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
C. Duchet a, b, Th. Caquet b, E. Franquet c, C. Lagneau a, L. Lagadic b, *
a
b
c
Entente Interdépartementale de Démoustication du Littoral Méditerranéen, 165 avenue Paul-Rimbaud, Montpellier F-34184, France
INRA, UMR985 Écologie et Santé des Écosystèmes, Équipe Écotoxicologie et Qualité des Milieux Aquatiques, 65 rue de Saint Brieuc, Rennes F-35042, France
Université Paul Cézanne, Institut Méditerranéen d’Écologie et de Paléoécologie, Faculté des Sciences et Techniques Saint Jérôme, 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:
Received 10 August 2009
Received in revised form
23 October 2009
Accepted 4 November 2009
The present study was undertaken to assess the impact of a candidate mosquito larvicide, spinosad (8, 17
and 33 mg L1) on a field population of Daphnia magna under natural variations of water temperature and
salinity, using Bti (0.16 and 0.50 mL L1) as the reference larvicide. Microcosms (125 L) were placed in
a shallow temporary marsh where D. magna was naturally present. The peak of salinity observed during
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 microcosms treated with spinosad which caused a sharp decrease of D. magna abundance within the first two
days following treatment whereas Bti had no effect. These results suggest that it may be difficult for
a field population of daphnids to cope simultaneously with natural (water salinity and temperature) and
anthropogenic (larvicides) stressors.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Daphnia
Biopesticide
Salinity
Temperature
Stressor
In situ microcosms
1. Introduction
Mediterranean coastal wetlands are characterized by large
spatial and temporal variations of many environmental parameters
(Comin and Valiela, 1993; Nuccio et al., 2003). Aquatic invertebrates
living in these ecosystems may therefore be exposed to changes in
water level, temperature or salinity. Under some circumstances
(e.g., duration or intensity of exposure to extreme values of a given
factor), the stress associated with these variations may have an
impact on the physiological integrity of organisms, leading to
a decrease in their overall fitness (Smolders et al., 2005). These
ecosystems are also highly suitable habitats for numerous insect
species, including mosquitoes (Diptera: Culicidae), which
frequently exhibit mass occurrences that may become a great
nuisance (Becker et al., 2003). Therefore, these ecosystems are
target areas for mosquito control using chemicals that may have an
impact on non-target aquatic invertebrate species. Combating the
poisoning effects of toxic compounds has also a metabolic cost for
these organisms, and this has implications for linking physiological
* Corresponding author. Tel.: þ33 223 485 237; fax: þ33 223 485 440.
E-mail address: [email protected] (L. Lagadic).
0269-7491/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2009.11.008
stress responses observed at the level of individuals to populationlevel effects (Calow, 1991). Some laboratory experiments showed
that environmental factors such as water salinity, hardness or
temperature may interfere with the effects of toxicants on daphnids (Semsari and Haı̈t-Amara, 2001; Heugens et al., 2006; de la Paz
Gomez-Diaz and Martinez-Jeronimo, 2009). Anthropogenic and
natural stressors may not simply act in additive ways; rather
multiplicative interactions occur either increasing (synergistic) or
dampening (antagonistic) the effects of stressors (Salbu et al., 2005;
Hames et al., 2006). However, little is known on the possible
interaction between toxicants and natural changes in environmental parameters in the field.
A number of natural products have been proposed as ‘environment-friendly’ insecticides and some of them exhibit selectivity
towards certain insect taxa which promotes their use for mosquito
control. Among these compounds, the bacterial larvicide Bacillus
thuringiensis subspecies israelensis (Bti), which is well-known for its
selectivity for Nematocera dipterans, in particular Culicidae
(mosquitoes), Simuliidae (black flies) and Chironomidae (nonbiting midges; Boisvert and Boisvert, 2000), is widely used for
mosquito control all over the world (Lacoursière and Boisvert,
2004). Spinosad, a mixture of spinosyns A and D known as
fermentation products of a soil bacterium (Saccharopolyspora
1826
C. Duchet et al. / Environmental Pollution 158 (2010) 1825–1833
spinosa, Actinomycetes; Crouse et al., 2001), is a neurotoxic
biological insecticide that is also a potential candidate for mosquito
control (Cetin et al., 2005). However, some studies indicated that it
may be toxic to beneficial or non-target species (Nasreen et al.,
2000; Tillman and Mulrooney, 2000; Consoli et al., 2001). The
Lethal Dose which caused 50% mortality (LD50) for the bee (Apis
mellifera) was estimated at 0.057 mg/bee in the case of an oral
exposure (WHO, 2007). The No Observed Effect Concentration
(NOEC) in chronic toxicity was estimated at 1.6 mg L1 for Chironomus riparius and 8 mg L1 for Daphnia magna (WHO, 2007).
Furthermore, adverse effects of spinosad have been demonstrated
for the zooplankton crustacean Daphnia pulex (Crustacea, Cladocera) under laboratory conditions (Stark and Vargas, 2003) and in
field microcosms (Duchet et al., 2008).
In a field study performed in a shallow temporary oligohaline
marsh located in Western France (Duchet et al., 2008), we showed
that spinosad applied at concentrations ranging from 8 to 33 mg L1
had a negative impact on D. pulex survival and population size
structure. At the lowest concentration tested, daphnid population
recovered after the first week, demonstrating the recovery potential
of these organisms to spinosad exposure under natural environmental conditions. However, no significant changes in water
temperature or salinity were observed during the study period.
Therefore, the present study was undertaken to assess the impact of
spinosad on a Mediterranean coastal wetland population of
D. magna under natural variations of water temperature and salinity,
using Bti as a reference larvicide. Water temperature and salinity
were selected because they are not supposed to vary following
larvicide treatment and because they usually exhibit the highest
variation in mediterranean wetlands (Waterkeyn et al., 2008). The
experiment was carried out using in situ microcosms (enclosures).
These systems give the opportunity to integrate the effects of the
exposure to larvicides and to natural changes in physicochemical
parameters, which is not possible with single-species laboratory
toxicity tests (van den Brink et al., 2005). D. magna population-level
effects were assessed on the basis of population density and sizestructure analysis for increasing levels of larvicide exposure.
2. Materials and methods
2.1. Study site and microcosms
The study was performed in a shallow temporary oligohaline marsh located
in Les Saintes-Maries-de-la-Mer (Bouches-du-Rhône, Camargue, France; 43 290 36.9800 N–4 230 31.8300 E) where D. magna populations are naturally present. The
microcosms were 0.125 m3 cube-shaped bottomless plexiglas enclosures
(50 50 50 cm). They were pushed into the sediment surface (5–10 cm depth)
to avoid leaking of contaminated water from the microcosms where the larvicides
were applied.
2.2. Experimental design
Thirty microcosms were used to enclose fractions of the natural daphnid population. Ten microcosms were treated with Bti (VectobacÒ 12AS; Valent Biosciences,
Libertyville, IL, USA), 15 microcosms were used for the treatment with spinosad
(Spinosad 120SCÒ; Dow AgroSciences, Indianapolis, IN, USA), and 5 microcosms
remained as untreated controls. Microcosms were allowed to stabilize for 24 h
before larvicide application. Treatments were randomly assigned to the microcosms
using a random number table (R for Windows Version 2.7.0). VectobacÒ 12AS was
applied at 0.8 and 2.5 L ha1 (nominal concentration for 30 cm water depth: 0.16 and
0.50 mL L1, respectively), each concentration being applied to 5 microcosms
(replicates). These concentrations correspond to the minimum recommended and
the maximum registered rates for terrestrial and aerial treatments, respectively
(ACTA, 2008). Spinosad 120SC was applied as a suspension concentrate formulation
containing 120 g active substance per litre at 25, 50 and 100 g ha1 (nominal
concentration for 30 cm water depth: 8, 17 and 33 mg L1, respectively). The treatment rates were chosen in order to encompass the rate of 50 g ha1 which would be
the mean presumed recommended rate for field application. Five replicates were
used for each spinosad concentration. The treatments were performed on August 10,
2005. Each larvicide was diluted into tap water before spraying at the water surface,
using a portable spraying apparatus as described elsewhere (Duchet et al., 2008).
Monitoring started just before the treatments (Day 0), and was carried out until 21
days after insecticide spraying. Sampling was performed on Day 0, 2, 4, 7, 14 and 21.
2.3. Water quality parameters
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
surface, using portable apparatuses (Wissenschaftlich-Technische-Werkstätten –
WTW, Champagne au Mont d’Or, France). Water level was measured to the nearest
1 mm in every microcosm using a graduated aluminium gauge. Measurements were
always made between 10:00 and 12:00 AM to ensure consistency among data
relative to possible circadial influence. Suspended Matter (SM) concentration was
determined in 250 mL water samples filtered through pre-weighted oven-dried (2 h
at 500 C) Whatman GF/C fiberglass filters (1.2-mm mesh size; Whatman International, Maidstone, UK) that were weighted again after 48 h at 105 C according to the
AFNOR (1996) method. Chlorophyll a concentration in water was measured in
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)
mixture. Chlorophyll a was quantified spectrophotometrically (Prim Advanced,
SECOMAM, Domont, France) according to Lorenzen (1967).
2.4. Sampling procedures and measurement of endpoints in daphnids
Water samples were collected using PVC tube samplers (70 cm length, 6 cm
inner diameter) equipped with a 2 4 mm mesh screen-covered one-way valve at
the bottom (Roucaute and Quemeneur, 2007). Samples were collected from twenty
regularly spaced locations within each microcosm, in order to reduce the effects of
plankton patchiness (Stephenson et al., 1984; SETAC, 1991), and grouped into
a beaker. The resulting composite sample (mean SE volume ¼ 54 23 mL,
depending on the water level in the microcosm) was filtered through 30-mm mesh
nylon net. The retained organisms (daphnids and some other pelagic invertebrates)
were transferred to a 500 mL plastic vial and preserved using neutral aqueous
formaldehyde/sucrose (4%, v/v; 40 g L1) that contained 250 mg L1 Bengal pink dye.
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,
Zeiss, Thornwood, NY, USA) and their body length was measured from the eye to
base of the tail spine using an ocular micrometer (Boronat and Miracle, 1997).
Abundances of D. magna were expressed as the number of individuals per litre based
on the volume of the composite samples collected in the microcosms.
2.5. Data analysis
The normality of physicochemical data was tested using Shapiro–Wilks test, and
the homogeneity of variances between treatments was tested using Bartlett’s test.
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). Logarithmic (y0 ¼ log(y þ 1)) and square root (y0 ¼ y0.5) transformations were tested.
For normally-distributed data (either raw or transformed data), a two-way
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
there was a significant difference between the treatments, a one-way ANOVA was
performed for each sampling date. Dunnet’s post-hoc test was used to identify which
treatments were different from the control.
When data transformation failed, non-parametric Friedman’s test was used to
check for heterogeneity in the temporal dynamics of the different parameters
between the microcosms. To evaluate the influence of larvicide treatment on the
various environmental parameters, a Kruskal–Wallis test was performed for each
sampling date, followed by the appropriate post-hoc test (kruskalmc function from R
package pgirmess).
The effects of larvicides on the population density of D. magna were analysed for
the whole study period and on each sampling date using a negative binomial
Generalized Linear Model (GLM). A Dunnet’s post-hoc test was used to test for
differences between control and treated systems.
The influence of water temperature and salinity on the effects of the two
larvicides on the population density of D. magna was checked using a three factor
negative binomial GLM. Two categories, ‘low’ and ‘high’, were defined for water
temperature and salinity values. Values inferior to the median values computed for
temperature or salinity for the whole study period were categorized as ‘low’,
whereas values superior to these median values were categorized as ‘high’.
Preliminary investigations showed that D. magna length data clearly exhibited
a non-normal right-skewed distribution and that neither log, nor square-root
transformation was able to normalize the data or homogenize the variances.
Therefore, mean length values computed for the various treatments were compared
at each sampling date using a Kruskal–Wallis non-parametric test followed by
a non-parametric multiple comparison post-hoc test. Length frequency distributions
were constructed by counting the relative number of individuals in successive
0.5 mm width classes. For each sampling date, values of the relative abundance of
a given length class were compared using a proportion comparison test. When the
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 ¼ 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
pH
Control
Bti 0.16 mL L1
Bti 0.50 mL L1
Spd 8 mg L1
Spd 17 mg L1
Spd 33 mg L1
7.8
7.9
7.9
7.9
7.9
7.8
0.10
0.05
0.06
0.04
0.04
0.04
8.4
8.6
8.5
8.9
9.0
9.1
0.07
0.10
0.06
0.08*
0.08**
0.06**
9.0
9.4
9.3
9.3
9.7
9.6
0.18
0.06
0.14
0.12
0.12**
0.10*
9.5
9.6
9.4
9.6
9.7
9.8
0.14
0.10
0.17
0.20
0.25
0.09
8.4
8.4
8.5
8.7
8.7
8.8
0.03
0.08
0.05
0.03*
0.06**
0.04***
[Dissolved O2] (mg L1)
Control
Bti 0.16 mL L1
Bti 0.50 mL L1
Spd 8 mg L1
Spd 17 mg L1
Spd 33 mg L1
5.3
5.9
6.2
5.0
5.6
4.9
0.4
0.4
0.3
0.3
0.5
0.4
6.1
7.4
7.3
8.1
10.1
9.1
0.3
0.2
0.2
0.9
0.8***
0.6**
8.8
9.9
9.4
9.3
10.7
9.9
0.3
0.2
0.3
0.4
0.3**
0.6
10.5
11.6
10.7
10.5
11.2
10.8
0.2
0.4
0.5
0.5
0.4
0.6
5.7
6.7
6.7
6.6
7.9
7.0
[Chlorophyll a] (mg L1)
Control
Bti 0.16 mL L1
Bti 0.50 mL L1
Spd 8 mg L1
Spd 17 mg L1
Spd 33 mg L1
64.8
48.3
52.7
35.6
51.5
37.9
19.2
3.5
6.8
9.6
12.8
11.2
49.0
60.8
60.0
85.4
77.5
92.5
5.6
3.1
3.9
12.9**
3.1*
10.5***
34.8
37.5
58.4
64.9
45.0
59.3
12.4
11.2
11.8
9.7
6.4
8.6
44.9
51.9
68.0
75.2
48.7
105.7
14.6
22.0
13.1
10.4
15.0
29.2
36.8
36.2
38.7
50.8
48.4
51.0
[Suspended Matter] (mg L1)
Control
Bti 0.16 mL L1
Bti 0.50 mL L1
Spd 8 mg L1
Spd 17 mg L1
Spd 33 mg L1
15.7
19.7
19.2
18.9
16.3
12.2
1.5
4.6
4.6
0.8
1.9
3.8
12.7
14.2
10.1
17.6
40.5
28.7
0.7
2.3
4.2
2.6
16.0*
6.9
19.8
22.2
21.8
36.1
57.0
40.3
3.6
3.2
4.1
5.8
21.6*
5.0*
32.0
31.2
46.2
87.7
87.0
110.2
4.3
4.7
7.9
44.9
44.1
24.2**
37.7
63.6
53.0
38.6
48.8
33.7
Day 2
Day 4
the control and for each treatment were compared using proportion comparison
tests for two values.
All tests were performed using R for Windows Version 2.9.0 (R foundation for
Statistical Computing). Significance was accepted at a ¼ 0.05 for all tests excepted
for proportion comparison tests for two values for which a Bonferroni correction
was applied based on the number of tests performed.
3. Results
3.1. Environmental parameters
Table 1 gives the mean values (SE) of the environmental
parameters which significantly varied between control and treated
enclosures at the different sampling dates in the different types of
microcosms. Only raw water temperature and square root-transformed chlorophyll a concentration data met the requirements of
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 differences were observed (p ¼ 0.27). Time also had a significant effect on
chlorophyll a concentration in water (two-way RM-ANOVA on
square root transformed data, p < 0.001). Concentrations of chlorophyll a increased in all the microcosms during the first 14 days of
the experiment, and then gradually decreased. A significant treatment effect on chlorophyll a concentration in water was shown on
Day 2 and 21 (one-way ANOVA on square root-transformed values,
p ¼ 0.0016 and p ¼ 0.0028, respectively). On both dates, chlorophyll
a concentration values were significantly higher in spinosadtreated microcosms than in the control systems, irrespective of
larvicide concentration (Dunnet’s post-hoc test).
No adequate data transformation method was found for the
other parameters. The results of Friedman’s test indicate a significant between-microcosms heterogeneity for all these parameters
(p < 0.001). Water level gradually decreased in all the microcosms
during the study (Fig. 2). No significant between-treatment difference was identified with the date-by-date analysis (Kruskal–Wallis
Day 7
Day 14
Day 21
8.5
8.7
8.7
9.1
9.4
9.5
0.07
0.10
0.10
0.05*
0.12**
0.11***
0.3
0.2
0.2
0.6
0.6
0.5
7.1
8.2
8.5
8.1
10.4
9.0
0.4
0.6
0.5
0.4
0.6***
0.6
6.0
7.4
3.3
3.1
4.1
9.2
15.1
26.2
30.5
41.1
50.4
42.6
5.4
6.4
2.9
7.4**
8.1***
5.1**
13.1
6.9
14.7
12.1
15.2
8.1
18.1
18.1
19.0
25.9
28.5
27.3
1.9
2.2
2.7
0.8
2.1**
1.2*
test). Water salinity increased from Day 0 to Day 7, and showed
further decline until the end of the experiment (Fig. 2). No significant between-treatment difference was shown by the date-bydate analysis (Kruskal–Wallis test).
A significant effect of treatment on suspended matter concentration values was observed on Day 2, 4, 7 and 21 (Kruskal–Wallis
test, p ¼ 0.021, p ¼ 0.017, p ¼ 0.020 and p ¼ 0.002, respectively).
Non-parametric multiple comparison tests showed that the values
were sometimes significantly higher in the microcosms treated
with the intermediate or high spinosad concentration, but no clear
pattern was observed. A significant effect of treatment on water pH
was shown on Day 2, 4, 14 and 21 (Kruskal–Wallis test, p ¼ 0.0004,
p ¼ 0.029, p ¼ 0.0003 and p ¼ 0.0002, respectively). Non-
25
24
Temperature (°C)
Day 0
23
22
21
20
19
0
3
6
9
12
15
18
21
Sampling date (day after treatment)
Fig. 1. Change in mean values (SE; n ¼ 5) of temperature (expressed in C) for all the
microcosms.
C. Duchet et al. / Environmental Pollution 158 (2010) 1825–1833
Water Level
Salinity
Table 2
Results of the fitting of a three factor negative binomial GLM model to the population density of Daphnia magna (Chi-square p values indicate the level of significance of the various factors in the model).
6
5
Factor
Chi-square p value
4
Treatment
Salinity
Temperature
Treatment Salinity
Treatment Temperature
Temperature Salinity
Treatment Salinity Temperature
2.7 1010
0.0048
2 108
0.0014
1.95 106
0.0002
0.0747
20
-1
Water level (cm)
25
Salinity (g.L )
1828
15
3
2,0
10
0
3
6
9
12
15
18
2
21
Sampling date (day after treatment)
Fig. 2. Change in mean values (SE; n ¼ 5) of water level (expressed in cm) and
salinity (expressed in g L1) for all the microcosms.
parametric multiple comparison tests showed that pH values were
significantly higher in spinosad-treated microcosms than in control
systems, with a clear positive concentration–effect relation. A
significant effect on dissolved oxygen concentration in water was
shown on Day 2, 4 and 21 (Kruskal–Wallis test, p ¼ 0.0031,
p ¼ 0.037 and p ¼ 0.011, respectively). Non-parametric multiple
comparison tests showed that dissolved oxygen concentration
values were significantly higher, as compared to control, on these
three dates in the microcosms treated with the intermediate spinosad concentration and in the microcosms treated with the
highest spinosad concentration on Day 2.
3.2. D. magna abundance and size
D. magna largely dominated the zooplankton community. Only
a few Simocephalus sp. individuals (n ¼ 17 for the whole study
period) were found in the samples. The mean values of D. magna
density just before the treatment were not statistically different as
Abundance of Daphnia magna
(number of individuals per liter)
9000
Control
Bti, 0.16 µL.L-1
-1
Bti, 0.50 µL.L
Spinosad, 8 µg.L-1
Spinosad, 17 µg.L-1
Spinosad, 33 µg.L-1
6000
3000
***
0
0
3
***
***
6
***
9
12
**
15
18
21
Sampling date (day after treatment)
Fig. 3. Change in mean values (þSE; n ¼ 5) of Daphnia magna abundance (expressed as
the number of individuals per litre) in the control microcosms, the microcosms treated
with Bti at 0.16 and 0.50 mL L1, and the microcosms treated with spinosad at 8, 17 and
33 mg L1. (Significantly different from control, Dunnet’s post-hoc test following
negative binomial GLM fitting: **: 0.01 > p > 0.001; ***: p < 0.001).
shown by GLM analysis (Fig. 3). Mean D. magna population density
increased in control and Bti-treated microcosms from the beginning of the experiment to Day 4, and then decreased until the end
of the experiment. A sharp decrease in mean D. magna density was
observed in the microcosms treated with spinosad, except on Day 7
when a peak of density was observed in the microcosms treated
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, respectively. No recovery was observed during the study in the microcosms treated with spinosad. Negative binomial GLM fitted to the
D. magna density data for the whole study period showed a significant overall effect of the treatments (p ¼ 1.5 109). All the tested
spinosad concentrations had a significant effect (Dunnet’s post-hoc
test; p ¼ 0.001, p < 0.001 and p < 0.001 for spinosad nominal
concentrations of 8, 17 and 33 mg L1, respectively). In contrast, no
effect of Bti treatments was shown although the p value was close
to the significance threshold for the highest Bti concentration
(Dunnet’s post-hoc test; p ¼ 0.991 and p ¼ 0.056 for Bti nominal
concentrations of 0.16 and 0.5 mL L1, respectively).
GLM analysis performed for each sampling date (Fig. 3) showed
that D. magna densities were not different between the treatments
before introduction of the larvicides (p ¼ 0.352) whereas significant
differences were continuously observed for all the subsequent
dates (all p values inferior to 0.001 from Day 2 to the end of the
study). Dunnet’s post-hoc test showed a negative effect (p < 0.001)
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
concentration on D. magna density was also observed (Dunnet’s
post-hoc test, p ¼ 0.003).
Negative binomial GLM including treatment, salinity and water
temperature as explanatory variables was fitted to the D. magna
density values (Table 2). The minimal adequate model contained
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.
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. Nonparametric multiple comparison tests showed that the length of
daphnids was slightly higher in treated than in control microcosms
before treatment. This is confirmed by the statistical analysis of
length class frequencies that showed a higher relative abundance of
small daphnids (body length comprised between 0.5 and 1 mm) in
control than in treated microcosms on Day 0. Accordingly, the
relative frequencies of medium-size daphnids (body length
C. Duchet et al. / Environmental Pollution 158 (2010) 1825–1833
Mean density of Daphnia magna
(Number of individuals per liter)
250
High temperature
Low temperature
200
150
significant reproduction occurred after Day 2. This is corroborated
by the changes with time of mean population density values
(Fig. 3). On Day 7 and 14, the frequency of daphnids measuring less
than 1.5 mm was higher in Bti-treated microcosms, suggesting
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 spinosad. This result should be considered cautiously because the
abundance of daphnids in these systems was very low after Day 4.
100
4. Discussion
50
0
Bti
Control
Bti
0.16 µL.L-1 0.50 µL.L-1
Spd
Spd
Spd
8 µg.L-1
17 µg.L-1
33 µg.L-1
Treatment
300
Mean density of Daphnia magna
(Number of individuals per liter)
1829
High salinity
Low salinity
250
200
150
100
50
0
Bti
Control
Bti
0.16 µL.L-1 0.50 µL.L-1
Spd
Spd
Spd
8 µg.L-1
17 µg.L-1
33 µg.L-1
Treatment
Fig. 4. Interaction plots between larvicide treatment and water temperature (upper
panel) or water salinity (lower panel), with the mean density of daphnids as the
dependent variable. Bti: Bacillus thuringiensis subspecies israelensis; Spd: spinosad.
comprised between 1.5 and 2.5 mm) were higher in the treated
microcosms. Between Day 0 and Day 2, there was an increase in the
frequency of small individuals (body length inferior to 1 mm) for all
treatments, suggesting that adults reproduced at the beginning of
the study period. In all the microcosms, including the controls,
mean body length of the sampled individuals increased with time,
and the shape of length frequency distributions suggests that no
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 Mediterranean climate (Comin and Valiela, 1993; Nuccio et al., 2003).
Hydroperiod duration has been identified as the main factor in
determining the faunal composition and structure of aquatic
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
water habitats requires specific physiological and life history traits
such as rapid development, migratory/colonizing ability (strong
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 parthenogenetic species capable of both asexual and sexual reproduction.
Their survival strategy in sexual reproduction is to produce sufficient 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
the most well-represented crustaceans in temporary ponds and
wetlands (Metge, 1986; Lake et al., 1989). As such, they constitute
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
various environmental parameters measured during the survey of
the microcosms were within the range of values usually found for
Mediterranean coastal wetlands (see Table 1), as shallow coastal
lagoon pH ranges from 7.9 to 8.2 (Dromgoole, 1978) except at the
end of summer when it can rise to 9 (Menéndez et al., 2001).
Furthermore, Munari et al. (2003) measured chlorophyll a concentrations close to 40 mg L1 and O2 concentrations less than 6 mg L1
in August in the Po River deltaic area (northern Italy). In our
microcosms, water level gradually decreased as a consequence of
the hydraulic management of the site where the study was performed. The wetland was artificially flooded on mid-July by water
pumped in the Petit-Rhône River to provide suitable habitats for
ducks (the wetland is used as a hunting site in autumn). Afterwards,
water level was maintained through regular inflow of water from
the river. Evaporation, which is intense in this area in summer,
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
Bti 0.16 mL L1
Bti 0.50 mL L1
Spinosad 8 mg L1
Spinosad 17 mg L1
Spinosad 33 mg L1
1.536 (0.026; 914)
1.629* (0.032; 628)
1.701*** (0.035; 529)
1.771*** (0.027; 641)
1.684*** (0.027; 853)
1.793*** (0.032; 540)
1.139 (0.017; 1692)
1.105 (0.019; 1390)
1.134 (0.027; 671)
0.880*** (0.049; 117)
1.083 (0.061; 114)
0.846 (0.068; 25)
1.063 (0.012; 2040)
1.020*** (0.011; 2382)
0.978*** (0.019; 724)
0.794* (0.057; 18)
0.604*** (0.061; 14)
0.751 (0.112; 5)
1.375 (0.019; 946)
1.368 (0.021; 736)
1.207*** (0.031; 343)
1.243*** (0.019; 648)
0.847* (0.202; 5)
–
1.834 (0.032; 346)
1.663*** (0.040; 306)
1.708** (0.040; 96)
1.965 (0.103; 3)
1.664 (NC; 1)
–
1.613 (0.118; 63)
2.284*** (0.097; 60)
1.939 (0.255; 9)
–
–
–
1830
C. Duchet et al. / Environmental Pollution 158 (2010) 1825–1833
1
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
*
* *
1
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
1
0.5
1.5
2
2.5
3
3.5
*
*
*
*
*
1
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
0.5
1
1.5
2
2.5
3
3.5
*
*
*
*
*
0
0.5
1
1.5
2
2.5
3
3.5
4
Length class (mm)
Control
Bti 0.16 µL.L-1
Bti 0.50 µL.L-1
*
0.5
1
*
1.5
*
*
2
**
**
2.5
**
*
3
3.5
4
3
3.5
4
3
3.5
4
Day 7
*
*
*
*
*
**
0
F
*
*
1
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
4
Day 14
Day 2
0
D
Day 4
*
1
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
4
Frequency
Frequency
Frequency
*
*
0
E
Frequency
*
**
0
C
B
Day 0
Frequency
Frequency
A
1
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
0.5
1
1.5
2
Day 21
*
2.5
*
*
*
0
0.5
1
1.5
2
2.5
Length class (mm)
Spinosad 8 µg.L-1
Spinosad 17 µg.L-1
Spinosad 33 µg.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.
Mean water salinity varied between 3.2 and 5.6 g L1 with a clear
unimodal pattern of change with time, and maximum values were
recorded on Day 4 and 7.
Both water temperature and salinity had a significant effect on
the mean abundance of D. magna in the microcosms. A peak in
D. magna density was observed in control and Bti-treated microcosms when water temperature was low but it is not clear whether
there is a direct relationship between these two parameters, or if
this is a temporal coincidence. D. magna has been shown to have
genetic ability to tolerate temperatures higher than those recorded
during this study (up to 25 C; Stephenson and Watts, 1984;
MacIsaac et al., 1985; Lagerspetz, 2000) and therefore changes of
population density with time are probably the result of natural
population dynamics in the study site. Statistical analysis of density
data showed an interaction between water temperature and spinosad effect, with higher density values at high temperature. Once
again, this result is more probably linked to a temporal correlation
between water temperature and population density rather than to
a causal relationship. Indeed, in spinosad-treated microcosms,
D. magna population density sharply decreased after the treatment
and never recovered. Therefore, the highest density values in these
C. Duchet et al. / Environmental Pollution 158 (2010) 1825–1833
systems were measured at the beginning of the experiment, when
water temperatures were also high.
Like other cladocerans, D. magna mostly inhabits freshwater
ecosystems. However, natural populations living in oligohaline to
mesohaline habitats also exist (Arnér and Koivisto, 1993). Schuytema et al. (1997) showed that D. magna could survive and reproduce well in water with salinity below 4 g L1. According to
Lagerspetz (1955 in Arnér and Koivisto, 1993), D. magna has been
found in brackish ponds up to 12.5 g L1 but its occurrence declines
abruptly when salinity was above 4 g L1. This is consistent with the
48-h LC50 of 5.48 g L1 for NaCl determined for D. magna by
Martinez-Jeronimo and Martinez-Jeronimo (2007). Several authors
have shown that salinity may have sublethal effects on Daphnia life
history traits. Cowgill and Milazzo (1991) experimentally demonstrated that reproduction, population growth rate, and survival of
D. magna are inversely related to salinity. Studying life history traits
of D. magna in laboratory experiments under freshwater and
brackish conditions, Teschner (1995) showed a negative effect of
salinity (5 g L1) on growth and reproduction, which was attributed
to an alteration of the molt cycle. Martinez-Jeronimo and MartinezJeronimo (2007) also demonstrated that average lifespan, life
expectancy at birth, longevity, average clutch size, total progeny,
number of clutches, net reproductive rate and intrinsic rate of
population growth were significantly reduced by an increasing
NaCl concentration. This is consistent with the results obtained in
field studies by Green et al. (2005) who concluded that although
D. magna may be considered as a euryhaline species which is able to
colonize brackish water environments, its reproductive and/or
survival rates are reduced at higher water salinity. In the present
study, a significant effect of water salinity on D. magna population
density was observed. Results presented in Fig. 4 suggest that there
was a positive effect of high salinity values in the control microcosms. This may reflect the fact that the peak of salinity occurred
just after a reproduction period in daphnids. Therefore, the possible
negative effect of high salinity on D. magna density was probably
delayed. This could explain why there was a subsequent decrease in
population density and no reproduction even in control microcosms during the rest of the experiment.
D. magna survival was affected by all the spinosad treatments. In
the microcosms treated with 8 mg L1 spinosad, D. magna density
measured within the first four days of exposure was significantly
less than the control but a peak of density was observed on Day 7,
suggesting that the exposed population began to recover from the
exposure to the larvicide. However, recovery was not confirmed by
the data obtained later in the study. At 17 and 33 mg L1, spinosad
affected the whole population and did not selectively impact
a particular size class. No recovery of the D. magna population
occurred in the microcosms treated with these spinosad concentrations. Chronic spinosad NOEC values for daphnids have been
estimated at 6.7 and 8 mg L1 in static and semi-static laboratory
tests, respectively (National Registration Authority for Agricultural
and veterinary Chemicals, 1998; WHO, 2007). In the present study,
8 mg L1 spinosad produced a significant mortality in D. magna, and
recovery was not observed whereas in a comparable field study, D.
pulex recovered after the first week following a treatment at the
same concentration (Duchet et al., 2008). There was a significant
interaction between spinosad treatment and salinity (Fig. 4). This
may be due to the high mortality caused by the treatment, in
combination with the increase of water salinity. Such an interaction
between salinity and pesticide toxicity has already been demonstrated on copepods by Hall et al. (1994) and Staton et al. (2002).
Any factor that disrupts the physiological integrity of exposed
organisms will induce defence and repair mechanisms, which
depend on energy-requiring processes such as active transport (e.g.,
exclusion of chemical stressors) and synthetic activity (e.g.,
1831
synthesis of stress proteins). As such, combating stress is likely to be
energetically costly for stressed organisms (Calow, 1991; De Coen
and Janssen, 2003). Stress will increase the energy expenditure of
organisms and, as a consequence, the energy status of an organism
at any given time will affect its capacity to cope with additional
stress. Consequently, animals with a high energy status (organisms
living in non-stressful environment) are more successful in dealing
with anthropogenic stress than animals with a low energy status
(organisms living in stressful conditions) (Smolders et al., 2005). In
the present study, the energetic cost of spinosad metabolisation and
exclusion may have reduced the energy available for osmoregulation of daphnids and affected their capacity to cope with salinity
stress, leading to limited reproduction and growth, and ultimately
to death. This could explain the absence of recovery in the microcosms treated with spinosad at 8 mg L1.
The between-microcosm heterogeneity detected by Friedman’s
test indicates that each test system exhibited its own dynamics.
Nevertheless, the combination of this test performed for the whole
study period with a date-by-date analysis allowed identifying the
effects of the treatments on some environmental parameters.
Significant differences in water pH, dissolved oxygen and chlorophyll a concentration were frequently observed between control
and spinosad-treated microcosms, with higher values in the
treated systems. These differences are probably due to an indirect
effect of the larvicide associated with the drastic decrease in D.
magna population density in the spinosad-treated microcosms.
Disappearance of daphnids contributed to decrease the grazing
pressure on phytoplankton resulting in an increase of algal
biomass leading to a subsequent increase in water pH and dissolved oxygen concentration through an enhancement of photosynthesis (Axelsson, 1988; Shiraiwa et al., 1993; Rigobello-Masini
et al., 2003). Such effects have frequently been reported as indirect
consequences of insecticide treatment in aquatic ecosystems
(Caquet et al., 1992; Fleeger et al., 2003; Hanson et al., 2007). A
reduction of dissolved oxygen consumption directly associated
with daphnid disappearance cannot be excluded, although its
contribution to the difference between control and spinosadtreated microcosms is probably far less important than the indirect effect associated with phytoplankton bloom. The absence of
significant differences for pH, dissolved oxygen and chlorophyll
a concentration on Day 4 and 7 may be a consequence of the stress
on the phytoplankton community induced by the increase in
water salinity. A salinity of ca. 5 g L1 forms a lethal barrier for
most planktonic algae living in brackish waters (Flöder and Burns,
2004). At this salinity level, freshwater and marine species exhibit
a severe osmotic stress (Kies, 1997) and planktonic species diversity is minimal (Flöder and Burns, 2004). Therefore, the increase in
water salinity may have temporarily hampered the development
of planktonic algae in spinosad-treated microcosms. Once the
salinity has decreased to its original value, algae were again able to
proliferate.
Effects of spinosad on D. magna were compared to those of Bti,
applied at 0.16 and 0.50 mL L1. Bti treatments did not have any
effect on the various environmental parameters measured during
the study period, and it had almost no effect on D. magna survival
(the only significant effect was observed at Day 21 for the highest
Bti concentration) confirming a number of previous studies (Boisvert and Boisvert, 2000; Lacoursière and Boisvert, 2004). In
particular, Ali (1981) and Miura et al. (1981) showed that Ephemeroptera, Amphipoda, Copepoda and Cladocera were not affected
by Bti. Results concerning body length (Table 3) did not allow
concluding about the effect of Bti on the size of D. magna, as there
was heterogeneity in individual sizes before treatment. However, in
a previous field study (Duchet et al., 2008), no effect of Bti on the
size structure of D. pulex was observed.
1832
C. Duchet et al. / Environmental Pollution 158 (2010) 1825–1833
5. Conclusion
Unlike Bti, spinosad had a strong lethal effect on D. magna
population at presumed recommended rates for field application.
Our results suggest that it may be difficult for a natural daphnid
population to cope simultaneously with natural (water salinity and
temperature) and anthropogenic (larvicides) stressors, and this
may directly affect the recovery potential of the population. The
experiment also allowed observing indirect effects of spinosad, in
particular an increase of phytoplankton abundance and photosynthetic activity (as indicated by elevated pH and dissolved oxygen
concentration) due to the decreasing abundance of primary
consumers.
Acknowledgements
Financial support for this work was provided by the French
Ministry for Ecology, Sustainable Development and Spatial Planning through the National Programme for Ecotoxicology (PNETOX).
The authors wish to thank Dow AgroSciences for the generous gift
of spinosad 120SC and Mr. Girand for giving access to the study site.
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