i An update to this article is included at the end

Science of the Total Environment 502 (2015) 91–102

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Science of the Total Environment

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Biomarkers and energy reserves in the isopod Porcellionides pruinosus:

The effects of long-term exposure to dimethoate
Nuno G.C. Ferreira ⁎, Rui Morgado, Miguel J.G. Santos, Amadeu M.V.M. Soares, Susana Loureiro ⁎
Department of Biology & CESAM, University of Aveiro, 3810-193 Aveiro, Portugal

H I G H L I G H T S

• Dimethoate field application dose induced low to moderate toxicity to isopods.

• Dimethoate also generates oxidative stress leading to high lipid peroxidation rates.
• Terrestrial isopods increase dimethoate’s degradation rates on soils.

a r t i c l e i n f o a b s t r a c t

Article history: Terrestrial isopods from the species Porcellionides pruinosus were exposed to the recommended field dose appli-
Received 19 March 2014 cation (0.4 mg/kg soil) and a sublethal concentration (10 mg/kg soil) of dimethoate at two temperatures that can
Received in revised form 13 August 2014 be generally found in several countries (20 °C and 25 °C) and are commonly used as reference temperatures. The
Accepted 19 August 2014
organisms were exposed for 28 days and sampled at the following time points: 24 h, 48 h, 96 h, 7 days, 14 days,
Available online 20 September 2014
21 days, 28 days; organisms were then changed to clean soil for a recovery period of 14 days during which organ-
Editor: Mark Hanson isms were sampled on day 35 and 42. For each sampling time, the enzyme activities of acetylcholinesterase
(AChE), glutathione-S-transferases (GST), catalase (CAT), lactate dehydrogenase (LDH) were determined as
Keywords: well as the following: total lipid, carbohydrate and protein content; energy available (Ea); energy consumption
Integrated biomarker response (Ec); cellular energy allocation (CEA) and lipid peroxidation rate (LPO). The integrated biomarker response (IBR)
Oxidative stress was calculated for each sampling time and for each of the above parameters. Mortality was also recorded during
Neurotoxicity the study.
Combined effects The results obtained showed that dimethoate causes toxicity by several mechanisms. This study found evidence
Temperature changes
for the inhibition of the acetylcholinesterase enzyme, which has been previously reported, and also evidence of
oxidative stress, which altered the levels of GST, CAT or LPO. In addition, the study showed that the two concen-
trations used of dimethoate caused the activation of different general detoxification mechanisms, and also that
the same concentration at different temperatures induced different toxicity responses.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction on the acquisition of a new homeostasis status (Morgan et al., 1999),

or they just do not represent any major life-changing effects. Neverthe-
The potential impact of a stressor in ecosystems requires the obser- less, they can provide crucial information on stressors’ modes of action,
vation of effects at different levels of biological organization, starting at which improve the knowledge on their related effects. Depletion of
the molecular level and ending at the population/community level energy reserves and energy metabolic costs can be used as another
(Moore et al., 2004). At a molecular level, several biomarkers have parameter to evaluate chemical exposure effects. Due to the stress
been used as efficient tools due to their sensitivity, quickness and accu- induced by xenobiotics, metabolic changes can induce the depletion
rate relationship between toxicant exposure and respective biological of energy reserves especially under long time exposures, negatively
response (Morgan et al., 1999). However, results from molecular affecting individuals’ growth or reproduction, and finally impairing
approaches may have limited information if they are not related to population dynamics and structures (de Coen and Janssen, 2003).
higher and more complex biological levels of organization. Indeed, the In the soil compartment, organisms play an important role on de-
effects at lower organizational levels may not necessarily be observed composition and fragmentation processes, and their exposure to xeno-
or be meaningful at superior biological levels whenever they consist biotics may change overall soil functions, causing a decrease in soil
quality and soil services (MEA, 2005). There is a wide range of xenobi-
⁎ Corresponding authors. Tel.: +351 234 370 350; Fax: 234 372 587. otics that can appear in the terrestrial compartment. Organophospho-
E-mail addresses: nunoferreira@ua.pt (N.G.C. Ferreira), sloureiro@ua.pt (S. Loureiro). rous compounds (OP) are one of the most extensively used pesticides

http://dx.doi.org/10.1016/j.scitotenv.2014.08.062
0048-9697/© 2014 Elsevier B.V. All rights reserved.
92 N.G.C. Ferreira et al. / Science of the Total Environment 502 (2015) 91–102

in agriculture practices. One of the most commonly applied is dimetho- cultures. In culture, isopods were fed ad libitum with alder leaves
ate, which successfully combines a selective toxicity to insects through a (Alnus glutinosa) and maintained at two different temperatures (20 ±
systemic action. By acting on the enzyme AChE, this pesticide inhibits 1 °C and 25 ± 1 °C), with a 16:8 h (light:dark) photoperiod. Twice a
the degradation of acetylcholine thereby producing extensive choliner- week cultures were sprayed with water and food provided. Only adult
gic stimulation and neurotoxicity (de Coen and Janssen, 2003). When organisms (15–25 mg wet weight) were used in the experiments and
looking at xenobiotics’ exposures, the overall conditions of exposure no distinction between sexes was made, although pregnant females
can also bring additional stress or induce changes in physiological re- were excluded.
sponses of organisms when they are dealing with chemical exposure.
Terrestrial isopods are macrodecomposers that play an important 2.2. Soil spiking
role in the decomposition processes, vegetal litter fragmentation and
re-cycling process of nutrients (Ferreira et al., 2010; Loureiro et al., LUFA 2.2 soil (Speyer, Germany) was spiked with two different
2006; Zimmer, 2002; Zimmer et al., 2003). The terrestrial isopod species concentrations of dimethoate (0.4 and 10 mg dimethoate/kg soil),
Porcellionides pruinosus has been described as a good test-organism to with a final moisture content equivalent to 50% of the soil water holding
evaluate soil contamination or other environmental changes in their capacity. The concentration of 0.4 mg dimethoate/kg soil represents
habitat (Jansch et al., 2005; Loureiro et al., 2005, 2009; Takeda, 1980; the recommended field dose for dimethoate application (TitanAG,
Vink et al., 1995). Several individual parameters have been chosen as 2010) and the 10 mg dimethoate/kg soil was used based on the study
indicators of isopod health status but also as parameters tightly related by Fischer et al. (1997) that found in LUFA 2.2 soil for the isopod
to their function in soils. Feeding activities, including excretion rates, Porcellio scaber, a EC50 value of 17.5 mg dimethoate/kg soil for growth;
reproduction, growth and behaviour are amongst the most used param- 16.8 mg dimethoate/kg soil for mancae/surviving females and 15.4 mg
eters in isopods ecotoxicological tests. Along with these, the use of dimethoate/kg soil for pregnant/surviving females. Therefore, the con-
neurotoxicological (acetylcholinesterase), detoxification (glutathione centration of 10 mg dimethoate/kg soil was chosen as a sublethal value.
S-transferases), oxidative stress (catalase, glutathione peroxidase, and
lipid peroxidation), energy related (lactate dehydrogenase) biomarkers’ 2.3. Experimental procedure
basal activities and energy reserves (total lipid, carbohydrate and pro-
tein content) can be used as good evaluation tools that will provide use- Toxicity tests were performed in plastic boxes (26 length × 18 width ×
ful information and a connection between these two ecological levels. In 7.5 height cm), containing approx. 2 cm height of natural LUFA 2.2 soil
previous studies, these biomarkers have been used to determine the (Speyer, Germany) and 40 isopods (per box). Test organisms were collect
basal levels on organisms from well-established lab cultures (Ferreira from culture boxes, weighted (15–25 mg) and placed in each test-box.
et al., 2010) and they have also shown to respond to the short term Organisms with abnormalities, moulting characteristics or pregnant
exposure of pesticides (e.g. Jemec et al., 2009, 2012). females were excluded from trial. Although food was provided ad libitum
Time of exposure is one of the key factors to improve ecological rel- in the form of alder leaf disks (Ø 10 mm), it was made available consider-
evance. In isopod bioassays, tests are usually carried out for 48 h, when ing also that a soil coverage by leaves could influence isopods’ exposure to
considering the avoidance behaviour test (Loureiro et al., 2002), 14 or the spiked soil. Therefore, food was added in small quantities but contin-
28 days when regarding feeding inhibition tests (Loureiro et al., 2006), uously reintroduced throughout the test period. Organisms were exposed
14 days for survival (Calhôa et al., 2012; Santos et al., 2010), 21 days to 0.4 and 10 mg dimethoate/kg soil in a 16:8 h (light:dark) photoperiod,
for bioaccumulation tests (Sousa et al., 2000), or more than one at two different temperatures: 20 °C and 25 °C. Both temperatures are
month when reproduction is being evaluated (Calhôa et al., 2012). relevant temperatures for Mediterranean countries, and can be found
Long term exposure tests are advisable when a more comprehensive elsewhere during a year time and are widely used in ecotoxicological
approach is required in order to integrate chemical fate and changes assays with several species of terrestrial isopods (Calhôa et al., 2012;
in bioavailability with time, but also to consider the ability of organisms Dailey et al., 2009; Loureiro et al., 2006; Morgado et al., 2013; Ribeiro
to recover when exposure ends. et al., 1999; Santos et al., 2011).
Therefore, the main goal of this study was to evaluate and under- A total of five replicates were performed for each concentration and
stand the long-term effects of dimethoate using several enzymatic bio- temperature. Four organisms from each box/replicate were collected at
markers and energy reserves in the terrestrial isopod Porcellionides the following time points: 0 h, 24 h, 48 h, 96 h, 7 days, 14 days, 21 days,
pruinosus. Organisms were exposed to two dimethoate concentrations 28 days (exposure period) and 35 days, 42 days (recovery period). In
(a recommended field dose application and a concentration below the results section, the 35 and 42 days of test duration will be
EC50 level) and two different exposure temperatures (20 °C and denominated as 7 and 14 days of post-exposure.
25 °C) during a 28 day exposure period followed by a 14 day recovery The enzymatic biomarkers glutathione S-transferases (GST), gluta-
period. The results were then combined using the integrated biomarker thione peroxidase (GPx), catalase (CAT) and lipid peroxidation (LPO)
response index (IBR). were measured using a pool of two full-body organisms per replicate.
The mode of action (MoA) analysis of dimethoate to the terrestrial Another organism was divided into head and body to analyse acetylcho-
isopod P. pruinosus was determined in several ways: 1) the toxicity linesterase (AChE) and lactate dehydrogenase (LDH) activity, respec-
and inherent effects of two dimethoate concentrations; 2) the toxicity tively, each part corresponding also to a replicate. All chemicals used
effect of dimethoate combined with temperature; 3) response patterns in these experiments were obtained from Sigma-Aldrich Europe, except
at the different times of exposure and 4) differences between the expo- the Bradford reagent, which was purchased from Bio-Rad (Germany),
sure and recovery period. Within this approach, dimethoate degradation and hydrogen peroxide from Fluka.
in soils was also integrated in the results in the presence or absence of For the energy reserves (total lipid, carbohydrate and protein
terrestrial isopods. content) and electron transport system, only one organism was used
as a replicate.
2. Materials and methods At each sampling time, the number of dead organisms were recorded
and removed from the test boxes.
2.1. Test organism and culture procedure
2.4. Measured parameters and IBR
The organisms used in this study belong to the species Porcellionides
pruinosus Brandt (1833), and were previously collected from a horse The protocol used to process samples was previously described by
manure heap and maintained for several generations in laboratory Ferreira et al. (2010) and is extensively described in the supplementary
 N.G.C. Ferreira et al. / Science of the Total Environment 502 (2015) 91–102 93

data. The lipid peroxidation (LPO) assay was adapted from the methods and Janssen (1997). The Ea, Ec and CEA value were calculated as
described by Bird and Draper (1984) and Ohkawa et al. (1979) to a mi- described by Verslycke et al. (2004):
croplate format. The glutathione S-transferases (GST) and glutathione
peroxidase (GPx) activities were determined as described by Habig et al. Eaðavailable energyÞ ¼ carbohydrates þ lipids þ proteinsðmJ=mg org:Þ
(1974) and Mohandas et al. (1984), respectively. Catalase (CAT) activity Ecðenergy consumptionÞ ¼ ETS activityðmJ=mg org:=hÞ
was determined based on the method described by Clairborne (1985) CEAðcellular energy allocationÞ ¼ Ea=Ecð=hÞ
and adapted to a microplate format. Lactate dehydrogenase (LDH) activ-
ities were measured using the method described by Vassault (1983), To integrate results from the different biomarkers and understand
adapted to microplate format by Diamantino et al. (2001) and the acetyl- global/general responses, the integrated biomarker response (IBR)
cholinesterase (AChE) activity according to the Ellman method (Ellman was calculated according to Beliaeff and Burgeot (2002); details can
et al., 1961), adapted to microplate format described by Guilhermino also be found in the supplementary data.
et al. (1996). For all biomarkers, protein concentration was determined
according to the Bradford method (Bradford, 1976), adapted from 2.5. Chemical analysis
BioRad's Bradford micro-assay set up in a 96 well flat bottom plate,
using bovine γ-globuline as a standard. Determination of total dimethoate concentration per kilogram of
Considering energy parameters, to determine total protein, car- soil was performed by the Marchwood Scientific Services Ltd. The
bohydrate and lipid contents, oxygen consumption rate in the electron lower detection limit for dimethoate was 0.4 μg/kg soil. The method
transport system (energy consumption - Ec), energy available (Ea) and used to analyse soil spiked with dimethoate involved air drying and
cellular energy allocation (CEA) protocols were adapted from de Coen grinding the samples. Then 0.5 gram of sample was used for extraction

Fig. 1. Cumulative total number of dead organism from the species Porcellionides
pruinosus during the exposure and recovery period in the control and exposed to Fig. 2. Dimethoate decay curve for soil spiked with 0.4 mg dimethoate/kg soil (A) and
0.4 mg dimethoate/kg soil and 10 mg dimethoate/kg soil. A total of 200 organisms were 10 mg dimethoate/kg soil (B) at 25 °C exposure in the presence and absence of terrestrial
exposed per treatment. A – organisms exposed to 20 °C, B – organisms exposed to 25 °C. isopods.
94 N.G.C. Ferreira et al. / Science of the Total Environment 502 (2015) 91–102

with acidified acetonitrile. The sample was then filtered and the filtrate 3. Results
used for analysis by Liquid Chromatography-Tandem Mass Spectrome-
try following a pre-treatment buffering stage. The instrument used for Organisms exposed to dimethoate under the two different tempera-
the analysis was an Agilent 6410 Triple quad LCMS-MS. Standards ture regimes (20 °C and 25 °C) showed different mortality patterns. In
were prepared in solvents at 7 levels with recoveries in the range of Fig. 1 it is presented the number of dead organisms and the cumulative
80–120%. The water sample analysis passed for all the samples except number at a given sampling time. In the 20 °C exposure regime, due to
the drying and grinding stages. the mortality observed at the highest concentration, on the 14th day of
Soil samples analysed consisted of a pool of 5 soil replicates for each recovery it was not possible to analyse the energy related parameters
concentration used to expose isopods. Similarly, soil spiked with the (proteins, lipids, carbohydrates, Ec, Ea and CEA). Similarly in the 25 °C
same concentrations of dimethoate but with no isopods was also sam- exposure regime, the high mortality observed for the highest concen-
pled. Samples were taken at the beginning of the exposure, and at tration did not allow the analysis of the energy related parameters
days 7, 14, 21 and 28. (proteins, lipids, carbohydrates, Ec, Ea and CEA) for the 14th day of
exposure and all parameters forward.
The decay rate for the lower and higher concentrations of dimetho-
2.6. Data analysis ate in soil at 20 °C with isopods present was respectively 0.32/d (y =
0.4e− 0.3216x; r 2 = 0.9994) and 0.13/d (y = 9.9e− 0.1277x). For the
A one-way analysis of variance (ANOVA) or a Student test (t test) exposure at 20 °C where no isopods were present, no data is available
was performed to compare differences between treatments at each due to analytical constraints.
sampling time and Dunnett’s comparison test was carried out to dis- The decay rates of dimethoate in soil at 25 °C are presented in Fig. 2.
criminate statistically different treatments from the control (SPSS, At the lower concentration, the decay rates for soil with and without
1999). When possible, data transformation was used to achieve normal- isopods were respectively 0.37/day (y = 0.4e−0.3665x r 2 = 0.9987)
ity. When data did not show a normal distribution or homoscedasticity, and 0.33/day (y = 0.4e−0.3259x; r 2 = 1.0000). For the higher con-
the non-parametric test Kruskal-Wallis One Way Analysis of Variance centration, those rates were 0.29/day (y = 10e−0.2921x; r2 = 1.0000)
on Ranks was used. and 0.13/day (y = 9.9e−0.1315x; r 2 = 0.9903), for soil with and without
Data values that were higher or lower than the mean value, plus or isopods.
minus two times the standard deviation, were considered outliers, and
withdrawn from analysis (Rousseeuw and Croux, 1993). Whenever
there was enough data (n N 3, due to high mortality rates), a two-way 3.1. Biomarkers activity and energy reserve content
analysis of variance (two-way ANOVA) was performed to check for in-
teractions between time and concentration. The two-way ANOVA was The activity of the biomarkers and energy reserves of organisms
performed separately for the exposure and the recovery period. The exposed at 20 °C and 25 °C during the exposure and the recovery period
one-way ANOVA and two-way ANOVA with significance of α = 0.05. is presented in Figs. 1SD and 2SD (suppplentarry data) and in Table 1.
Due to the mortality observed within each temperature, under 20 °C a Details relative to the significant differences found between treatments
two-way ANOVA could only be performed for the exposure period and control are presented as supplementary data (Table 2SD).
and for the 25 °C exposure for the recovery period. The main target enzyme of the pesticide dimethoate (AChE) showed
Dimethoate decay on time was calculated using non-linear regres- significant differences mainly at the higher concentration (10 mg di-
sion curves at the two different temperatures, with and without the methoate/kg soil) for both temperatures. The lower exposure concen-
presence of isopods, and calculated as an exponential single decay tration only showed significant differences at 20 °C after 48 h of


curve with 2 parameters C t ¼ C 0 e−K 0 t , where Ct is the dimethoate exposure. Regarding the oxidative stress related biomarkers (LPO,
concentration in soil (mg/kg soil), C0 is the initial concentration of GST, CAT and GPx), significant differences were mainly observed at
dimethoate in soil, K0 the decay rate of dimethoate in soil (/day), and t 25 °C exposure and after 48 h/96 h of exposure or during the recovery
the time (days). period (more evident at the higher exposure concentration). Finally

Table 1
Significant differences found for biomarkers and energy reserves between control organisms from the species Porcellionides pruinosus and those exposed to dimethoate at 20 °C and 25 °C.
Data refers to a 28 day exposure period followed by a 14 day recovery period. Red boxes denote deleterious effects, green boxes denote positive effects and grey boxes represent parameters
that could not be measure due to the lack of organisms. ↗ denotes significant increase, ↘ denotes significant decrease; one-way ANOVA, ANOVA on ranks or Student’s t-test, p ≤ 0.05.

AChE GST LPO CAT LDH Lipids Carbohydr. Proteins Ea Ec CEA

20oC 25oC 20oC 25oC 20oC 25oC 20oC 25oC 20oC 25oC 20oC 25oC 20oC 25oC 20oC 25oC 20oC 25oC 20oC 25oC 20oC 25oC
0.4mg/kg
24h
10mg/kg
0.4mg/kg
48h
10mg/kg
0.4mg/kg
96h
10mg/kg
0.4mg/kg
Exposure 7d
10mg/kg
0.4mg/kg
14d
10mg/kg
0.4mg/kg
21d
10mg/kg
0.4mg/kg
28d
10mg/kg
0.4mg/kg
7d
10mg/kg
Post-exposure
0.4mg/kg
14d
10mg/kg
 N.G.C. Ferreira et al. / Science of the Total Environment 502 (2015) 91–102
Exposure/Recovery: 20ºC

Fig. 3. Star plots for each sampling time of Porcellionides pruinosus exposed to 20 °C (exposure period: 0 h, 24 h, 48 h, 96 h, 7 days, 14 days, 21 days, 28 days; recovery period: 35 days, 42 days). AChE = acetylcholinesterase, GST = glutathione S-
transferases, LPO = lipid peroxidation, CAT = catalase, LDH = lactate dehydrogenase, Ea = available energy, Ec = energy consumption, CEA = cellular energy allocation.

95
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N.G.C. Ferreira et al. / Science of the Total Environment 502 (2015) 91–102
Exposure/Recovery: 25ºC

Fig. 4. Star plots for each sampling time of Porcellionides pruinosus exposed to 25 °C (exposure period: 0 h, 24 h, 48 h, 96 h, 7 days, 14 days, 21 days, 28 days; recovery period: 35 days, 42 days). AChE = acetylcholinesterase, GST = glutatione S-
transferases, LPO = lipid peroxidation, CAT = catalase, LDH = lactate dehydrogenase, Ea = available energy, Ec = energy consumption, CEA = cellular energy allocation.
 N.G.C. Ferreira et al. / Science of the Total Environment 502 (2015) 91–102 97

Fig. 5. Integrated biomarker response(IBR) represented by starplot and histogram of Porcellionides pruinosus in the control and exposed to dimethoate (0.4 and 10 mg/kg soil) at 20 °C.
Exposure period: 0 h, 24 h, 48 h, 96 h, 7 days, 14 days, 21 days, 28 days; recovery period: 35 days, 42 days.

the energy related enzyme LDH presented significant differences at lower and higher values) obtained for each parameter are summarized
20 °C for both concentrations during the 7/14 days of exposure. in Table 1SD (supplementary data).
The significant differences observed at 25 °C on the energy related
parameters were mainly found after 96 h of exposure and mostly
consisted on positive effects (except for the energy consumption – 3.2.1. Exposure 20 °C: IBR and IBR/n analysis
Ec), whereas for 20 °C negative effects were registered, in particular The IBR analysis showed frequently worse scores (higher values) for
after 21 days of exposure (except for CEA after 48 h and 96 h of the highest concentration (10 mg dimethoate/kg soil) than the other
exposure). treatments (Figs. 5 and 6). When analysing the IBR according to the
At 20 °C, significant interactions between time of exposure and sampling time (Fig. 5), one can see that only at sampling times 24 h
dimethoate concentrations were observed for the energy consumption and 28 days of exposure this situation did not occur. Moreover, apart
(Ec), energy available and CEA in the exposure period (two-way from day 7 and day 28 of exposure, the lower exposure concentration
ANOVA, ln transformation, F14,75 = 2.967; p = 0.001; two-way (0.4 mg dimethoate/kg soil) showed always worse scores than the con-
ANOVA, ln transformation, F14,79 = 2.355; p = 0.009 and two-way trol. When changed to clean soil for recovery this effect disappears and
ANOVA, F14,68 = 3.586; p b 0.001, respectively) and for catalase in the the lower concentration always presented better scores than the con-
recovery period (two-way ANOVA, F4,24 = 5.180; p = 0.004). trol. The absence of an IBR for the highest concentration at day 14 of
At 25 °C, significant interactions between time of exposure and the recovery phase is due to the aforementioned lack of organisms to
dimethoate concentrations were only observed in the recovery period measure the complete set of parameters used in the remaining sam-
for LPO, GST, LDH and lipids content (respectively two-way ANOVA, pling times (high mortality). Organisms in the control treatment always
F2,20 = 5.727; p = 0.011; two-way ANOVA, F2,24 = 5.727; p = 0.009; showed better scores except for the 14th day of recovery. A statistical
Two Way ANOVA, F2,22 = 9.343; p = 0.001 and two-way ANOVA, analysis showed that only the highest concentration (10 mg dimetho-
F2,21 = 3.959; p = 0.035). ate/kg soil) is significantly different from the control (one-way ANOVA,
F2,26 =3.678; p = 0.039).
When analysing the parameters individually, organisms in the
3.2. Integrated biomarker response (IBR) control treatment always showed better scores, except for LPO and
CEA. For the lower concentration the parameters that exhibit the highest
The IBR starplot is presented in Figs. 3 and 4, and includes the scores toxicity when compared to the control were energy consumption (Ec –
of each measured parameter and each sampling time during the expo- 11.3× higher), GST (9.6× higher) and protein content (4.7× higher). For
sure and post-exposure period. Better or worse scores (respectively the highest concentration of exposure (10 mg dimethoate/kg soil),
98 N.G.C. Ferreira et al. / Science of the Total Environment 502 (2015) 91–102

Fig. 6. Integrated biomarker response represented by starplot and histogram of Porcellionides pruinosus in control and exposed to dimethoate (0.4 and 10 mg/kg soil) at 20 °C during the
exposure and recovery period. AChE = acetylcholinesterase, GST = glutathione S-transferases, LPO = lipid peroxidation, CAT = catalase, LDH = lactate dehydrogenase, Ea = available
energy, Ec = energy consumption, CEA = cellular energy allocation.

higher toxicity was observed for the energy consumption (Ec – 21.1× pathways are triggered by stressors, singly applied or as mixtures
higher), AChE (16.7× higher) and GST (13.4× higher). (e.g. Santos et al., 2010, 2011; Jemec et al., 2009, 2012; Stanek et al.,
2006). In these studies, the number of biomarkers used is normally
3.2.2. Exposure 25 °C: IBR and IBR/n high so the interpretation and integration of all their effects is difficult
As in the 20 °C exposure, the IBR analysis showed always worse and further consequences for the organism are hard to determine.
scores for the highest concentration used (10 mg dimethoate/kg soil) This analysis brings several problems in terms of interpretation, since
than the other treatments (Figs. 7 and 8). Also and except for the 24 h nonexistence of significant differences to the control in one or several
and day 21 of exposure the lower exposure concentration (0.4 mg di- biomarkers may still be indicative of stress. In fact, an organism that
methoate/kg soil) showed always worse scores than the control, show- does not show significant alterations from the control in all biomarkers
ing again a deterioration effect that increases with the exposure from a specific pathway may be in a similar state to an organism that
concentration (Fig. 7). But, contrary to the 20 °C exposure, the change shows a significant difference in only one of the biomarkers. The use
of organisms to clean soil for recovery did not show any positive effect. of IBR for a laboratory exposure and not as a tool to evaluate a specific
As it can be seen in Fig. 7, the IBR for the highest dimethoate concentra- field scenario has only been reported by Morgado et al. (2013). In the
tion could not be assessed for sampling times from day 14 onwards of present study, the use of star-plots helped to identify patterns of toxicity
the exposure phase due to the increased mortality found at 25 °C. The that were not so clear when significant differences were analysed. And
statistical analysis also showed that only the highest concentration it proved to be a robust tool, as it could identify significant results be-
(10 mg dimethoate/kg soil) is significantly different from the control tween control and exposures in almost all cases, except one (CEA,
(Kruskal-Wallis, H =8.348; d.f. = 2; p = 0.015). 24 h, 0.4 mg dimethoate/kg soil, 25 °C) representing 0.30% of total
For the lower concentration the parameters that exhibit the comparisons. An example of this was the AChE activity in organisms
highest toxicity when compared to the control were CAT (11.0 × exposed to the lowest concentration at 20 °C. When looking only to sig-
higher), GST (7.4 × higher) and LDH (6.7 × higher) (Fig. 8). For the nificant differences between the treatment and control, organisms may
highest concentration of exposure, the parameters that exhibits the be considered in “good conditions”, but when analysing the IBR results
highest toxicity were CAT (11.5 × higher), AChE (10.8 × higher) and an inherent toxicity pattern was observed.
LDH (7.6 × higher). In our results, one should highlight that organisms exposed to the
highest concentration used (10 mg dimethoate/kg soil) showed a
4. Discussion higher mortality at 25 °C. For the 20 °C exposure, mortality was not so
high and therefore almost all sampling times could be fulfilled; in addi-
The use of biomarkers to assess toxicity has already been used for tion the observed mortality was very similar to the one found for the
a large number of species, scenarios and as a tool to determine which lower concentration (0.4 mg dimethoate/kg soil). These differences
 N.G.C. Ferreira et al. / Science of the Total Environment 502 (2015) 91–102 99

Fig. 7. Integrated biomarker response(IBR) represented by starplot and histogram of Porcellionides pruinosus in the control and exposed to dimethoate (0.4 and 10 mg/kg soil) at 25 °C.
Exposure period: 0 h, 24 h, 48 h, 96 h, 7 days, 14 days, 21 days, 28 days; recovery period: 35 days, 42 days.

may be explained by faster degradation of dimethoate at 25 °C, which on fungi leads to the release of faecal pellets that are enriched with
can lead to an increase of metabolites that can be more toxic than di- bacteria present in their gut. This will increase the soil microbiome
methoate (Lucier and Menzer, 1970; Martikainen, 1996). activity leading in a final step to a possible faster degradation of
Secondly another highlight from the present study was the strong these organic compounds (Loureiro et al., 2002; Zimmer and Topp,
inhibition of the enzyme acetylcholinesterase in the 10 mg dimetho- 1999).
ate/kg exposure. It is widely known that this enzyme is the main target Regarding biomarkers, the enzyme GST, which is involved in the
of dimethoate, and its inhibition was expected during the exposure. But detoxification process, did not show any significant differences during
an inhibition N90% was observed for the highest concentration for all the exposure and recovery period at 20 °C and at the lowest dimethoate
sampling times at both temperatures. Such inhibition is generally con- concentration at 25 °C; on the other hand, the highest dimethoate con-
sidered to cause severe problems to the organisms and in some cases centration exposure at 25 °C induced effects on this enzymatic activity.
even death (Guimarães et al., 2007; Lucier and Menzer, 1970). The inhi- At 25 °C, after 14 days of recovery the decrease on GST activity for the
bition of the AChE activity was also observed in a recent work for two lower concentration cannot be considered an inhibition response, but
other species of earthworms, exposed to dimethoate, where concentra- a basal value according to Ferreira et al. (2010). However, the value
tions corresponding to 25% of the field recommended dose inhibited up for the control showed a significant increase during the test, higher
to 60% of this enzyme activity (Velki and Hackenberger, 2012). In addi- than basal values reported (Ferreira et al., 2010) and higher than values
tion, diazinon exposure to isopods via food led to approximately 50% from organisms sampled before the starting of the test for the same
and 90% inhibition, in adults and juveniles, respectively (Stanek et al., temperature. These differences observed for the control are unclear,
2006). The negative effects of dimethoate in terrestrial isopods based since a return to basal levels were observed after 14 days of exposure.
on the locomotion impairment have been reported by Engenheiro An often reported effect of OPs is the induction of oxidative stress,
et al. (2005), where an AChE inhibition of ~ 60% was correlated with a by generating reactive oxygen species (ROS) as well as alterations with-
shorter path length travelled and more stops per path. Also some previ- in the antioxidant and scavenging system (Karami-Mohajeri and
ous works using dimethoate and terrestrial isopods showed a higher Abdollahi, 2011). The study carried out on the biomarkers GST, LPO
AChE inhibition and high mortality (Santos et al., 2010, 2011). and CAT showed also some degree of oxidative stress as a result of the
This study also highlights the influence of isopods on the increase of exposure to dimethoate. LPO and CAT activity showed small responses
dimethoate’s degradation rate. This has been reported in a previous to dimethoate exposure, although an increase on LPO was found at
work by Loureiro et al. (2002) for the degradation of lindane in soil. the 7 days of exposure at 25 °C (5x higher than control) that could be
The mechanism that underlies this faster rate of degradation of di- related to the high mortality observed. So, oxidative stress can be
methoate in the presence of isopods may be related to their role in found as a result of dimethoate exposure during an adaptation period
ecosystems. Their feeding on decaying vegetal matter and grazing of 96 h/7 days and also related to possible effects induced by the higher
100 N.G.C. Ferreira et al. / Science of the Total Environment 502 (2015) 91–102

Fig. 8. Integrated biomarker response represented by starplot and histogram of Porcellionides pruinosus in control and exposed to dimethoate (0.4 and 10 mg/kg soil) at 25 °C during the
exposure and recovery period. AChE = acetylcholinesterase, GST = glutathione S-transferases, LPO = lipid peroxidation, CAT = catalase, LDH = lactate dehydrogenase, Ea = available
energy, Ec = energy consumption, CEA = cellular energy allocation.

exposure temperature (25 °C). In fact, previous works have shown dimethoate toxicity even indicating a possible interaction within the
situations of oxidative stress for exposure to increment temperatures two different temperatures. Whereas for the 20 °C exposure, almost
(e.g. Lesser, 2006; Lesser and Kruse, 2004), mainly leading to elevated no increase was found in the energy reserve content for the experimen-
dehydration (França et al., 2007). In addition CAT activity has been tal duration, the 25 °C exposure presented not only a great content
also reported to decrease as a result of the increase of superoxide anions increase, but also around the 7/14 days of exposure these reserves
(Velki and Hackenberger, 2012). reached a plateau, indicating an equilibrium in the organism physiology.
Energy response biomarker LDH apparently responded to dimetho- In addition, it was also possible to depict small variations after reaching
ate exposure at 20 °C, independently from concentration. After 7 days of the plateau. The results obtained between the 96 h and 7 days of expo-
exposure and 7 days of recovery this biomarker showed differences at sure at 25 °C indicated an increase in carbohydrate, lipid and the protein
the highest concentration under both temperatures. Although a clear content leading into a new plateau, which might also indicate some
response was not obtained by using this biomarker one can hypothesise physiological or behavioural changes that the organisms underwent to
that dimethoate may be interfering in the glycogen cycle and some prevent stress. In this case it seems also plausible to transpose all
of the effects found for this enzyme might also be associated with de- these results on the interaction between temperature and dimethoate
creases in carbohydrates content (Moorthy et al., 1983). into a disturbance in the moulting cycle. Along with the protein in-
Therefore, one main finding was that there were different responses crease, it was also expected an increase in the other energy reserves
for each energy reserve within each temperature of exposure. For lipids, as observed within our experiment. This impairment in the moulting
an increase was observed in some of the time points and for both tem- cycle, which seems to happen when organisms are exposed to 25 °C,
peratures of exposure, but in general dimethoate did not seem to affect may also be a behaviour strategy to increase its feeding rate, so they
the lipid content. The carbohydrates presented a similar pattern of could endure the stress for a longer time period. Previous works have
decrease for both temperatures within the first 7 days of exposure, already reported an increase in the muscle groups located in the anterior
after which the effects of dimethoate seemed to disappear and values and posterior segments, which can be directly related to the protein
tended to the basal levels. As expected, total protein did not present content, and also that the moult, which is biphasic, and can occur with
significant changes, as it is known that they are the last energy reserve hours or days (Whiteley and El Haj, 1997). This could be that organisms
to change or be used upon stress exposure. As in the previous case, an exposed to 20 °C had a total protein content that did not increase in
increase was also observed after 96 h exposure at 25 °C. Lower energy quantity, but stayed constant, and the increment in the energy reserves
contents were observed mainly at 20 °C and for the lowest concentra- resulted from carbohydrate and lipid changes. These results are
tion. In general, energy reserves presented essential data to understand contradicted in several previous works where a decrease in feeding
 N.G.C. Ferreira et al. / Science of the Total Environment 502 (2015) 91–102 101

behaviour could be observed for terrestrial isopods exposed to stressors consumption, CEA, can indicate the impairment of key functions essential
(Abdel-Lateif et al., 1998; Donker et al., 1998). Nevertheless one must for the maintenance of these organisms individually, and be transposed
consider that these studies have been performed with contaminated to the population level due to a possible effect on their reproduction
food, and although small amounts of pesticide must had been adsorbed behaviour/pattern.
by the leaves in our study, a possible avoidance was due to the effects on
the organisms and not by changes in palatability. 5. Conclusions
Finally, in this study the available energy as a parameter did not
seem to be a good indicator of the organisms’ status, since little variation As previously shown by other studies, dimethoate affected the
in contents was observed. The same happened with the CEA since differ- enzyme AChE but also other biomarkers such as the detoxification
ences in Ea values were low, and the organisms’ behaviour (represented enzyme, GST and the damage related biomarker, LPO. The increase of
by the Ec values, where higher consumption ratios relates to organisms’ LPO also seemed to be related to the high mortality observed in 25 °C
higher activities) were not intense enough to cause an impact. Whenever exposure.
a decline in CEA was observed, it was indicating either a reduction The organisms exposed to the lower concentration of 0.4 mg di-
in available energy or a higher energy expenditure, both resulting in methoate/kg soil, simulating a field application dose, presented low to
a lower amount of energy available for growth, reproduction or basal moderate toxicity which was in accordance with the work of Fischer
activity (de Coen and Janssen, 1997). et al. (1997) and therefore was used as baseline for this study. However,
Within the two temperatures used, differences were observed in in the organisms exposed to the highest concentration (10 mg dimeth-
terms of the total available energy, energy consumption and subse- oate/kg soil), toxicity was mainly due to the inhibition of AChE and the
quently the CEA. In fact the increase of Ec and decrease in Ea for the degradation of dimethoate that can possibly lead to the formation of
exposed organisms at 20 °C is contrary to the patterns shown at the highly toxic metabolites and also reactive oxygen species and oxidative
25 °C exposure, which may suggest that at 20 °C organisms did not stress that caused high LPO rates.
slow their metabolism. However, at 20 °C, temperature did not seem This study showed an increase in the energy reserve contents within
to be the dominant factor influencing the energy-related parameters, the first 7 days of exposure that could be linked to an impairment of
but dimethoate, as can be seen by the patterns of Ea and Ec, as a result the moult cycle or an increase in the feeding behaviour. This study
of the detoxification process. Contrary, for the organisms exposed to highlights also a possible strategy of recovery for these organisms
25 °C, it did not seem that any of the stressors had a preponderance with an increase in total lipid content, which was the only energy
effect over the other, as organisms decreased significantly their Ec and reserve that returned to baseline levels within the recovery period.
allocated energy to the detoxification process. At this temperature, the Generally, long term experiments using realistic concentrations
Ea increase might be explained by an increase in isopods’ feeding activ- should be performed to understand the mechanisms of toxicity of
ities. This type of behaviour has not yet been reported and new studies stressors, such as dimethoate, and to develop a baseline for future
regarding the combined effects of temperature and dimethoate on feed- studies. This will allow better understanding of the specific mechanisms
ing rates would provide further information. underlying the toxicity process and detoxification pathways. In addi-
The recovery period did also provide some important and interest- tion, the results from soil chemical analysis highlight the possibility
ing remarks on organisms previously exposed to dimethoate. From that isopods can increase the decay rates of dimethoate, as also reported
previous works with other non-target organisms, such as earthworms, for other pesticides.
a slow recovery rate in AChE has also been reported (Aamodt et al.,
2007). In the present work at 20 °C and at the highest dimethoate con- Acknowledgment
centration, the AChE inhibition was in accordance with studies that
show that it can become an irreversible process (Ranjbar et al., 2005). This study was supported by funding FEDER through COMPETE-
The organisms exposed to 20 °C followed a similar pattern when re- Programa Operacional Factores de Competitividade, and by National
garding oxidative stress biomarkers where a slow recovery was ob- funding through FCT-Fundação para a Ciência e Tecnologia within the
served, although the LPO rates, that indicate damage from ROS, research project CLIMAFUN- CLImate Changes and Potential Impact on
reached similar levels of those in the control after the end of the test pe- Soil FUNctional Ecology (FCOMP-01-0124-FEDER-008656) and through
riod. For the organisms exposed to 25 °C although a similar pattern can a PhD grant (SFRH/BD/65739/2009) awarded to Nuno G. C. Ferreira by
be observed, the LPO rates continued to be significantly higher than FCT. The authors would like to thank for the technical support and
the control, which may indicate that this temperature may influence advice given by Dr. Abel Ferreira and Dr. Ceri Moris.
the recovery of the organisms even after 14 days. The fluctuation of the
energy related parameters in both the 20 °C and the 25 °C exposure are Appendix A. Supplementary data
in accordance with results from biomarkers. In fact it can be
hypothesised that the initial fluctuation (96 h/7 days) that could be Supplementary data to this article can be found online at http://dx.
seen in the exposure period which was more pronounced in the recovery doi.org/10.1016/j.scitotenv.2014.08.062.
period, was a possible adaptation of organisms to reach homeostasis
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 Update
Science of the Total Environment
Volume 511, Issue , 1 April 2015, Page 829

DOI: https://doi.org/10.1016/j.scitotenv.2015.01.010
 Science of the Total Environment 511 (2015) 829

Contents lists available at ScienceDirect

Science of the Total Environment

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

Corrigendum

Corrigendum to “Biomarkers and energy reserves in the isopod

Porcellionides pruinosus: The effects of long-term exposure to
dimethoate” [Science of the Total Environment 502 (2015) 91–102]
Nuno G.C. Ferreira ⁎, Rui Morgado, Miguel J.G. Santos, Amadeu M.V.M. Soares, Susana Loureiro ⁎
Department of Biology & CESAM, University of Aveiro, 3810-193 Aveiro, Portugal

The authors regret an error in “Fig. 1: Cumulative total number of recovery period in the control and exposed to 0.4 mg dimethoate/kg
dead organism from the species Porcellionides pruinosus during the soil and 10 mg dimethoate/kg soil. A total of 200 organisms were
exposure and recovery period in the control and exposed to 0.4 mg exposed per treatment. A — organisms exposed to 25 °C, B — organisms
dimethoate/kg soil and 10 mg dimethoate/kg soil. A total of 200 exposed to 20 °C”. The authors would like to apologize for any inconve-
organisms were exposed per treatment. A — organisms exposed to nience caused.
20 °C, B — organisms exposed to 25 °C” of the original publication. The
figure should state “Fig. 1: Cumulative total number of dead organism
from the species Porcellionides pruinosus during the exposure and

DOI of original article: http://dx.doi.org/10.1016/j.scitotenv.2014.08.062.

⁎ Corresponding authors.
E-mail addresses: nunoferreira@ua.pt (N.G.C. Ferreira), sloureiro@ua.pt (S. Loureiro).