet , Th. Caquet , E. Franquet , C. Lagneau , L. Lagadic
a
Entente Interd�partementale de D�moustication du Littoral M�diterran�en, 165 avenue Paul-Rimbaud, Montpellier F-34184, France
b
INRA, UMR985 �cologie et Sant� des �cosystŁmes, �quipe �cotoxicologie et Qualit� des Milieux Aquatiques, 65 rue de Saint Brieuc, Rennes F-35042, France
c
Universit� Paul C�zanne, Institut M�diterran�en d �cologie et de Pal�o�cologie, Facult� des Sciences et Techniques Saint J�r�me, C31, Marseille F-13397, France
Significant interaction between salinity and spinosad exposure impairs the recovery of a natural population of Daphnia magna.
a r t i c l e i n f o a b s t r a c t
Article history:
The present study was undertaken to assess the impact of a candidate mosquito larvicide, spinosad (8, 17
Received 10 August 2009
and 33 mgL 1) on a field population of Daphnia magna under natural variations of water temperature and
Received in revised form
salinity, using Bti (0.16 and 0.50 mL L 1) as the reference larvicide. Microcosms (125 L) were placed in
23 October 2009
a shallow temporary marsh where D. magna was naturally present. The peak of salinity observed during
Accepted 4 November 2009
the 21-day observation period may have been partly responsible for the decrease of daphnid population
density in all the microcosms. It is also probably responsible for the absence of recovery in the micro-
Keywords:
cosms treated with spinosad which caused a sharp decrease of D. magna abundance within the first two
Daphnia
days following treatment whereas Bti had no effect. These results suggest that it may be difficult for
Biopesticide
a field population of daphnids to cope simultaneously with natural (water salinity and temperature) and
Salinity
anthropogenic (larvicides) stressors.
Temperature
Stressor Ó 2009 Elsevier Ltd. All rights reserved.
In situ microcosms
1. Introduction stress responses observed at the level of individuals to population-
level effects (Calow, 1991). Some laboratory experiments showed
Mediterranean coastal wetlands are characterized by large that environmental factors such as water salinity, hardness or
spatial and temporal variations of many environmental parameters temperature may interfere with the effects of toxicants on daph-
(Comin and Valiela, 1993; Nuccio et al., 2003). Aquatic invertebrates nids (Semsari and Ha�t-Amara, 2001; Heugens et al., 2006; de la Paz
living in these ecosystems may therefore be exposed to changes in Gomez-Diaz and Martinez-Jeronimo, 2009). Anthropogenic and
water level, temperature or salinity. Under some circumstances natural stressors may not simply act in additive ways; rather
(e.g., duration or intensity of exposure to extreme values of a given multiplicative interactions occur either increasing (synergistic) or
factor), the stress associated with these variations may have an dampening (antagonistic) the effects of stressors (Salbu et al., 2005;
impact on the physiological integrity of organisms, leading to Hames et al., 2006). However, little is known on the possible
a decrease in their overall fitness (Smolders et al., 2005). These interaction between toxicants and natural changes in environ-
ecosystems are also highly suitable habitats for numerous insect mental parameters in the field.
species, including mosquitoes (Diptera: Culicidae), which A number of natural products have been proposed as  environ-
frequently exhibit mass occurrences that may become a great ment-friendly insecticides and some of them exhibit selectivity
nuisance (Becker et al., 2003). Therefore, these ecosystems are towards certain insect taxa which promotes their use for mosquito
target areas for mosquito control using chemicals that may have an control. Among these compounds, the bacterial larvicide Bacillus
impact on non-target aquatic invertebrate species. Combating the thuringiensis subspecies israelensis (Bti), which is well-known for its
poisoning effects of toxic compounds has also a metabolic cost for selectivity for Nematocera dipterans, in particular Culicidae
these organisms, and this has implications for linking physiological (mosquitoes), Simuliidae (black flies) and Chironomidae (non-
biting midges; Boisvert and Boisvert, 2000), is widely used for
mosquito control all over the world (LacoursiŁre and Boisvert,
2004). Spinosad, a mixture of spinosyns A and D known as
* Corresponding author. Tel.:�33 223 485 237; fax:�33 223 485 440.
E-mail address: [e-mail] (L. Lagadic). fermentation products of a soil bacterium (Saccharopolyspora
0269-7491/$  see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2009.11.008
1826 C. Duchet et al. / Environmental Pollution 158 (2010) 1825 1833
Monitoring started just before the treatments (Day 0), and was carried out until 21
spinosa, Actinomycetes; Crouse et al., 2001), is a neurotoxic
days after insecticide spraying. Sampling was performed on Day 0, 2, 4, 7, 14 and 21.
biological insecticide that is also a potential candidate for mosquito
control (Cetin et al., 2005). However, some studies indicated that it
2.3. Water quality parameters
may be toxic to beneficial or non-target species (Nasreen et al.,
2000; Tillman and Mulrooney, 2000; Consoli et al., 2001). The
On each sampling date, the water temperature, dissolved oxygen concentration,
salinity, and pH were measured in every microcosm at ca. 5 cm below the water
Lethal Dose which caused 50% mortality (LD50) for the bee (Apis
surface, using portable apparatuses (Wissenschaftlich-Technische-Werkst�tten 
mellifera) was estimated at 0.057 mg/bee in the case of an oral
WTW, Champagne au Mont d Or, France). Water level was measured to the nearest
exposure (WHO, 2007). The No Observed Effect Concentration
1 mm in every microcosm using a graduated aluminium gauge. Measurements were
(NOEC) in chronic toxicity was estimated at 1.6 mg L 1 for Chiro-
always made between 10:00 and 12:00 AM to ensure consistency among data
nomus riparius and 8 mg L 1 for Daphnia magna (WHO, 2007). relative to possible circadial influence. Suspended Matter (SM) concentration was
determined in 250 mL water samples filtered through pre-weighted oven-dried (2 h
Furthermore, adverse effects of spinosad have been demonstrated
at 500 C) Whatman GF/C fiberglass filters (1.2-mm mesh size; Whatman Interna-
for the zooplankton crustacean Daphnia pulex (Crustacea, Clado-
tional, Maidstone, UK) that were weighted again after 48 h at 105 C according to the
cera) under laboratory conditions (Stark and Vargas, 2003) and in
AFNOR (1996) method. Chlorophyll a concentration in water was measured in
field microcosms (Duchet et al., 2008).
250 mL water samples filtered through Whatman GF/C fiberglass filters. Pigments
were extracted overnight using 5 mL of an acetone/distilled water (90/10, v/v)
In a field study performed in a shallow temporary oligohaline
mixture. Chlorophyll a was quantified spectrophotometrically (Prim Advanced,
marsh located in Western France (Duchet et al., 2008), we showed
SECOMAM, Domont, France) according to Lorenzen (1967).
that spinosad applied at concentrations ranging from 8 to 33 mgL 1
had a negative impact on D. pulex survival and population size
2.4. Sampling procedures and measurement of endpoints in daphnids
structure. At the lowest concentration tested, daphnid population
Water samples were collected using PVC tube samplers (70 cm length, 6 cm
recovered after the first week, demonstrating the recovery potential
inner diameter) equipped with a 2 4 mm mesh screen-covered one-way valve at
of these organisms to spinosad exposure under natural environ-
the bottom (Roucaute and Quemeneur, 2007). Samples were collected from twenty
mental conditions. However, no significant changes in water
regularly spaced locations within each microcosm, in order to reduce the effects of
temperature or salinity were observed during the study period.
plankton patchiness (Stephenson et al., 1984; SETAC, 1991), and grouped into
Therefore, the present study was undertaken to assess the impact of a beaker. The resulting composite sample (mean SE volume ź 54 23 mL,
depending on the water level in the microcosm) was filtered through 30-mm mesh
spinosad on a Mediterranean coastal wetland population of
nylon net. The retained organisms (daphnids and some other pelagic invertebrates)
D. magna under natural variations of water temperature and salinity,
were transferred to a 500 mL plastic vial and preserved using neutral aqueous
using Bti as a reference larvicide. Water temperature and salinity
formaldehyde/sucrose (4%, v/v; 40 g L 1) that contained 250 mgL 1 Bengal pink dye.
were selected because they are not supposed to vary following
All the daphnids found in the samples were identified to the species level using the
key of Amoros (1984). They were counted using a stereomicroscope (Stemi SV 6,
larvicide treatment and because they usually exhibit the highest
Zeiss, Thornwood, NY, USA) and their body length was measured from the eye to
variation in mediterranean wetlands (Waterkeyn et al., 2008). The
base of the tail spine using an ocular micrometer (Boronat and Miracle, 1997).
experiment was carried out using in situ microcosms (enclosures).
Abundances of D. magna were expressed as the number of individuals per litre based
These systems give the opportunity to integrate the effects of the
on the volume of the composite samples collected in the microcosms.
exposure to larvicides and to natural changes in physicochemical
2.5. Data analysis
parameters, which is not possible with single-species laboratory
toxicity tests (van den Brink et al., 2005). D. magna population-level
The normality of physicochemical data was tested using Shapiro Wilks test, and
effects were assessed on the basis of population density and size-
the homogeneity of variances between treatments was tested using Bartlett s test.
structure analysis for increasing levels of larvicide exposure.
When one of these tests failed, data were transformed in order to meet the
requirements of parametric one-way analysis of variance (one-way ANOVA). Loga-
2. Materials and methods
rithmic (y0źlog(y�1)) and square root (y0źy0.5) transformations were tested.
For normally-distributed data (either raw or transformed data), a two-way
2.1. Study site and microcosms
Repeated Measures Analysis of variance (RM-ANOVA) was performed, in order to
identify overall effects of the treatments. When two-way RM-ANOVA indicated that
The study was performed in a shallow temporary oligohaline marsh located
there was a significant difference between the treatments, a one-way ANOVA was
in Les Saintes-Maries-de-la-Mer (Bouches-du-Rh�ne, Camargue, France; 43 290- performed for each sampling date. Dunnet s post-hoc test was used to identify which
36.9800N 4 23031.8300E) where D. magna populations are naturally present. The
treatments were different from the control.
microcosms were 0.125 m3 cube-shaped bottomless plexiglas enclosures When data transformation failed, non-parametric Friedman s test was used to
(50 50 50 cm). They were pushed into the sediment surface (5 10 cm depth)
check for heterogeneity in the temporal dynamics of the different parameters
to avoid leaking of contaminated water from the microcosms where the larvicides
between the microcosms. To evaluate the influence of larvicide treatment on the
were applied.
various environmental parameters, a Kruskal Wallis test was performed for each
sampling date, followed by the appropriate post-hoc test (kruskalmc function from R
2.2. Experimental design package pgirmess).
The effects of larvicides on the population density of D. magna were analysed for
Thirty microcosms were used to enclose fractions of the natural daphnid pop- the whole study period and on each sampling date using a negative binomial
ulation. Ten microcosms were treated with Bti (Vectobac� 12AS; Valent Biosciences, Generalized Linear Model (GLM). A Dunnet s post-hoc test was used to test for
Libertyville, IL, USA), 15 microcosms were used for the treatment with spinosad differences between control and treated systems.
(Spinosad 120SC�; Dow AgroSciences, Indianapolis, IN, USA), and 5 microcosms The influence of water temperature and salinity on the effects of the two
remained as untreated controls. Microcosms were allowed to stabilize for 24 h larvicides on the population density of D. magna was checked using a three factor
before larvicide application. Treatments were randomly assigned to the microcosms negative binomial GLM. Two categories,  low and  high , were defined for water
using a random number table (R for Windows Version 2.7.0). Vectobac� 12AS was temperature and salinity values. Values inferior to the median values computed for
applied at 0.8 and 2.5 L ha 1 (nominal concentration for 30 cm water depth: 0.16 and temperature or salinity for the whole study period were categorized as  low ,
0.50 mL L 1, respectively), each concentration being applied to 5 microcosms whereas values superior to these median values were categorized as  high .
(replicates). These concentrations correspond to the minimum recommended and Preliminary investigations showed that D. magna length data clearly exhibited
the maximum registered rates for terrestrial and aerial treatments, respectively a non-normal right-skewed distribution and that neither log, nor square-root
(ACTA, 2008). Spinosad 120SC was applied as a suspension concentrate formulation transformation was able to normalize the data or homogenize the variances.
containing 120 g active substance per litre at 25, 50 and 100 g ha 1 (nominal Therefore, mean length values computed for the various treatments were compared
concentration for 30 cm water depth: 8, 17 and 33 mg L 1, respectively). The treat- at each sampling date using a Kruskal Wallis non-parametric test followed by
ment rates were chosen in order to encompass the rate of 50 g ha 1 which would be a non-parametric multiple comparison post-hoc test. Length frequency distributions
the mean presumed recommended rate for field application. Five replicates were were constructed by counting the relative number of individuals in successive
used for each spinosad concentration. The treatments were performed on August 10, 0.5 mm width classes. For each sampling date, values of the relative abundance of
2005. Each larvicide was diluted into tap water before spraying at the water surface, a given length class were compared using a proportion comparison test. When the
using a portable spraying apparatus as described elsewhere (Duchet et al., 2008). test indicated a significant between-treatment difference, the values obtained for
C. Duchet et al. / Environmental Pollution 158 (2010) 1825 1833 1827
Table 1
Mean standard-error (n ź 5) values of the environmental parameters which significantly varied between control and treated enclosures at each sampling date
(significantly different from the control using a non-parametric multiple comparison test for pH, [Dissolved O2] and [Suspended Matter], and Dunnet s post-hoc test for
[Chlorophyll a]: *: 0.05 > p > 0.01, **: 0.01 > p > 0.001, ***: p < 0.001). Bti: Bacillus thuringiensis subspecies israelensis; Spd: spinosad.
Environmental parameter Treatment Sampling date
Day 0 Day 2 Day 4 Day 7 Day 14 Day 21
pH Control 7.8 0.10 8.4 0.07 9.0 0.18 9.5 0.14 8.4 0.03 8.5 0.07
Bti 0.16 mL L 1 7.9 0.05 8.6 0.10 9.4 0.06 9.6 0.10 8.4 0.08 8.7 0.10
Bti 0.50 mL L 1 7.9 0.06 8.5 0.06 9.3 0.14 9.4 0.17 8.5 0.05 8.7 0.10
Spd 8 mg L 1 7.9 0.04 8.9 0.08* 9.3 0.12 9.6 0.20 8.7 0.03* 9.1 0.05*
Spd 17 mg L 1 7.9 0.04 9.0 0.08** 9.7 0.12** 9.7 0.25 8.7 0.06** 9.4 0.12**
Spd 33 mg L 1 7.8 0.04 9.1 0.06** 9.6 0.10* 9.8 0.09 8.8 0.04*** 9.5 0.11***
[Dissolved O2] (mg L 1) Control 5.3 0.4 6.1 0.3 8.8 0.3 10.5 0.2 5.7 0.3 7.1 0.4
Bti 0.16 mL L 1 5.9 0.4 7.4 0.2 9.9 0.2 11.6 0.4 6.7 0.2 8.2 0.6
Bti 0.50 mL L 1 6.2 0.3 7.3 0.2 9.4 0.3 10.7 0.5 6.7 0.2 8.5 0.5
Spd 8 mg L 1 5.0 0.3 8.1 0.9 9.3 0.4 10.5 0.5 6.6 0.6 8.1 0.4
Spd 17 mg L 1 5.6 0.5 10.1 0.8*** 10.7 0.3** 11.2 0.4 7.9 0.6 10.4 0.6***
Spd 33 mg L 1 4.9 0.4 9.1 0.6** 9.9 0.6 10.8 0.6 7.0 0.5 9.0 0.6
[Chlorophyll a] (mg L 1) Control 64.8 19.2 49.0 5.6 34.8 12.4 44.9 14.6 36.8 6.0 15.1 5.4
Bti 0.16 mL L 1 48.3 3.5 60.8 3.1 37.5 11.2 51.9 22.0 36.2 7.4 26.2 6.4
Bti 0.50 mL L 1 52.7 6.8 60.0 3.9 58.4 11.8 68.0 13.1 38.7 3.3 30.5 2.9
Spd 8 mg L 1 35.6 9.6 85.4 12.9** 64.9 9.7 75.2 10.4 50.8 3.1 41.1 7.4**
Spd 17 mg L 1 51.5 12.8 77.5 3.1* 45.0 6.4 48.7 15.0 48.4 4.1 50.4 8.1***
Spd 33 mg L 1 37.9 11.2 92.5 10.5*** 59.3 8.6 105.7 29.2 51.0 9.2 42.6 5.1**
[Suspended Matter] (mg L 1) Control 15.7 1.5 12.7 0.7 19.8 3.6 32.0 4.3 37.7 13.1 18.1 1.9
Bti 0.16 mL L 1 19.7 4.6 14.2 2.3 22.2 3.2 31.2 4.7 63.6 6.9 18.1 2.2
Bti 0.50 mL L 1 19.2 4.6 10.1 4.2 21.8 4.1 46.2 7.9 53.0 14.7 19.0 2.7
Spd 8 mg L 1 18.9 0.8 17.6 2.6 36.1 5.8 87.7 44.9 38.6 12.1 25.9 0.8
Spd 17 mg L 1 16.3 1.9 40.5 16.0* 57.0 21.6* 87.0 44.1 48.8 15.2 28.5 2.1**
Spd 33 mg L 1 12.2 3.8 28.7 6.9 40.3 5.0* 110.2 24.2** 33.7 8.1 27.3 1.2*
the control and for each treatment were compared using proportion comparison
test). Water salinity increased from Day 0 to Day 7, and showed
tests for two values.
further decline until the end of the experiment (Fig. 2). No signif-
All tests were performed using R for Windows Version 2.9.0 (R foundation for
icant between-treatment difference was shown by the date-by-
Statistical Computing). Significance was accepted at aź0.05 for all tests excepted
date analysis (Kruskal Wallis test).
for proportion comparison tests for two values for which a Bonferroni correction
was applied based on the number of tests performed. A significant effect of treatment on suspended matter concen-
tration values was observed on Day 2, 4, 7 and 21 (Kruskal Wallis
test, pź0.021, pź0.017, pź0.020 and pź0.002, respectively).
3. Results
Non-parametric multiple comparison tests showed that the values
were sometimes significantly higher in the microcosms treated
3.1. Environmental parameters
with the intermediate or high spinosad concentration, but no clear
pattern was observed. A significant effect of treatment on water pH
Table 1 gives the mean values ( SE) of the environmental
was shown on Day 2, 4, 14 and 21 (Kruskal Wallis test, pź0.0004,
parameters which significantly varied between control and treated
p ź 0.029, p ź 0.0003 and p ź 0.0002, respectively). Non-
enclosures at the different sampling dates in the different types of
microcosms. Only raw water temperature and square root-trans-
formed chlorophyll a concentration data met the requirements of
25
parametric methods of analysis. Two-way RM-ANOVA showed that
water temperature varied during the study in all the microcosms
(p < 0.001; Fig. 1), and no significant between-treatment differ- 24
ences were observed (pź0.27). Time also had a significant effect on
chlorophyll a concentration in water (two-way RM-ANOVA on
23
square root transformed data, p < 0.001). Concentrations of chlo-
rophyll a increased in all the microcosms during the first 14 days of
22
the experiment, and then gradually decreased. A significant treat-
ment effect on chlorophyll a concentration in water was shown on
Day 2 and 21 (one-way ANOVA on square root-transformed values,
21
pź0.0016 and pź0.0028, respectively). On both dates, chlorophyll
a concentration values were significantly higher in spinosad-
20
treated microcosms than in the control systems, irrespective of
larvicide concentration (Dunnet s post-hoc test).
No adequate data transformation method was found for the
19
other parameters. The results of Friedman s test indicate a signifi-
0 3 6 9 12 15 18 21
cant between-microcosms heterogeneity for all these parameters
Sampling date (day after treatment)
(p<0.001). Water level gradually decreased in all the microcosms
during the study (Fig. 2). No significant between-treatment differ-
Fig. 1. Change in mean values ( SE; nź5) of temperature (expressed in C) for all the
ence was identified with the date-by-date analysis (Kruskal Wallis microcosms.
Temperature (�C)
1828 C. Duchet et al. / Environmental Pollution 158 (2010) 1825 1833
Table 2
Water Level Salinity
25 6
Results of the fitting of a three factor negative binomial GLM model to the pop-
ulation density of Daphnia magna (Chi-square p values indicate the level of signifi-
cance of the various factors in the model).
Factor Chi-square p value
5
Treatment 2.7 10 10
20
Salinity 0.0048
Temperature 2 10 8
4
Treatment Salinity 0.0014
Treatment Temperature 1.95 10 6
Temperature Salinity 0.0002
15
Treatment Salinity Temperature 0.0747
3
shown by GLM analysis (Fig. 3). Mean D. magna population density
10 2,0 2
increased in control and Bti-treated microcosms from the begin-
0 3 6 9 12 15 18 21
ning of the experiment to Day 4, and then decreased until the end
Sampling date (day after treatment)
of the experiment. A sharp decrease in mean D. magna density was
observed in the microcosms treated with spinosad, except on Day 7
Fig. 2. Change in mean values ( SE; nź5) of water level (expressed in cm) and
when a peak of density was observed in the microcosms treated
salinity (expressed in g L 1) for all the microcosms.
with the lowest spinosad concentration. D. magna populations
went to extinction in the microcosms treated with the two lowest
and the highest spinosad concentration on Day 21 and 4, respec-
parametric multiple comparison tests showed that pH values were
tively. No recovery was observed during the study in the micro-
significantly higher in spinosad-treated microcosms than in control
cosms treated with spinosad. Negative binomial GLM fitted to the
systems, with a clear positive concentration effect relation. A
D. magna density data for the whole study period showed a signif-
significant effect on dissolved oxygen concentration in water was
icant overall effect of the treatments (pź1.5 10 9). All the tested
shown on Day 2, 4 and 21 (Kruskal Wallis test, p ź 0.0031,
spinosad concentrations had a significant effect (Dunnet s post-hoc
pź0.037 and pź0.011, respectively). Non-parametric multiple
test; pź0.001, p < 0.001 and p < 0.001 for spinosad nominal
comparison tests showed that dissolved oxygen concentration
concentrations of 8, 17 and 33 mg L 1, respectively). In contrast, no
values were significantly higher, as compared to control, on these
three dates in the microcosms treated with the intermediate spi- effect of Bti treatments was shown although the p value was close
to the significance threshold for the highest Bti concentration
nosad concentration and in the microcosms treated with the
(Dunnet s post-hoc test; pź0.991 and pź0.056 for Bti nominal
highest spinosad concentration on Day 2.
concentrations of 0.16 and 0.5 mL L 1, respectively).
GLM analysis performed for each sampling date (Fig. 3) showed
3.2. D. magna abundance and size
that D. magna densities were not different between the treatments
before introduction of the larvicides (pź0.352) whereas significant
D. magna largely dominated the zooplankton community. Only
differences were continuously observed for all the subsequent
a few Simocephalus sp. individuals (nź17 for the whole study
dates (all p values inferior to 0.001 from Day 2 to the end of the
period) were found in the samples. The mean values of D. magna
study). Dunnet s post-hoc test showed a negative effect (p<0.001)
density just before the treatment were not statistically different as
of all spinosad concentration on Day 2, 4, 14 and 21. On Day 7, only
the two highest spinosad concentrations had a significant negative
effect. On Day 21, a significant negative effect of the highest Bti
9000
Control
concentration on D. magna density was also observed (Dunnet s
Bti, 0.16 �L.L-1
post-hoc test, pź0.003).
Bti, 0.50 �L.L-1
Negative binomial GLM including treatment, salinity and water
Spinosad, 8 �g.L-1
Spinosad, 17 �g.L-1 temperature as explanatory variables was fitted to the D. magna
Spinosad, 33 �g.L-1
density values (Table 2). The minimal adequate model contained
6000
the three variables and the three second-order interactions. The
third-order interaction was not significant. Interaction plots
between treatments and the two environmental variables (Fig. 4)
clearly show the interaction between spinosad treatment and the
two environmental variables.
3000
Table 3 summarizes the mean values of D. magna length
measured in the microcosms, and Fig. 5 shows the frequency of the
different length classes within the different populations on each
sampling date. The results of Kruskal Wallis tests indicated that
there was always a difference (p<0.001) in the mean body length
***
***
***
*** **
of daphnids between the microcosms including on Day 0. Non-
0
0 3 6 9 12 15 18 21
parametric multiple comparison tests showed that the length of
Sampling date (day after treatment) daphnids was slightly higher in treated than in control microcosms
before treatment. This is confirmed by the statistical analysis of
Fig. 3. Change in mean values (�SE; nź5) of Daphnia magna abundance (expressed as
length class frequencies that showed a higher relative abundance of
the number of individuals per litre) in the control microcosms, the microcosms treated
small daphnids (body length comprised between 0.5 and 1 mm) in
with Bti at 0.16 and 0.50 mL L 1, and the microcosms treated with spinosad at 8, 17 and
control than in treated microcosms on Day 0. Accordingly, the
33 mg L 1. (Significantly different from control, Dunnet s post-hoc test following
negative binomial GLM fitting: **: 0.01 > p > 0.001; ***: p < 0.001). relative frequencies of medium-size daphnids (body length
-1
Salinity (g.L )
Water level (cm)
Abundance of
Daphnia magna
(number of individuals per liter)
C. Duchet et al. / Environmental Pollution 158 (2010) 1825 1833 1829
significant reproduction occurred after Day 2. This is corroborated
250
High temperature
by the changes with time of mean population density values
Low temperature (Fig. 3). On Day 7 and 14, the frequency of daphnids measuring less
than 1.5 mm was higher in Bti-treated microcosms, suggesting
200
a possible delayed effect of the treatments on growth of these
organisms. From Day 2 to Day 7, daphnids were frequently smaller
in larvicide-treated microcosms, especially those treated with spi-
150
nosad. This result should be considered cautiously because the
abundance of daphnids in these systems was very low after Day 4.
100
4. Discussion
50
The composition and dynamics of the communities inhabiting
coastal wetlands are influenced by the duration of the hydroperiod
and by seasonality (Boix et al., 2001), especially under Mediterra-
0
nean climate (Comin and Valiela, 1993; Nuccio et al., 2003).
Bti Bti
Control Spd Spd Spd
Hydroperiod duration has been identified as the main factor in
0.16 �L.L-1 0.50 �L.L-1 8 �g.L-1 17 �g.L-1 33 �g.L-1
determining the faunal composition and structure of aquatic
Treatment
communities in these systems (McLachlan, 1985; Jeffries, 1994),
and drought is often the major mortality factor for insects in
temporary pools (Batzer and Resh, 1992). Living in variable saline
300
High salinity
water habitats requires specific physiological and life history traits
such as rapid development, migratory/colonizing ability (strong
Low salinity
250
ability of dissemination), as well as resistant or dormant life stages
to spend the drought period in situ (Herbst, 2001). Daphnids
present most of these favourable traits. They are cyclic partheno-
200
genetic species capable of both asexual and sexual reproduction.
Their survival strategy in sexual reproduction is to produce suffi-
150
cient numbers of resting eggs (ephippia) that enter a diapause stage
and hatch in spring as parthenogenetic females (Baer and Owens,
1999). These features explain why cladocerans are probably among
100
the most well-represented crustaceans in temporary ponds and
wetlands (Metge, 1986; Lake et al., 1989). As such, they constitute
50
putative sentinel organisms for assessment of the effects of natural
and anthropogenic stress on these ecosystems.
This study was performed in summer and the values of the
0
various environmental parameters measured during the survey of
Bti Bti
Control Spd Spd Spd
the microcosms were within the range of values usually found for
0.16 �L.L-1 0.50 �L.L-1 8 �g.L-1 17 �g.L-1 33 �g.L-1
Mediterranean coastal wetlands (see Table 1), as shallow coastal
Treatment
lagoon pH ranges from 7.9 to 8.2 (Dromgoole, 1978) except at the
end of summer when it can rise to 9 (Men�ndez et al., 2001).
Fig. 4. Interaction plots between larvicide treatment and water temperature (upper
Furthermore, Munari et al. (2003) measured chlorophyll a concen-
panel) or water salinity (lower panel), with the mean density of daphnids as the
trations close to 40 mgL 1 and O2 concentrations less than 6 mg L 1
dependent variable. Bti: Bacillus thuringiensis subspecies israelensis; Spd: spinosad.
in August in the Po River deltaic area (northern Italy). In our
microcosms, water level gradually decreased as a consequence of
comprised between 1.5 and 2.5 mm) were higher in the treated
the hydraulic management of the site where the study was per-
microcosms. Between Day 0 and Day 2, there was an increase in the
formed. The wetland was artificially flooded on mid-July by water
frequency of small individuals (body length inferior to 1 mm) for all
pumped in the Petit-Rh�ne River to provide suitable habitats for
treatments, suggesting that adults reproduced at the beginning of
ducks (the wetland is used as a hunting site in autumn). Afterwards,
the study period. In all the microcosms, including the controls,
water level was maintained through regular inflow of water from
mean body length of the sampled individuals increased with time,
the river. Evaporation, which is intense in this area in summer,
and the shape of length frequency distributions suggests that no
induced a further decrease of water level leading to an increase in
Table 3
Daphnia magna mean body length (standard error; n) in mm on each sampling date (significantly different for the control using a non-parametric multiple comparison
test: *: 0.05 > p > 0.01, **: 0.01 > p > 0.001, ***: p < 0.001; NC: not computable;  : no individuals in the samples).
Treatment Sampling date
Day 0 Day 2 Day 4 Day 7 Day 14 Day 21
Control 1.536 (0.026; 914) 1.139 (0.017; 1692) 1.063 (0.012; 2040) 1.375 (0.019; 946) 1.834 (0.032; 346) 1.613 (0.118; 63)
Bti 0.16 mL L 1 1.629* (0.032; 628) 1.105 (0.019; 1390) 1.020*** (0.011; 2382) 1.368 (0.021; 736) 1.663*** (0.040; 306) 2.284*** (0.097; 60)
Bti 0.50 mL L 1 1.701*** (0.035; 529) 1.134 (0.027; 671) 0.978*** (0.019; 724) 1.207*** (0.031; 343) 1.708** (0.040; 96) 1.939 (0.255; 9)
Spinosad 8 mg L 1 1.771*** (0.027; 641) 0.880*** (0.049; 117) 0.794* (0.057; 18) 1.243*** (0.019; 648) 1.965 (0.103; 3) 
Spinosad 17 mg L 1 1.684*** (0.027; 853) 1.083 (0.061; 114) 0.604*** (0.061; 14) 0.847* (0.202; 5) 1.664 (NC; 1) 
Spinosad 33 mg L 1 1.793*** (0.032; 540) 0.846 (0.068; 25) 0.751 (0.112; 5)   
(Number of individuals per liter)
Mean density of
Daphnia magna
Mean density of
Daphnia magna
(Number of individuals per liter)
1830 C. Duchet et al. / Environmental Pollution 158 (2010) 1825 1833
1 1
Day 0
A B Day 2
0,9 0,9
0,8 0,8
0,7 0,7
0,6 0,6
*
0,5 0,5
*
*
0,4 * 0,4
*
*
0,3 0,3
*
*
0,2 0,2
*
*
*
* * * *
*
* *
0,1 0,1
*
*
0 0
0 0.5 1 1.5 2 2.5 3 3.5 4 0 0.5 1 1.5 2 2.5 3 3.5 4
1 1
C D Day 7
Day 4
0,9 0,9
0,8 0,8
*
*
0,7 0,7
0,6 0,6
*
*
* *
0,5 0,5
*
0,4 0,4
*
0,3 0,3
*
*
*
0,2 0,2
0,1 0,1 **
*
0 0
0 0 0.5 1 1.5 2 2.5 3 3.5 4
0.5 1 1.5 2 2.5 3 3.5 4
1 1
EF Day 21
Day 14
*
0,9 0,9
0,8 0,8
0,7 0,7
* *
0,6 0,6
0,5 0,5
*
*
0,4 * 0,4
*
0,3 0,3
*
0,2 0,2
*
0,1 0,1
0 0
0 0.5 1 1.5 2 2.5 3 3.5 4 0 0.5 1 1.5 2 2.5 3 3.5 4
Length class (mm) Length class (mm)
Control Spinosad 8 �g.L-1
Bti 0.16 �L.L-1 Spinosad 17 �g.L-1
Bti 0.50 �L.L-1 Spinosad 33 �g.L-1
Fig. 5. Frequency of the different classes of Daphnia magna length observed on each sampling date for the various treatments (*: significantly different from control, proportion
comparison tests for two values with Bonferroni correction, p < 0.05/m where m is the number of comparisons).
water salinity a few days after the beginning of the experiment. during this study (up to 25 C; Stephenson and Watts, 1984;
Mean water salinity varied between 3.2 and 5.6 g L 1 with a clear MacIsaac et al., 1985; Lagerspetz, 2000) and therefore changes of
unimodal pattern of change with time, and maximum values were population density with time are probably the result of natural
recorded on Day 4 and 7. population dynamics in the study site. Statistical analysis of density
Both water temperature and salinity had a significant effect on data showed an interaction between water temperature and spi-
the mean abundance of D. magna in the microcosms. A peak in nosad effect, with higher density values at high temperature. Once
D. magna density was observed in control and Bti-treated micro- again, this result is more probably linked to a temporal correlation
cosms when water temperature was low but it is not clear whether between water temperature and population density rather than to
there is a direct relationship between these two parameters, or if a causal relationship. Indeed, in spinosad-treated microcosms,
this is a temporal coincidence. D. magna has been shown to have D. magna population density sharply decreased after the treatment
genetic ability to tolerate temperatures higher than those recorded and never recovered. Therefore, the highest density values in these
Frequency
Frequency
Frequency
Frequency
Frequency
Frequency
C. Duchet et al. / Environmental Pollution 158 (2010) 1825 1833 1831
systems were measured at the beginning of the experiment, when synthesis of stress proteins). As such, combating stress is likely to be
water temperatures were also high. energetically costly for stressed organisms (Calow, 1991; De Coen
Like other cladocerans, D. magna mostly inhabits freshwater and Janssen, 2003). Stress will increase the energy expenditure of
ecosystems. However, natural populations living in oligohaline to organisms and, as a consequence, the energy status of an organism
mesohaline habitats also exist (Arn�r and Koivisto, 1993). Schuy- at any given time will affect its capacity to cope with additional
tema et al. (1997) showed that D. magna could survive and repro- stress. Consequently, animals with a high energy status (organisms
duce well in water with salinity below 4 g L 1. According to living in non-stressful environment) are more successful in dealing
Lagerspetz (1955 in Arn�r and Koivisto, 1993), D. magna has been with anthropogenic stress than animals with a low energy status
found in brackish ponds up to 12.5 g L 1 but its occurrence declines (organisms living in stressful conditions) (Smolders et al., 2005). In
abruptly when salinity was above 4 g L 1. This is consistent with the the present study, the energetic cost of spinosad metabolisation and
48-h LC50 of 5.48 g L 1 for NaCl determined for D. magna by exclusion may have reduced the energy available for osmoregula-
Martinez-Jeronimo and Martinez-Jeronimo (2007). Several authors tion of daphnids and affected their capacity to cope with salinity
have shown that salinity may have sublethal effects on Daphnia life stress, leading to limited reproduction and growth, and ultimately
history traits. Cowgill and Milazzo (1991) experimentally demon- to death. This could explain the absence of recovery in the micro-
strated that reproduction, population growth rate, and survival of cosms treated with spinosad at 8 mg L 1.
D. magna are inversely related to salinity. Studying life history traits The between-microcosm heterogeneity detected by Friedman s
of D. magna in laboratory experiments under freshwater and test indicates that each test system exhibited its own dynamics.
brackish conditions, Teschner (1995) showed a negative effect of Nevertheless, the combination of this test performed for the whole
salinity (5 g L 1) on growth and reproduction, which was attributed study period with a date-by-date analysis allowed identifying the
to an alteration of the molt cycle. Martinez-Jeronimo and Martinez- effects of the treatments on some environmental parameters.
Jeronimo (2007) also demonstrated that average lifespan, life Significant differences in water pH, dissolved oxygen and chloro-
expectancy at birth, longevity, average clutch size, total progeny, phyll a concentration were frequently observed between control
number of clutches, net reproductive rate and intrinsic rate of and spinosad-treated microcosms, with higher values in the
population growth were significantly reduced by an increasing treated systems. These differences are probably due to an indirect
NaCl concentration. This is consistent with the results obtained in effect of the larvicide associated with the drastic decrease in D.
field studies by Green et al. (2005) who concluded that although magna population density in the spinosad-treated microcosms.
D. magna may be considered as a euryhaline species which is able to Disappearance of daphnids contributed to decrease the grazing
colonize brackish water environments, its reproductive and/or pressure on phytoplankton resulting in an increase of algal
survival rates are reduced at higher water salinity. In the present biomass leading to a subsequent increase in water pH and dis-
study, a significant effect of water salinity on D. magna population solved oxygen concentration through an enhancement of photo-
density was observed. Results presented in Fig. 4 suggest that there synthesis (Axelsson, 1988; Shiraiwa et al., 1993; Rigobello-Masini
was a positive effect of high salinity values in the control micro- et al., 2003). Such effects have frequently been reported as indirect
cosms. This may reflect the fact that the peak of salinity occurred consequences of insecticide treatment in aquatic ecosystems
just after a reproduction period in daphnids. Therefore, the possible (Caquet et al., 1992; Fleeger et al., 2003; Hanson et al., 2007). A
negative effect of high salinity on D. magna density was probably reduction of dissolved oxygen consumption directly associated
delayed. This could explain why there was a subsequent decrease in with daphnid disappearance cannot be excluded, although its
population density and no reproduction even in control micro- contribution to the difference between control and spinosad-
cosms during the rest of the experiment. treated microcosms is probably far less important than the indi-
D. magna survival was affected by all the spinosad treatments. In rect effect associated with phytoplankton bloom. The absence of
the microcosms treated with 8 mg L 1 spinosad, D. magna density significant differences for pH, dissolved oxygen and chlorophyll
measured within the first four days of exposure was significantly a concentration on Day 4 and 7 may be a consequence of the stress
less than the control but a peak of density was observed on Day 7, on the phytoplankton community induced by the increase in
suggesting that the exposed population began to recover from the water salinity. A salinity of ca. 5 g L 1 forms a lethal barrier for
exposure to the larvicide. However, recovery was not confirmed by most planktonic algae living in brackish waters (Fl�der and Burns,
the data obtained later in the study. At 17 and 33 mg L 1, spinosad 2004). At this salinity level, freshwater and marine species exhibit
affected the whole population and did not selectively impact a severe osmotic stress (Kies, 1997) and planktonic species diver-
a particular size class. No recovery of the D. magna population sity is minimal (Fl�der and Burns, 2004). Therefore, the increase in
occurred in the microcosms treated with these spinosad concen- water salinity may have temporarily hampered the development
trations. Chronic spinosad NOEC values for daphnids have been of planktonic algae in spinosad-treated microcosms. Once the
estimated at 6.7 and 8 mg L 1 in static and semi-static laboratory salinity has decreased to its original value, algae were again able to
tests, respectively (National Registration Authority for Agricultural proliferate.
and veterinary Chemicals, 1998; WHO, 2007). In the present study, Effects of spinosad on D. magna were compared to those of Bti,
8 mgL 1 spinosad produced a significant mortality in D. magna, and applied at 0.16 and 0.50 mL L 1. Bti treatments did not have any
recovery was not observed whereas in a comparable field study, D. effect on the various environmental parameters measured during
pulex recovered after the first week following a treatment at the the study period, and it had almost no effect on D. magna survival
same concentration (Duchet et al., 2008). There was a significant (the only significant effect was observed at Day 21 for the highest
interaction between spinosad treatment and salinity (Fig. 4). This Bti concentration) confirming a number of previous studies (Bois-
may be due to the high mortality caused by the treatment, in vert and Boisvert, 2000; LacoursiŁre and Boisvert, 2004). In
combination with the increase of water salinity. Such an interaction particular, Ali (1981) and Miura et al. (1981) showed that Ephem-
between salinity and pesticide toxicity has already been demon- eroptera, Amphipoda, Copepoda and Cladocera were not affected
strated on copepods by Hall et al. (1994) and Staton et al. (2002). by Bti. Results concerning body length (Table 3) did not allow
Any factor that disrupts the physiological integrity of exposed concluding about the effect of Bti on the size of D. magna, as there
organisms will induce defence and repair mechanisms, which was heterogeneity in individual sizes before treatment. However, in
depend on energy-requiring processes such as active transport (e.g., a previous field study (Duchet et al., 2008), no effect of Bti on the
exclusion of chemical stressors) and synthetic activity (e.g., size structure of D. pulex was observed.
1832 C. Duchet et al. / Environmental Pollution 158 (2010) 1825 1833
Daphnia magna and corresponding population characteristics. Environmental
5. Conclusion
Toxicology and Chemistry 22, 1632 1641.
de la Paz Gomez-Diaz, M., Martinez-Jeronimo, F., 2009. Modification of the acute
Unlike Bti, spinosad had a strong lethal effect on D. magna
toxic response of Daphnia magna Straus 1820 to Cr(VI) by the effect of varying
saline concentrations (NaCl). Ecotoxicology 18, 81 86.
population at presumed recommended rates for field application.
Dromgoole, F.I., 1978. The effect of pH and inorganic carbon on photosynthesis and
Our results suggest that it may be difficult for a natural daphnid
dark respiration of Carpophyllum (Fucales, Phaeophyceae). Aquatic Botany 4,
population to cope simultaneously with natural (water salinity and
11 22.
temperature) and anthropogenic (larvicides) stressors, and this Duchet, C., Larroque, M., Caquet, Th., Franquet, E., Lagneau, C., Lagadic, L., 2008.
Effects of spinosad and Bacillus thuringiensis israelensis on a natural population
may directly affect the recovery potential of the population. The
of Daphnia pulex in field microcosms. Chemosphere 74, 70 77.
experiment also allowed observing indirect effects of spinosad, in
Fleeger, J.W., Carman, K.R., Nisbet, R.M., 2003. Indirect effects of contaminants in
particular an increase of phytoplankton abundance and photosyn- aquatic ecosystems. The Science of the Total Environment 317, 207 233.
Fl�der, S., Burns, C.W., 2004. Phytoplankton diversity of shallow tidal lakes: influ-
thetic activity (as indicated by elevated pH and dissolved oxygen
ence of periodic salinity changes on diversity and species number of a natural
concentration) due to the decreasing abundance of primary
assemblage. Journal of Phycology 40, 54 61.
consumers.
Green, A.J., Fuentes, C., Moreno-Ostos, E., Rodrigues da Silva, S.L., 2005. Factors
influencing cladoceran abundance and species richness in brackish lakes in
Eastern Spain. Annales de Limnologie  International Journal of Limnology 41,
Acknowledgements
73 81.
Hall, L.W.J., Ziegenfuss, M.C., Anderson, R.D., Spittler, T.D., Leightweis, H.C., 1994.
Influence of salinity on atrazine toxicity to a Chesapeake Bay copepod (Eur-
Financial support for this work was provided by the French
ytemora affinis) and fish (Cyprinodon variegatus). Estuaries 17, 181 186.
Ministry for Ecology, Sustainable Development and Spatial Plan-
Hames, R.S., Lowe, J.D., Barker Swarthout, S., Rosenberg, K.V., 2006. Understanding
the risk to neotropical migrant bird species of multiple human-caused
ning through the National Programme for Ecotoxicology (PNETOX).
stressors: elucidating processes behind the patterns. Ecology and Society 11, 24
The authors wish to thank Dow AgroSciences for the generous gift
[online] URL: http://www.ecologyandsociety.org/vol11/iss21/art24/.
of spinosad 120SC and Mr. Girand for giving access to the study site.
Hanson, M.L., Graham, D.W., Babin, E., Azam, D., Coutellec, M.-A., Knapp, C.W.,
Lagadic, L., Caquet, Th., 2007. Influence of isolation on the recovery of pond
mesocosms from the application of an insecticide. I. Study design and plank-
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