Toxicity of Chlorpyrifos to Larval Rana dalmatina: Acute and Chronic Effects on Survival,...

Toxicity of Chlorpyrifos to Larval Rana dalmatina: Acuteand Chronic Effects on Survival, Development, Growth and GillApparatus

Ilaria Bernabo • Emilio Sperone • Sandro Tripepi •

Elvira Brunelli

Received: 3 September 2010 / Accepted: 7 February 2011 / Published online: 23 February 2011

� Springer Science+Business Media, LLC 2011

Abstract Chlorpyrifos [O,O-diethyl-O-(3,5,6-trichloro-

2-pyridyl)phosphorothioate] is a widely used non-systemic

organophosphorus insecticide frequently detected in sur-

face waters around the world. The goal of this study is to

evaluate the acute and chronic effects of this insecticide on

Rana dalmatina tadpoles. To assess the sensitivity of this

species, the LC50 value (i.e. the concentration at which

50% of tadpoles die) was determined after 96 h. Our results

showed that 5.174 mg L-1 chlorpyrifos caused 50% mor-

tality in tadpoles at Gosner stage 25. Chronic toxicity tests

were also conducted to evaluate the sublethal effects of

chlorpyrifos; tadpoles were exposed to three ecologically

relevant concentrations (0.025, 0.05 and 0.1 mg L-1) in

static renewal tests from Gosner stage 25 (tadpoles shortly

after hatching) until completed metamorphosis (Gosner

stage 46). No significant reduction was observed in sur-

vival, larval growth (mass), snout–vent length, stage

development or number metamorphosed. In contrast,

chlorpyrifos exhibited significant chronic toxic effects on

larval development, manifested as the appearance of

abnormalities, including tail flexure, skeletal and muscle

defects in later stages of development in tadpoles exposed

to all tested concentrations. We also evaluated the chronic

effects of chlorpyrifos on gill morphology and ultrastruc-

ture. Tadpoles were sacrificed after 8 days and 30 days of

exposure. Observations by both scanning (SEM) and

transmission electron microscopy (TEM) showed consid-

erable morphological and ultrastructural changes. The main

gill effects recorded were mucous secretion, epithelium

detachment and a degeneration phenomenon. Comparing

these results with our previous findings, we demonstrate

that the first effect of chlorpyrifos on R. dalmatina is gill

alteration, thus supporting the role of a morphological

approach in toxicological studies.

Keywords Chlorpyrifos � Gills � Tail � Amphibia �Tadpoles � Ultrastructure

Pesticides are commonly used to control pest species for

health and economic benefits, but worldwide use has led to

increased contamination of aquatic habitats (Davidson

et al. 2002; Jones et al. 2009; Houlahan et al. 2000; Hou-

lahan and Findlay 2003). As suggested by Relyea and

Hoverman (2006), understanding and predicting the

impacts of agrochemicals on non-target organisms is a

challenging proposition.

The concept that amphibians are particularly sensitive to

the action of pollutants due to their biological and ana-

tomical characteristics is widely accepted (Richards and

Kendall 2002; Venturino et al. 2003), also considering that

larvae may be exposed many times to residues of agricul-

tural contaminants during development (Bridges and

Boone 2003). For these reasons, amphibians are broadly

used as typical targets in evaluating the effects of chemi-

cals on aquatic and agricultural ecosystems (Bernabo et al.

2008; Pollet and Bendell-Young 2000; Schuytema and

Nebeker 1996; Venturino et al. 2003).

It is generally recognised that agrochemicals can seri-

ously affect local populations and the community structure

of amphibians, reducing survival, altering feeding and

swimming activity, causing a high incidence of deformities

and decreasing growth and development of larvae (Bonin

et al. 1997; Boone et al. 2001; Bridges 2000; Brunelli et al.

I. Bernabo � E. Sperone � S. Tripepi � E. Brunelli (&)

Department of Ecology, University of Calabria, Via P. Bucci,

87036 Rende (Cosenza), Italy

e-mail: [email protected]

123

Arch Environ Contam Toxicol (2011) 61:704–718

DOI 10.1007/s00244-011-9655-1

2009; Peltzer et al. 2008; Relyea 2005; Rohr et al. 2003;

Semlitsch et al. 1995; Taylor et al. 2005).

Organophosphorus pesticides (OPs) are increasingly

used in agriculture and have in many cases replaced

organochlorine and carbamate insecticides, as they have

high efficiency and lower persistence; currently they are

used at the highest rates for both domestic and agricultural

uses (Kiely et al. 2004). Chlorpyrifos [O,O-diethyl-O-

(3,5,6-trichloro-2-pyridyl)phosphorothioate] is one of the

most widely employed organophosphates in both agricul-

tural and urban environments (NRA 2000), posing a risk of

contamination to nearby aquatic environments via runoff,

spray drift or spillage (Pablo et al. 2008). The US Envi-

ronmental Protection Agency (EPA) imposed a ban on its

sale for residential use (EPA 2000, 2002, 2006), whereas in

Europe, chlorpyrifos is one of the top-selling insecticides

without restrictions on its use (Eurostat 2007; Bjørling-

Poulsen et al. 2008). Chlorpyrifos concentrations in stream

and water bodies adjacent to agricultural lands range from

0.01 to 0.1 mg L-1 (Barron and Woodburn 1995; CCME

2008; Carriger and Rand 2008; Gilliom et al. 2006;

Jergentz et al. 2005; Mazanti et al. 2003; Moore et al. 2002;

EPA 2002, 2006; UNEP 1991).

In vertebrates, chlorpyrifos elicits a number of effects

including hepatic dysfunction, immunological abnormali-

ties, embryo toxicity, genotoxicity, teratogenicity, and

neurochemical and neurobehavioural changes (Ali et al.

2009; Dam et al. 2000; Gomes et al. 1999; Rahman et al.

2002; Ricceri et al. 2006; Song et al. 1998). The effects of

chlorpyrifos on amphibians have not been extensively

studied (Giesy et al. 1999; Richards and Kendall 2002,

2003), and the toxicity of this pesticide has been examined

mainly on Xenopus laevis, which is a widely used experi-

mental model. Similar studies in other anurans are limited

to a few species and, as suggested by Richards and Kendall

(2002, 2003), more research needs to be conducted on

indigenous species that have high potential to be exposed

to agricultural pesticide.

The toxic effect of pesticides may be observed at the

population level (mortality), at the single-organism level

(i.e. changed body mass, physical malformations) or at the

sub-organismal level (i.e. different structure of organs,

ultrastructural changes etc.) (Adamski and Ziemnicki

2004; McCarthy and Shugart 1990). In ecotoxicology, a

traditional approach has been to assess the direct toxic

effects of pollutants by estimating the LC50 value (i.e. the

concentration expected to kill 50% of a population).

Hence, it is necessary to conduct LC50 tests on amphib-

ians, because they are not tested as part of the registration

process for the vast majority of pesticides (Hopkins 2007).

In addition, chronic toxicity studies are important to assess

more subtle and indirect effects of chemicals on several

endpoints.

Nevertheless, there are few chlorpyrifos LC50 data for

amphibians and insufficient data on the toxicity after long-

term exposure to sublethal concentrations in both Teleostei

and other aquatic vertebrates (Barron and Woodburn 1995;

CCME 2008; EPA 2002). To our knowledge, only a few

studies have considered morphological endpoints and no

previous studies have examined the effects of OPs on

amphibian gill morphology. For these purposes, we

investigated the effect of acute and chronic exposures of

chlorpyrifos on Rana dalmatina tadpoles. To assess the

sensitivity of the species, we first determined the direct

toxic effects of pollutants by estimating the LC50 value.

We also evaluated the effects of chronic larval exposure to

ecologically relevant concentrations of chlorpyrifos (0.025,

0.05 and 0.1 mg L-1) on mortality, growth and develop-

ment. In addition, the present paper focusses attention on

establishing morphological endpoints for detection of

chlorpyrifos effects on R. dalmatina tadpoles.

Histopathological studies are especially appropriate

when analysing sublethal effects of water-borne pollutants

on target organs such as gills and skin (Bernabo et al. 2008;

Brunelli and Tripepi 2005; Brunelli et al. 2008, 2010a, b).

As outlined by Au (2004), histocytological responses are

relatively easy to determine, and can be related to health

and fitness of individuals, which in turn allows further

extrapolation to population/community effects. In this

study, we therefore evaluated the chronic effects of

chlorpyrifos on gill apparatus, one of the key compartments

of amphibians. Since literature data reveal that OPs elicit

severe malformations, such as abnormal tail flexure, con-

torted posture and abnormal notochord, in various species

of frog embryos (Bonfanti et al. 2004; Colombo et al. 2005;

Richards and Kendall 2002), we also investigated histo-

logical alterations of the tail muscles.

Materials and Methods

Study Organism

The agile frog (R. dalmatina) is a common species in

Central and Southern Europe and SW Asia. The breeding

season occurs between February and March, and tadpoles

develop in various types of natural and artificial small

water bodies in agro-ecosystems. R. dalmatina completes

metamorphosis generally within 2–3 months, and sexual

maturity is reached at 3 or 4 years of age.

Animal Collection and Acclimation Period

Rana dalmatina tadpoles were obtained from eggs col-

lected from a pond situated in a location close to Cosenza

(Calabria, Southern Italy; 39�340N, 16�000E). We sampled

Arch Environ Contam Toxicol (2011) 61:704–718 705

123

a pond that has never been in contact with agrochemicals to

avoid possible biases due to adaptation. This pond has a

stable population of R. dalmatina. During the acclimation

period of 4 days, the tadpoles were housed at 22 ± 1�C in

50-L aerated tanks and were fed with boiled organic

spinach ad libitum. During the acclimation period, the

animals showed no signs of disease or stress. Determina-

tion of developmental stages was carried out according to

Gosner (1960).

Test Substance and Chemical Analysis

Test solutions were prepared by dissolving chlorpyrifos

(purity 99.5%; Chem Service Inc., West Chester, PA,

USA) in dechlorinated tap water to obtain the nominal

concentrations used for both acute and chronic tests.

Water samples were collected for chemical analysis at

the beginning and within 12 h of the renewal of test

solutions. Actual chlorpyrifos concentrations were verified

via gas chromatography (Varian Saturn 3800 GC NPD,

ECD and PFPD detectors). Procedures were in accordance

with the method described by Belden et al. (2000). Actual

pesticide concentrations were similar to nominal levels

(p [ 0.05); therefore, nominal concentrations are referred

to the present work.

Acute Exposure

The lethal concentration, LC50 (i.e. the concentration at

which 50% of tadpoles die), of chlorpyrifos was deter-

mined by exposing tadpoles at Gosner stage 25 for 96 h.

After a preliminary exposure to assess the range of pesti-

cide concentration, chlorpyrifos was dissolved in tap water

to obtain nominal concentrations of 1, 3, 4, 4.5, 5.5, 6, 7

and 15 mg L-1. For each experimental set, 10 tadpoles of

comparable body dimensions were placed in various 15-L

glass exposure tanks. The control group was kept in tap

water. Mortality after 96 h was recorded. A static exposure

system was used in accordance with standard procedure

guidelines (ASTM 1997). Throughout the experiment,

animals were maintained under a natural light/dark cycle,

median pH 7.3, and water temperature 22 ± 1�C. During

the 96-h exposure, the animals were not fed. During the

experimental period, the presence of mortality was moni-

tored, and dead animals removed. Three replicates were

used for each treatment and the control.

Test Design and Chronic Exposure Conditions

The three levels of chlorpyrifos selected for exposure trials

were based on field levels reported by Moore et al. (2002)

and Mazanti et al. (2003) and our LC50 values. Chronic

exposure was performed by dissolving chlorpyrifos in tap

water to obtain nominal concentrations of 0.025, 0.05 and

0.1 mg L-1, hereinafter referred to as low, medium and

high concentration, respectively. Individuals of comparable

body dimension (Gosner stage 25) were randomly placed

into 30-L glass tanks (1 tadpole/L) of the appropriate

treatment solution (control, low, medium and high), with

two replicates of 30 tadpoles per exposure. The control

group was kept in tap water. A static-renewal exposure

system was used in accordance with standard procedure

guidelines (ASTM 1997) with complete renewal of the

water volume every 3 days.

The exposure period was 57 days: from Gosner stage 25

(start of independent feeding and free swimming) to stage

46 (end of metamorphosis and complete tail resorption).

Throughout the experiment, all animals were held under

controlled laboratory conditions: water temperature of

22 ± 1�C, median pH of 7.3 and photoperiod of 12:12 h

light–dark cycles. Water quality parameters (pH, conduc-

tivity, temperature, dissolved oxygen alkalinity and hard-

ness) were recorded before and after renewal of test

solutions. Tadpoles were fed with boiled organic spinach

ad libitum three times a week throughout the exposure

period until the start of metamorphosis (Gosner stage 41).

After one forelimb had emerged (Gosner stage 42),

tadpoles were removed from exposure tanks to the final

stage of complete tail resorption and thus housed in small

aquaria containing 0.5 L treatment solution and a dry area.

At this time the animals were not fed, as metamorphosing

tadpoles live off of fat stored in their tails. Developmental

stage was determined weekly on a subsample of at least

five randomly selected tadpoles per tank. As an index of

growth, body weight (each tadpole was towel-dried and

weighed to the nearest milligram) and snout–vent length

(total tadpole length minus tail length) of all tadpoles was

measured at the beginning of the experiment and every

9 days during the whole test period. Once fully metamor-

phosed (Gosner stage 46), individual mass and date of

completed metamorphosis (from the first day of exposure)

were recorded for each individual.

During the experimental period, the presence of defor-

mities and mortality were monitored daily, and dead ani-

mals were removed. Every day, larvae exhibiting flexure

and bent tails, asymmetrical tails, bent axial skeleton,

oedema or abnormal swimming behaviours (defined as

lying on bottom, swimming upside down, swimming on its

side or swimming in circles) were recorded.

Morphological Analysis

Animals for morphological analysis were maintained in

parallel in separate tanks under the same experimental

conditions.

706 Arch Environ Contam Toxicol (2011) 61:704–718

123

Light-Microscopy Analysis of Tails

After 35 days, five randomly selected tadpoles per group

were euthanised with 2–4 g L-1 MS-222 (tricaine methane-

sulfonate; Sigma-Aldrich Chemical Co., St. Louis, MO,

USA). Tails were removed and fixed for 3 h in 4%

paraformaldehyde in 0.1 M phosphate buffered solution

(pH 7.4) at 4�C, dehydrated in graded ethanol and

embedded in Paraplast (Bio-Optica, Milan, Italy). Sec-

tions of 7 lm were cut using a Leica RM 2125 RT

microtome (Leica Microsystems, Wetzlar, Germany) and

mounted on slides. Sections were stained with haema-

toxylin and eosin (Bio-Optica, Italy) to visualise typical

morphological features.

Scanning (SEM) and Transmission Electron

Microscopy (TEM) of Gill Apparatus

After 8 (Gosner stage 27) and 30 days (Gosner stage 37),

five randomly selected tadpoles per group and time point

were analysed using SEM and TEM studies, respectively.

The animals were euthanised with 2–4 g L-1 MS-222

(tricaine methanesulphonate; Sigma-Aldrich Chemical Co.,

St. Louis, MO, USA), and the gills were removed using a

dissecting microscope.

The specimens were fixed in 3% glutaraldehyde in

phosphate buffer (0.1 M, pH 7.2) for 2 h at 4�C and then

post-fixed in 1% osmium tetroxide in the same buffer for

2 h at 4�C (Sigma-Aldrich Chemical Co., St. Louis, MO,

USA). Samples were dehydrated in increasing concentra-

tions of acetone and contrasted in block with aqueous

solution of 5% uranyl acetate for 2 h, and then embedded

in Epon-Araldite (Fluka Ag, Buchs, Switzerland). Ultrathin

sections (600–900 A) were cut using a LKB Nova ultra-

microtome and stained with uranyl acetate and lead citrate,

and then coated using an Edwards EM 400. Observations

and photographs were made using a Zeiss EM 900 trans-

mission electron microscope. Samples for the SEM study

were dehydrated and dried according to the critical-point

method, covered with gold, and observed using a Zeiss

DSM 940 scanning electron microscope. All analyses were

conducted blind, without knowledge of which exposure the

tadpole had been subjected to.

Statistical Analysis

LC50 values were determined using Finney’s (1971) probit

analysis LC50 determination method and version 1.00 of

the software developed by EPA (1999).

All data were analysed using Graph Pad Prism 5.00

(GraphPad Software Inc., San Diego, CA, USA), and a

level of significance of 0.05 was used for all statistical

tests. Data from the two replicates for all endpoints were

statistically compared using Mann–Whitney tests; because

no significant differences appeared, the data were pooled

into one data set per exposure group for further analyses.

Fisher’s exact probability test (one-way) was used to

compare chlorpyrifos exposure groups with control

groups with respect to mortality (number alive versus

number dead), the number of individuals that reached

and completed metamorphosis, and deformity incidence

(number normal versus number deformed). To evalu-

ate the effects of chlorpyrifos on developmental stage

(Gosner 1960), body weight and snout–vent length,

Kruskal–Wallis, followed by Dunn’s multiple-comparison

post-test was applied to test differences between the four

treatments.

Results

Acute Exposure

The nominal 96-h LC50 value of chlorpyrifos for R. dal-

matina tadpoles was found to be 5.174 mg L-1. Table 1

presents the relation between chlorpyrifos concentration

and mortality rate according to Finney’s probit analysis

using the EPA software. No mortality was observed in the

control group or 1, 3 and 4 mg L-1 chlorpyrifos concen-

tration groups. Estimated LC50 values and 95% confi-

dence limits for 96-h chlorpyrifos exposure are shown in

Table 2.

Chronic Exposure

Mortality, Growth, Development and Metamorphosis

During the experimental period, no significant differences

among chlorpyrifos-exposed groups and the untreated

control group (p [ 0.05) were observed in mortality or

number metamorphosed (Table 3). No differences in body

length were recorded (Fig. 1), and all experimental groups

reached metamorphosis (Gosner stage 42) with a compa-

rable dimension. On the contrary, a significant reduction in

body weight compared with control was observed from day

36 of exposure to day 45 (Fig. 2) in both medium- and

high-concentration groups; these differences were not

recognisable at metamorphosis.

Chlorpyrifos did not cause a concentration-related

reduction in developmental rate of R. dalmatina tadpoles.

Time to complete metamorphosis (Gosner stage 46) was

not markedly different among chlorpyrifos-exposed groups

and the untreated control group; all tadpoles completed

their metamorphosis between 42 and 57 days of exposure

(data not shown).


123

Morphological Abnormality of the Tail

Morphological features of tadpoles belonging to the control

group (Fig. 3a) did not differ from those expected for the

species at all time points of the experiment; none of the

tadpoles showed malformations (Table 3) or behavioural

abnormalities.

After 35 days of exposure, the appearance of deformi-

ties was recorded in all exposed groups; the incidence was

significant in both low- and medium-concentration groups

and highly significant in the high-concentration group,

compared with control (Table 3).

Physical abnormalities were mainly ascribed to skeletal

defect (Fig. 3b–f) and abnormal tail lateral flexure (Fig. 3e,

f); bloated heads and oedema (Fig. 3b–f) were also

detected. Irregular swimming was observed for those tad-

poles affected by deformities; in particular, they were not

able to balance their body during swimming phases.

Longitudinal tail tissue sections, obtained from tadpoles

after 35 days of exposure, were histologically analysed in

both control and each treatment group. In both control and

normal tails from chlorpyrifos-treated animals, each

Table 1 Relation between

chlorpyrifos concentration and

mortality rate of R. dalmatina

Concentration

(mg L-1)

Number

exposed

Number of

dead tadpoles

Death in

the bioassay

Expected

death

Estimated

death

1 30 0 0.0000 0.0000 0.0000

3 30 0 0.0000 0.0000 0.0152

4 30 0 0.0000 0.0000 0.1533

4.5 30 15 0.5000 0.5000 0.2896

5.5 30 21 0.7000 0.7000 0.5959

6 30 21 0.7000 0.7000 0.7219

7 30 24 0.8000 0.8000 0.8851

15 30 30 1.0000 1.0000 1.0000

Table 2 Estimated LC values and confidence limits for R. dalmatinaexposed to chlorpyrifos

Point Concentration (mg L-1) 95% confidence limits

Lower Upper

LC1.00 2.881 1.449 3.599

LC5.00 3.420 2.078 4.055

LC10.00 3.747 2.511 4.335

LC15.00 3.986 2.848 4.544

LC50.00 5.174 4.537 5.919

LC85.00 6.716 5.881 9.477

LC90.00 7.143 6.163 10.748

LC95.00 7.827 6.586 12.992

LC99.00 9.291 7.420 18.637

Table 3 Mortality, frequency of tadpoles metamorphosed (Gosner stage 46) and incidence of deformity (as percent of initial number of

tadpoles) in R. dalmatina tadpoles exposed to chlorpyrifos from Gosner stage 25 to 46 (n = 30 for each treatment)

Control

(0 mg L-1)

Low

(0.025 mg L-1)

p Medium

(0.05 mg L-1)

p High

(0.1 mg L-1)

p

Mortality 32% 33% ns 40% ns 45% ns

Deformity 0% 17% ** 15% ** 22% ***

Metamorphosed 68% 67% ns 60% ns 55% ns

Data from two replicates are shown

p-Value, when compared with the control group using the one-tailed Fisher’s exact probability test (ns not significant, * p \ 0.05, ** p \ 0.01,

*** p \ 0.001)

Fig. 1 Snout–vent length [mean ± S.D.] of R. dalmatina tadpoles of

Gosner stage 25 exposed to three concentrations of chlorpyrifos for

57 days (C = 0 mg L-1, L = 0.025 mg L-1, M = 0.05 mg L-1,

H = 0.1 mg L-1)


123

myotome was attached at regular intersomitic boundaries

and showed a regular myotomal structure with the myo-

cytes orientated parallel to the notochord occupying the

whole length of the myotomes (Fig. 4a, b). In several

chlorpyrifos-treated animals, notochord flexure was

observed (Fig. 4c, e) and an alteration of the tail muscular

structure was evident with the appearance of extracellular

spaces between myocytes and vacuolated regions (Fig. 4c,

d, f). Moreover, myotomes were distorted and the myo-

cytes revealed an uncorrected orientation with respect to

the notochord (Fig. 4e).

Gill Morphology

The general morphology of R. dalmatina has been previ-

ously described, therefore only aspects relevant to the

present paper will be briefly described.

Rana dalmatina internal gills are supported by cerato-

branchialia I–IV as in other anuran species; from branchial

arches arise dorsally the gill filters that play a role in the

feeding mechanism, and ventrally the gill tufts that are

involved in respiratory and osmoregulatory functions.

Observed by SEM, gill tufts (Fig. 5a) show a stem with

short ramifications, and their epithelial surface is made

from an almost continuous layer of polygonal pavement

cells (PVCs), with well-outlined boundaries; their surface

is characterised by the presence of microridges. Rarely,

cells with microvilli are interspersed between the pavement

cells (Fig. 5b).

Observed by TEM, gill tufts (Fig. 5c) appear to be

formed by a connective portion covered by a simple or

bilayered epithelium. Sometimes, ample intercellular

spaces could be observed between cell layers. The inner

layer is composed of the basal cells that separate the

connective tissue from the epithelial cells above. The

external layer is mainly composed by PVCs characterised

by numerous secretion granules located under the apical

plasmalemma and a wide lobated nucleus.

When observed by TEM, the cells provided with apical

microvilli (Fig. 5d) are easily recognisable as mitochon-

dria-rich cells (MRCs) the presence of a large number of

mitochondria that fill the whole cytoplasm. The nucleus is

wide, roundish and usually in the basal position.

Observations by SEM show that filters (Fig. 5e) are

constituted by a main flattened axis from which primary

and secondary lateral branches depart laterally.

Observed by TEM (Fig. 5f), such processes appear to be

constituted by one to three layers of epithelial cells. The

surface cells consist of flattened pavement cells which

represent the main cellular type; below these, cubic cells

with high nuclear–cytoplasmic ratio could be seen, in the

final portion of the filter.

Chlorpyrifos Exposed Groups, 8 Days

After 8 days of treatment with chlorpyrifos, histopatholo-

gical alterations were seen at all tested concentrations. The

first structural modifications that could be observed at

lowest concentration (Fig. 6a) was infolding at several

points of the gill tuft epithelium; epithelial surface kept its

own organisation, and junctional margins and microridges

could be seen. At both intermediate and high (Fig. 6b)

concentrations, gill tufts appeared covered with long

mucous cords; irregularities of epithelial surface were very

frequent and affected both stem and, to a minor extent, tuft

ramifications. The distal portion of tufts appeared heavily

dehydrated and collapsed at several points.

SEM examination did not show relevant modification in

the gill filters after exposure to the lowest concentration

(Fig. 6c), and the epithelium surface was still undamaged.

The extent of structural modifications increased with

increasing concentration. Macroscopic modifications

occurred in both medium- (Fig. 6d) and high-concentration

groups; epithelial projections departing from the margin of

the filter rows could be seen, and in the group exposed to

the highest concentration, a large amount of mucous

secretion covered the filter surface.

Ultrastructural analysis of the gills after 8 days con-

firmed the presence of epithelial damage at all concentra-

tions (Fig. 7a–d). Cell degeneration and hypertrophy of

pavement cells could be seen in the gill tuft (Fig. 7b, d); at

the highest concentration, the most conspicuous alterations

revealed by TEM were the detachment of external layer

from basal cells and the appearance of wide spaces and

lacunae (Fig. 7b); loss of contact between epithelium and

the connective tissue below and hypertrophy of endothelial

cells could also be observed (Fig. 7d).

Fig. 2 Body weight [mean ± S.D.] of R. dalmatina tadpoles of

Gosner stage 25 exposed to three concentrations of chlorpyrifos for

57 days (C = 0 mg L-1, L = 0.025 mg L-1, M = 0.05 mg L-1,

H = 0.1 mg L-1). Asterisks indicate treated groups that differ from

control; ***p \ 0.001


123

By TEM observation, the gill filter alterations seemed to

be less intense compared with those of the gill tufts, and the

epithelium maintained its organisation in the low-concen-

tration group; observing the irregularities of the cell sur-

face, previously revealed by SEM, in both medium- and

high-concentration groups we could note that they origi-

nate from PVCs which gave the appearance of deep in-

vaginations (Fig. 7a, c). At the highest concentration

(Fig. 7c), the main apparent effects on the ultrastructure of

epithelial cells were the increase of secretion granules in

PVC subapical cytoplasm.

Chlorpyrifos Exposed Groups, 30 days

SEM observations of the gill epithelium after 30 days of

exposure to chlorpyrifos showed an accentuated disruptive

phenomenon in all experimental groups. The extent of

Fig. 3 Morphological alterations of R. dalmatina tadpoles of Gosner

stage 25 exposed to three concentrations of chlorpyrifos for 57 days.

a Dorsal view of representative developmental stages and dimensions

of tadpoles from control (c) and low-concentration (l) groups after

8 days exposure. b, c Dorsal and ventral view of axis malformation

(arrow) and oedema (arrowhead) in tadpoles after 35 and 45 days

exposure to the low concentration, respectively. d Ventral view of

axis malformation (arrow) and oedema (arrowhead) of tadpoles from

medium-concentration group after 45 days exposure. (e, f) Dorsal

view of axis malformations and laterally deflected tails (arrow) and

oedema (arrowhead) of tadpoles from the high-concentration group

after 45 and 50 days exposure, respectively (bar = 0.5 cm)


123

structural modifications increased with chlorpyrifos con-

centration; the regular gill tuft arrangement was missing,

and the tufts appeared heavily dehydrated. Tuft ramifica-

tions appeared close to each other (Fig. 8a), and their

surface had an irregular corrugated appearance with lifting

of the superficial layer. At the two highest concentrations

(Fig. 8b), the collapse of apical portion was evident along

the whole tuft, and it was also possible to recognise PVCs

with irregularly formed microridges. Profuse amounts of

mucus were present on both filters and tuft surface

(Fig. 8a–d). Filter rows flattened, and their margins were

slightly enlarged at several points (Fig. 8c). The epithelial

surface appeared wrinkled, even though the PVCs were

still undamaged (Fig. 8d).

Fig. 4 Longitudinal histological sections of the tail in control and

chlorpyrifos-treated R. dalmatina tadpoles after 35 days of exposure.

a, b Myotomes (m) of tadpoles from control and low-concentration

groups with normal myocytes oriented in parallel to the notochord and

attached at regular intersomitic boundaries (arrowhead) (bars = 70

and 40 lm). c, d Bent notochord (asterisk) and myotomes (m) of

tadpoles from medium-concentration group showing extracellular

spaces between myocytes and vacuolated region (arrow); note the not

well-developed intersomitic boundaries (arrowhead) (bars = 60 lm

and 70 lm). e, f Notochord flexure (asterisk) and distorted myotomes

(m) from tadpoles exposed to the high concentration. Note the

uncorrected orientation to the notochord and the disorganisation of the

myotomes with the presence of hypertrophic areas and extracellular

spaces (arrow) (bars = 300 and 50 lm)


123

In both low- and medium-concentration groups

(Fig. 9a), the most conspicuous alterations of gill tuft

revealed by TEM were the infolding of external layer

with detachment from basal cells and appearance of wide

spaces and lacunae; the loss of contact between epithelium

and the connective tissue below involved the whole

tuft epithelium. The regular epithelium arrangement was

completely missing in the highest-concentration group

(Fig. 9c); some MRCs preserved an undamaged cytoplas-

mic content even if microvilli appeared degenerated; other

cell types showed signs of alteration, and hyperplasia of

endothelial cells became more pronounced. Structural

alterations of the gill filter were less evident in the

low-concentration group (Fig. 9b), whereas in both

Fig. 5 Scanning and transmission electron micrographs of R.dalmatina gill apparatus under control conditions. a General view

showing gill tuft ramifications (bar = 10 lm). b High-resolution

image of the gill tuft showing the presence of pavement cells (PVC)

and mitochondria-rich cells (MRC) (bar = 3 lm). c TEM image of

gill epithelium composed by basal cell (BC) and flattened pavement

cell (PVC) equipped with short microridges (arrowheads)

(bar = 2 lm). d Mitochondria-rich cell (MRC) characterised by an

electron-dense cytoplasm filled by numerous mitochondria (m) and

microvilli on the apex surface (arrowheads) (bar = 0.5 lm). e SEM

micrograph of gill filter (bar = 6 lm). f Ultrastructure of the gill

filter epithelium organisation. Apical pavement cells (PVC) and

underlying cubic cells (CUC) with high nuclear–cytoplasmic ratio

could be seen (bar = 2 lm)


123

medium- and high-concentration groups, epithelium

appeared markedly modified with enlargement of intercel-

lular spaces and degeneration of external cells (Fig. 9d).

Discussion

The purpose of this study is to evaluate the acute toxicity of

chlorpyrifos to R. dalmatina tadpoles, and to assess the

sublethal effects of this pesticide after chronic exposure.

Acute Toxicity Test (LC50)

The 96-h LC50 values range from 1 to 14 mg L-1 (Abbasi

and Soni 1991; Barron and Woodburn 1995; Richards and

Kendall 2002) in several anuran species; in addition 24-h

LC50 values were 3.005 mg L-1 in Rana boylii (Sparling

and Fellers 2007) and 0.177 mg L-1 in Rana tigrina

(Abbasi and Soni 1991). The LC50 value (5.174 mg L-1)

calculated for R. dalmatina is comparable to values

reported in literature. It is established that the acute toxicity

of pesticides varies depending on species and develop-

mental stage (Berrill et al. 1998; Bridges and Semlitsch

2000), and in some cases on differences in testing protocols

(Jones et al. 2009).

Chronic Exposure: Growth and Development

Concerning the chronic effects, application of statistical

analysis permitted us to demonstrate that ecologically rel-

evant concentrations (0.025, 0.05 and 0.1 mg L-1) of

chlorpyrifos did not affect mortality rate in R. dalmatina

tadpoles. Chlorpyrifos also showed no noxious effects on

growth, development or time to metamorphosis, even at the

highest concentration tested.

Rana boylii and Pseudacris regilla suffered no reduction

in survival, growth or development when exposed to

chlorpyrifos, but showed increased time to metamorphosis

at a concentration higher than used here (0.2 mg L-1)

(Sparling and Fellers 2009). Widder and Bidwell (2008)

reported in four North American species an effect also on

body weight and development after exposure to similar

concentrations (0.1 and 0.2 mg L-1).

Comparison of tolerance levels in different species is

complicated because of non-homogeneous protocols used

during the experimental phase and because of specimen

provenance from different environments.

Fig. 6 Scanning electron

micrographs of R. dalmatina gill

apparatus after 8 days of

exposure to chlorpyrifos. a Gills

after exposure to 0.025 mg L-1

chlorpyrifos. Note gill tuft

infolding at several points

(arrow = junctional margins)

(bar = 10 lm). b Gills after

exposure to 0.1 mg L-1

chlorpyrifos. Tuft surface is

irregular, and long mucous

cords (arrowhead) could be

seen (bar = 7 lm). c Gills after

exposure to 0.025 mg L-1

chlorpyrifos. Filters show a

normal morphological

arrangement (bar = 20 lm).

d Gills after exposure to

0.05 mg L-1 chlorpyrifos. Note

digitiform protrusions along

filter rows and mucous secretion

(bar = 10 lm)


123

Chronic Exposure: Morphological Effects

In the present study we used a chronic toxicity test that

lasted for the whole larval period; to our knowledge this is

the only report on chronic effects of this pesticide on

morphology, whereas only short-term exposure was pre-

viously reported regarding this topic (Bonfanti et al. 2004;

Colombo et al. 2005; Richards and Kendall 2003).

Despite the relative tolerance of R. dalmatina in terms of

mortality and developmental pattern, subsequent morpho-

logical evaluation revealed physical malformations such as

skeletal defects, flexure of the tail and oedema starting

from day 35 of exposure in all chlorpyrifos-treated groups.

Richards and Kendall (2002) reported, in two different

developmental stages of X. laevis after short-term exposure

(96 h) to several chlorpyrifos concentrations (range

1.7–0.5 mg L-1), consistent malformations such as spinal

abnormality, flexure of the tail and oedema. In the same

species, malformed embryos and larvae were reported also

by other authors (Bonfanti et al. 2004; Colombo et al.

2005). In Ambystoma mexicanum larvae exposed to

chlorpyrifos for 96 h, lateral tail flexure and injuries on

motor activity were detected at concentrations from 0.5 to

3 mg L-1 that did not induce mortality or delay in devel-

opment (Robles-Mendoza et al. 2009).

In the present paper we used chlorpyrifos concentrations

similar to those reported in both short- and long-term

studies (Bonfanti et al. 2004; Colombo et al. 2005; Rich-

ards and Kendall 2003; Sparling and Fellers 2009), and we

showed that early malformations appeared, starting from

day 35 of exposure. According to Richards and Kendall

(2002) it seems that, as development proceeds, malforma-

tions become more pronounced, thus suggesting that late

stages would be most sensitive to chlorpyrifos than early

stages.

After having demonstrated that the three chlorpyrifos

concentrations tested here might induce malformations,

subsequent studies using light microscopy (LM) showed

that chlorpyrifos induces severe alteration in myotome

arrangement and notochord curvature; these effects are

common injuries due to this pesticide (Bonfanti et al. 2004;

El-Merhibi et al. 2004; Richards and Kendall 2002; Ro-

bles-Mendoza et al. 2009).

It is well known that, in amphibians and in other non-

target organism, the predominant toxic role of organo-

phosphorus pesticides is linked to acetylcholinesterase

(AChE) inhibition (Fulton and Key 2001), which induces

typical effects such as complex posturing movements,

axial-muscular abnormalities and body shaking (Behra

et al. 2002; John et al. 2003; Karalliedde and Henry 1993).

Fig. 7 Transmission electron



exposure to 0.05 mg L-1 (a,

c) and 0.1 mg L-1 (b,

d) chlorpyrifos. a Long

projection originating from

pavement cells (PVC) could be

seen in the filter surface (arrow)

(bar = 3 lm). b The

appearance of large lacunae and

intercellular spaces (asterisk)

could be seen in gill tuft

epithelium (bar = 2.5 lm).

c The epithelial surface of filter

shows deep invaginations; in the

external layer, pavement cells

show a great number of

subapical granules

(bar = 3 lm). d Gills tufts

show conspicuous alterations;

note the hypertrophy of the

endothelial cells (arrow) and the

evident enlargement of the

intercellular space (asterisk)

(bar = 4 lm)


123

Our results were comparable to those reported by others on

X. laevis larvae exposed to chlorpyrifos (Bonfanti et al.

2004; Colombo et al. 2005; Richards and Kendall 2002,

2003); therefore, on the basis of our data, it is conceivable

that AChE inhibition may be involved in the morphological

alteration of tail muscles in R. dalmatina as a consequence

of uncontrolled and continuous contractions of the tail

musculature (Lien et al. 1997). Further studies are needed

to define the specific mechanism responsible for the subtle

toxicity of chlorpyrifos.

In the present paper we also evaluated, for the first time,

the chronic effects of chlorpyrifos on R. dalmatina gills;

our results were successful in showing that ecologically

relevant concentrations of chlorpyrifos induce severe

alteration in morphology and ultrastructure of this organ.

The morphological alterations induced by pesticide on

amphibian gill have not been investigated enough; available

information deals with acute effects of paraquat (Lajma-

novich et al. 1998) and both acute and chronic effects of

endosulfan (Bernabo et al. 2008; Brunelli et al. 2010a).

Gill damage and structural changes caused by OPs have

been reported for several fish species (Dutta et al. 1993;

Fanta et al. 2003; Richmonds and Dutta 1989; Rudnicki

et al. 2009) and appear starting from the first hour of

contamination (Fanta et al. 2003).

In R. dalmatina the degree of histopathological altera-

tions of gills is closely linked to pollutant concentrations

and duration of exposure and mainly involves the respira-

tory portion of gill apparatus (gill tufts). The first defence

mechanism in gills against exposure to chlorpyrifos is

secretion of mucus, followed by epithelium detachment

and cell degeneration. These are the most common effects

of pollutants and are not exclusive to chlorpyrifos, as

previously reported in some amphibian species. More

specific responses, such as the appearance of tubular-ves-

icle cells and lamellar bodies, have been previously

reported after both chronic and acute exposure to endo-

sulfan (Bernabo et al. 2008; Brunelli et al. 2010a), whereas

epithelium response to chlorpyrifos exposure seems to be a

defensive response to the aggression rather than an adap-

tive response. Such a general response led us to suppose

that epithelium is unable to react against the chlorpyrifos

exposure, thus reducing the gill’s capacity to adapt or

recover in prolonged treatment.

In R. dalmatina tadpoles chronically exposed to chlor-

pyrifos, the first pathological effects could be observed in

the gills after 8 days and preceded any other evident

alterations such as deformities or behavioural disorders.

As outlined by several authors, histopathological alter-

ations can be good indicators of toxicity of OPs in fish

Fig. 8 Scanning electron



exposure to chlorpyrifos. a,

b Gills after exposure to 0.05

and 0.1 mg L-1 chlorpyrifos.

Tuft ramifications are close to

each other, and the apical

portion collapsed. Note the

large amount of mucous

(bars = 15 and 10 lm). c,

d Gills after exposure to 0.05

and 0.1 mg L-1 chlorpyrifos.

Long mucous cords cover tuft

surface that is wrinkled

(bars = 10 and 5 lm)


123

(Fanta et al. 2003; Rodrigues and Fanta 1998); moreover, it

is well known that in fish biotransformation of insecticide

occurs in gills, causing intoxication and damaging the

structure (Fanta et al. 2003). Gills of amphibians are

physiologically complex and play a role in both respiration

and osmoregulation during aquatic larval stage (Brunelli

et al. 2004; Hourdry 1974; Lajmanovich et al. 1998;

Uchiyama and Yoshizawa 1992; Uchiyama et al. 1990),

also being an important target of pollutants in surrounding

water (Bernabo et al. 2008), thus it is not surprising that

earlier effects of xenobiotics would be histological altera-

tions of gill epithelium.

In summary, the results of the present investigation

allow evaluation of chlorpyrifos effects and potential

consequences for amphibians. In nature, the alterations that

we observed in the gills could result in at least respiratory

difficulties which may affect health and fitness of indi-

viduals; furthermore, muscular and skeletal damage could

result in motionless larvae unable to forage or avoid pre-

dation and in turn in reduced fitness, juvenile recruitment

and survival (Bernabo et al. 2008; Brunelli et al. 2010a;

Colombo et al. 2005; Rohr et al. 2003). Thus, we suggest

that evaluation of morphological parameters proved to be a

valuable toxicological tool, especially in terms of sensi-

tivity and easy identification and quantification.

Acknowledgments The authors would like to thank Regione

Calabria – Assessorato Ambiente for financial assistance (grant

number 171).

References

Abbasi SA, Soni R (1991) Evaluation of water quality criteria for four

common pesticides on the basis of computer-aided studies.

Indian J Environ Health 33:22–24

Adamski Z, Ziemnicki K (2004) Side effects of mancozeb on

Spodoptera exigua (Hubn.) larvae. J Appl Entomol 128:212–217

Ali D, Nagpure NS, Kumar S, Kumar R, Kushwaha B, Lakr WS

(2009) Assessment of genotoxic and mutagenic effects of

chlorpyrifos in freshwater fish Channa punctatus (Bloch) using

micronucleus assay and alkaline single-cell gel electrophoresis.

Food Chem Toxicol 47:650–656

Fig. 9 Transmission electron micrographs of R. dalmatina gill

apparatus after 30 days of exposure to chlorpyrifos. a Micrographs

of gill tuft after exposure to 0.05 mg L-1 chlorpyrifos showing

detachment of external layer from basal cells and appearance of wide

spaces and lacunae (asterisk). Note detachments of epithelium from

connective tissue (arrowhead) (bar = 5 lm). b Micrographs of gill

filter after exposure to 0.025 mg L-1. Note numerous subapical

granules (arrowhead) of pavement cells (bar = 2 lm). c Gill tuft

after exposure to 0.1 mg L-1 chlorpyrifos: detail of a mitochondria-

rich cell. Note wide spaces and degenerating cells. Endothelial cells

are hypertrophic (arrow) (bar = 4 lm). d Micrographs of gill filter

after exposure to 0.1 mg L-1. Note the enlargement of intercellular

spaces and degeneration of external cells (asterisk) (bar = 5 lm)


123

American Society for Testing Material (ASTM) (1997) Standard

practice for conducting acute toxicity tests with fishes, macro-

invertebrates, and amphibians. American society for testing and

materials standards, Philadelphia, pp E729–E790

Au DWT (2004) The application of histo-cytopathological biomark-

ers in marine pollution monitoring: a review. Mar Pollut Bull

48:817–834

Barron MG, Woodburn KB (1995) Ecotoxicology of chlorpyrifos.

Rev Environ Contam Toxicol 144:1–93

Behra M, Cousin X, Bertrand C, Vonesech JL, Biellmann D,

Chatonnet A, Strahle U (2002) Acetylcholinesterase is required

for neuronal and muscular development in the zebrafish embryo.

Neuroscience 5:111–118

Belden JB, Hofelt CS, Lydy MJ (2000) Analysis of multiple

pesticides in urban storm water using solid-phase extraction.

Arch Environ Contam Toxicol 38:7–10

Bernabo I, Brunelli E, Berg C, Bonacci A, Tripepi S (2008) Endosulfan

acute toxicity in Bufo bufo gills: ultrastructural changes and nitric

oxide synthase localization. Aquat Toxicol 86:447–456

Berrill M, Coulson D, McGillivray L, Pauli B (1998) Toxicity of

endosulfan to aquatic stage of anuran amphibians. Environ

Toxicol Chem 9:1738–1744

Bjørling-Poulsen M, Andersen HR, Grandjean P (2008) Potential

developmental neurotoxicity of pesticides used in Europe.

Environ Health 7:50

Bonfanti P, Colombo A, Orsi F, Pizzetto I, Andrioletti M, Bacchetta

R, Manteca P, Fascio U, Vailati G, Vismara C (2004)

Comparative teratogenicity of Chlorpyrifos and Malathion on

Xenopus laevis development. Aquat Toxicol 70:189–200

Bonin J, Ouellet M, Rodrigue J, Desgranges JL, Gagne F, Sharbel TF,

Lowcock LA (1997) Measuring the health of frogs in agricultural

habitats subjected to pesticides. In: Green DM (ed) Amphibians

in decline: Canadian studies of a global problem. Society for the

Study of Amphibians and Reptiles, St Louis, pp 246–257

Boone MD, Bridges CM, Rothermel BB (2001) Growth and

development of larval green frogs (Rana clamitans) exposed to

multiple doses of an insecticide. Oecologia 129:518–524

Bridges CM (2000) Long-term effects of pesticide exposure at various

life stages of the southern leopard frog (Rana sphenocephala).

Arch Environ Contam Toxicol 39:91–96

Bridges CM, Boone MD (2003) The interactive effects of UV-B and

insecticide exposure on tadpole survival, growth and develop-

ment. Biol Conserv 113:49–54

Bridges CM, Semlitsch RD (2000) Variation in pesticide tolerance of

tadpoles among and within species of Ranidae and patterns

amphibian decline. Conserv Biol 14:490–1499

Brunelli E, Tripepi S (2005) Effects of Low pH acute exposure on

survival and gill morphology in Triturus italicus larvae. J Exp

Zool A 303:946–957

Brunelli E, Perrotta E, Tripepi S (2004) Ultrastructure and develop-

ment of the gills in Rana dalmatina (Amphibia, Anura).

Zoomorphology 123:203–211

Brunelli E, Talarico E, Corapi B, Perrotta I, Tripepi S (2008) Effects

of a sublethal concentration of sodium lauryl sulphate on the

morphology and Na?/K? ATPase activity in the gill of the

ornate wrasse (Thalassoma pavo). Ecotoxicol Environ Saf 71:

436–445

Brunelli E, Bernabo I, Berg C, Lundstedt-Enkel K, Bonacci A,

Tripepi S (2009) Environmentally relevant concentrations of

endosulfan impair development, metamorphosis and behaviour

in Bufo bufo tadpoles. Aquat Toxicol 91:135–142

Brunelli E, Bernabo I, Sperone E, Tripepi S (2010a) Gill alterations as

biomarkers of chronic exposure to endosulfan in Bufo bufotadpoles. Histol Histopathol 25:1519–1529

Brunelli E, Mauceri A, Maisano M, Bernabo’ I, Giannetto A, De

Domenico E, Corapi B, Tripepi S, Fasulo S (2010b)

Ultrastructural and immunohistochemical investigation on the

gills of the teleost. Thalassoma pavo L., exposed to cadmium.

Acta Histochem 112:251–258

Carriger JF, Rand GM (2008) Aquatic risk assessment of pesticides in

surface waters in and adjacent to the Everglades and Biscayne

National Parks: II. Probabilistic analyses. Ecotoxicology

17:680–696

CCME (Canadian Council of Ministers of the Environment) (2008)

Canadian water quality guidelines for the protection of aquatic

life: chlorpyrifos. In: Canadian environmental quality guidelines,

1999, Canadian Council of Ministers of the Environment,

Winnipeg

Colombo A, Orsi F, Bonfanti P (2005) Exposure to the organophos-

phorus pesticide chlorpyrifos inhibits acetylcholinesterase activ-

ity and effects muscular integrity in Xenopus leavis larvae.

Chemosphere 61:1665–1671

Dam K, Seidler FJ, Slotkin TA (2000) Chlorpyrifos exposure during a

critical neonatal period elicits gender-selective deficits in the

development of coordination skills and locomotor activity. Brain

Res Dev Brain Res 121:179–187

Davidson C, Shaffer HB, Jennings MR (2002) Spatial tests of the

pesticide drift, habitat destruction, UV-B, and climate-change

hypotheses for California amphibian declines. Conserv Biol

16:1588–1601

Dutta HM, Richmonds CR, Zeno T (1993) Effects of diazinon on the

gills of bluegill sunfish Lepomis macrochirus. J Environ Pathol

12:219–227

El-Merhibi A, Kumar A, Smeaton T (2004) Role of piperonyl

butoxide in the toxicity of chlorpyrifos to Ceriodaphnia dubiaand Xenopus leavis. Ecotox Environ Safe 57:202–212

EPA (Environmental Protection Agency) (2000) Cancellation order

EPA (Environmental Protection Agency) (2002) Interim reregistra-

tion eligibility decision for chlorpyrifos. US Environ. Protection

Agency, Washington

EPA (Environmental Protection Agency) (2006) Reregistration Eli-

gibility Decision (RED) for Chlorpyrifos. U.S. Environmental

Protection Agency, Office of prevention, pesticides and toxic

substances, Office of pesticide programs, U.S. Government

Printing Office, Washington

Eurostat: The use of plant protection products in the European Union,

Data 1992–2003, Eurostat statistical books (2007)

Fanta E, Rios FS, Romao S, Vianna ACC, Freiberger S (2003)

Histopathology of the fish Corydoras paleatus contaminated

with sub lethal levels of organophosphorus in water and food.

Ecotoxicol Environ Saf 54:119–130

Finney DJ (1971) Probit Analysis, 3rd edn. Cambridge University

Press, New York, p 668

Fulton MH, Key PB (2001) Acetylcholinesterase inhibition in

estuarine fish and invertebrates as an indicator of organophos-

phorus insecticide exposure and effects. Environ Toxicol Chem

20:37–45

Giesy JP, Solomon KR, Coates JR, Dixon KR, Giddings JM, Kenaga

EE (1999) Chlorpyrifos: ecological risk assessment in North

American aquatic environments. Rev Environ Contam Toxicol

160:96–102

Gilliom RJ, Barbash JE, Crawford CG, Hamilton PA, Martin JD,

Nakagaki N, Nowell LH, Scott JC, Stackelberg PE, Thelin GP,

Wolock DM (2006) The Quality of Our Nation’s Waters-

Pesticides in the Nation’s Streams and Ground Water,

1992–2001. U.S. Geological Survey Circular 1291

Gomes J, Dawodu AH, Llyo O, Revitt DM, Anilal SV (1999) Hepatic

injury and disturbed amino acid metabolism in mice following

prolonged exposure to organophosphorus pesticides. Hum Exp

Toxicol 18:33–37

Gosner KL (1960) A simplified table for staging anuran embryos and

larvae with notes on identification. Herpetologica 16:183–190


123

Hopkins WA (2007) Amphibians as models for studying environ-

mental change. ILAR J 48:270–277

Houlahan JE, Findlay CS (2003) The effects of adjacent land use on

wetland amphibian species richness and community composi-

tion. Can J Fish Aquat Sci 60:1078–1094

Houlahan JE, Findlay CS, Schmidt BR, Meyer AH, Kuzmin SL

(2000) Quantitative evidence for global amphibian population

decline. Nature 404:752–755

Hourdry J (1974) Etude des branchies ‘‘internes’’ puis de leur

regression au moment de la metamorphose, chez la larve de

Discoglossus pictus (OTTH), Amphibien Anoure. J Microsc

Paris 20:165–182

Jergentz S, Mugni H, Bonetto C, Schulz R (2005) Assessment of

insecticide contamination in runoff and stream water of small

agricultural streams in the main soybean area of Argentina.


John M, Oommen A, Zachariah A (2003) Muscle injury in

organophosphorus poisoning and its role in the development of

intermediate syndrome. Neurotoxicology 24:43–53

Jones DK, Hammond JI, Relyea RA (2009) Very highly toxic effects

of endosulfan across nine species of tadpole: lag effects and

family level selectivity. Environ Toxicol Chem 28:1939–1945

Karalliedde L, Henry JA (1993) Effects of organophosphates on

skeletal muscle. Hum Exp Toxicol 12:289–296

Kiely T, Donaldson D, Grube A (2004) Pesticides industry sales and

usage. 2000 and 2001 market estimates. Washington, DC: U.S.

Environmental Protection Agency, Report No. EPA-733-R-99-001

Lajmanovich RC, Izaguirre MF, Casco VH (1998) Paraquat tolerance

and alteration of internal gill structure of Scinax nasica tadpoles

(Anura: Hylidae). Arch Environ Contam Toxicol 34:364–369

Lien NT, Adriaens D, Janssen CR (1997) Morphological abnor-

malities in African catfish (Clarias gariepinus) larvae exposed to

malathion. Chemosphere 35:1475–1486

Mazanti LE, Rice C, Bialek K, Sparling D, Stevenson C, Johnson

WE, Kangas P, Rheinstein J (2003) Aqueous-phase disappear-

ance of atrazine, metolachlor, and chlorpyrifos in laboratory

aquaria and outdoor macrocosms. Arch Environ Contamin

Toxicol 44:67–76

McCarthy F, Shugart LR (1990) Biomarkers of environmental

contamination. Lewis, Chelsea

Moore MT, Schulz R, Cooper CM, Smith S Jr, Rodgers JH Jr (2002)

Mitigation of chlorpyrifos runoff using constructed wetlands.


NRA (National Registration Authority) (2000) The NRA review of

chlorpyrifos. National Registration Authority, Canberra, Australia

Pablo F, Krassoi FR, Jones PRF, Colville AE, Hose GC, Lim RP

(2008) Comparison of the fate and toxicity of chlorpyrifos-

laboratory versus a coastal mesocosm system. Ecotoxicol

Environ Saf 71:219–229

Peltzer PM, Lajmanovich RC, Sanchez-Hernandez JC, Cabagna MC,

Attademo AM, Basso A (2008) Effects of agricultural pond

eutrophication on survival and health status of Scinax nasicustadpoles. Ecotoxicol Environ Saf 70:185–197

Pollet I, Bendell-Young LI (2000) Amphibians as indicators of

wetland quality in wetlands formed from oil sands effluent.

Environ Toxicol Chem 19:2589–2597

Rahman MF, Mahboob M, Danadevi K, Saleha B, Grover P (2002)

Assessment of genotoxicity effects of chlorpyrifos and aceta-

phate by the comet assay in mice leucocytes. Mutat Res

516:139–147

Relyea RA (2005) The impact of insecticides and herbicides on the

biodiversity and productivity of aquatic communities. Ecol Appl

15:618–627

Relyea RA, Hoverman JT (2006) Assessing the ecology in ecotoxi-

cology: a review and synthesis in freshwater systems. Ecol Lett

9:1157–1171

Ricceri L, Venerasi A, Capone F, Cometa MF, Lorenzini P, Fortuna

S, Calamandrei G (2006) Developmental neurotoxicity of

organophosphorous pesticides: fetal and neonatal exposure to

chlorpyrifos alters sex-specific behaviors at adulthood in mice.

Toxicol Sci 93:105–113

Richards SM, Kendall RJ (2002) Biochemical effects of chlorpyrifos

on two developmental stages of Xenopus laevis. Environ Toxicol

Chem 21:1826–1835

Richards SM, Kendall RJ (2003) Physical effects of chlorpyrifos on

two stages developmental stages of Xenopus laevis. J Toxicol

Environ Health A 66:75–91

Richmonds C, Dutta HM (1989) Histopathological changes induced

by malathion in the gills of bluegill Lepomis macrochirus. Bull

Environ Contam Toxicol 43:123–130

Robles-Mendoza C, Garcıa-Basilio C, Cram-Heydrich S, Hernandez-

Quiroz M, Vanegas-Perez C (2009) Organophosphorus pesti-

cides effect on early stages of the axolotl Ambystoma mexicanum(Amphibia: Caudata). Chemosphere 74:703–710

Rodrigues EL, Fanta E (1998) Liver histopathology of the fish

Brachydanio rerio after acute exposure to sublethal levels of the

organophosphate Dimetoato 500. Rev Bras Zool 15:441–450

Rohr JR, Elskus AA, Shepherd BS, Crowley PH, McCarthy TM,

Niedzwiecki JH, Sager T, Sih A, Palmer BD (2003) Lethal and

sublethal effects of atrazine, carbaryl, endosulfan, and octylphe-

nol on the streamside salamander, Ambystoma barbouri. Environ

Toxicol Chem 22:2385–2392

Rudnicki CAM, Melo GC, Donatti L, Kawall HG, Fanta E (2009)

Gills of juvenile fish Piaractus mesopotamicus as histological

biomarkers for experimental sub-lethal contamination with the

organophosphorus Azodrin�400. Braz Arch Biol Technol 52:

1431–1441 ‘‘in memoriam’’

Schuytema GS, Nebeker AV (1996) Amphibian toxicity data for

water quality criteria chemicals. EPA/600/R-96/124, U.S. EPA.

NHEERL/WED, Corvallis

Semlitsch RD, Foglia M, Mueller A (1995) Short-term exposure to

triphenyltin affects the swimming and feeding behaviour of

tadpoles. Environ Toxicol Chem 14:1419–1423

Song X, Violin JD, Seidler FJ, Slotkin TA (1998) Modelling the

developmental neurotoxicity of chlorpyrifos in vitro: macromol-

ecule synthesis in PC12 cells. Toxicol Appl Pharmacol

151:182–191

Sparling DW, Fellers G (2007) Comparative toxicity of chlorpyrifos,

diazinon, malathion and their oxon derivatives to larval Ranaboylii. Environ Pollut 147:535–539

Sparling DW, Fellers G (2009) Toxicity of two insecticides to

California, USA, Anurans and its relevance to declining

amphibian populations. Environ Toxicol Chem 28:1696–1703

Taylor B, Skelly D, Demarchis LK, Slade MD, Galusha D,

Rabinowitz PM (2005) Proximity to pollution sources and risk

of amphibian limb malformation. Environ Health Perspect

113:1497–1501

Uchiyama M, Yoshizawa H (1992) Salinity tolerance and structure of

external and internal gills in tadpoles of the crab-eating frog,

Rana cancrivora. Cell Tissue Res 267:35–44

Uchiyama M, Yoshizawa H, Wakasugi C, Oguro C (1990) Structure

of internal gills in tadpoles of the crab-eating frog, Ranacancrivora. Zool Sci 7:623–630

UNEP, FAO, WHO, IAEA (1991) Assessment of the state of

pollution of the Mediterranean Sea by organophosphorus

compounds. MAP Technical Reports Series no 58, UNEP,

Athens

Venturino A, Rosenbaum E, Caballero de Castro A (2003) Biomark-

ers of effect in toads and frogs. Biomarkers 8:167–186

Widder PD, Bidwell JR (2008) Tadpole size, cholinesterase activity,

and swim speed in four frog species after exposure to sub-lethal

concentrations of chlorpyrifos. Aquat Toxicol 88:9–18


123

Toxicity of Chlorpyrifos to Larval Rana dalmatina: Acute and Chronic Effects on Survival,...

Documents

Transcript of Toxicity of Chlorpyrifos to Larval Rana dalmatina: Acute and Chronic Effects on Survival,...