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Ecotoxicology and Environmental Safety 67 (2007) 218–226

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Highlighted article

The use of Chironomus riparius larvae to assess effects of pesticides fromrice fields in adjacent freshwater ecosystems

Mafalda S. Faria�, Antonio J.A. Nogueira, Amadeu M.V.M. Soares

CESAM & Departamento de Biologia da Universidade de Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal

Received 30 March 2006; received in revised form 10 November 2006; accepted 20 November 2006

Available online 16 January 2007

Abstract

A bioassay with Chironomus riparius larvae, using larval development and growth as endpoints, was carried out inside a rice field and

in the adjacent wetland channel in Portugal, during pesticide treatments (molinate, endosulfan and propanil) to determine impact caused

by pesticide contamination in freshwater ecosystems. The bioassay was also performed under laboratory conditions, to assess whether in

situ and laboratory bioassays demonstrated comparable results. Growth was inhibited by concentrations of endosulfan (2.3 and

1.9mgL�1 averages) in water from rice field in both the field and laboratory, and by concentrations of endosulfan (0.55 and 0.76 mgL�1

averages) in water from the wetland channel in the laboratory bioassay, while development was not affected. C. riparius larvae were not

affected by molinate and propanil concentrations. The results indicate that endosulfan treatments in rice fields may cause an ecological

impairment in adjacent freshwater ecosystems. The results also indicate that laboratory testing can be used to assess in situ toxicity

caused by pesticide contamination.

r 2006 Elsevier Inc. All rights reserved.

Keywords: Chironomus riparius; Bioassays; Endosulfan; Molinate; Propanil; Rice field; Ecological impact; Wetlands

1. Introduction

Several pesticides are globally used in agriculture andmay enter aquatic environments through spray drift orrunoff events, drainage or leaching, resulting in contamina-tion of surface and ground waters (Leonard et al., 1999;Schulz et al., 2001a, b; Cerejeira et al., 2003). Pesticides usedin Portuguese agricultural areas have been found in bothsurface and ground waters. Insecticides (e.g., endosulfan,lindane, chlorfenvinphos) and herbicides (e.g., propanil,molinate, atrazine) have been detected in the surface watersof three river basins (Tejo, Sado and Guadiana) from 1983to 1999, and in ground water samples, collected from wellsin seven different agricultural areas in the Tejo and Sadobasins between 1991 and 1998 (Cerejeira et al., 2003).Waters contaminated with pesticides can cause toxic effectsto aquatic flora and fauna (Forney and Davis, 1981; Mulla

e front matter r 2006 Elsevier Inc. All rights reserved.

oenv.2006.11.018

ing author. Fax: +351 234426408.

ess: faria.mafalda@gmail.com (M.S. Faria).

and Mian, 1981), and may affect human health (Buxtonand Stedfast, 1994; Sumner and McLaughlin, 1996).Rice, along with wheat and corn, is one of the three

crops on which many human subsists. Almost two billionpeople depend on rice for their staple diet (Ronald, 1997).It is also an important crop in terms of pesticide use,particularly herbicides. The use of pesticides during cerealgrowth may affect the quality of the surrounding aquaticenvironment. In Portugal, rice is an irrigated crop andwater is frequently discharged into surrounding waterbodies (Pereira et al., 2000). Studies designed to clarify thepotential toxic effects of rice-associated pesticides onaquatic ecosystems are therefore an important tool forbiological understanding and environmental management.However, the impact of pesticides on aquatic ecosystems isoften difficult to assess because they degrade very quickly(Barry and Logan, 1998), can be absorbed onto thesediments (Peterson and Batley, 1993; Hamer et al., 1999)and peak concentrations that coincide with storm runoffevents may be difficult to measure (Cooper, 1996).

ARTICLE IN PRESS

125 m0 m

Arzila Marsh

High

Contaminated

Site

Reference

Site

Low

Contaminated

Site

Wetland C

hannel

Rice F

ield

s

Fig. 1. Study sites where the in situ bioassay were performed.

M.S. Faria et al. / Ecotoxicology and Environmental Safety 67 (2007) 218–226 219

The aim of this study was to determine the effect ofpesticides used in rice treatments on a selected aquaticmacroinvertebrate and assess the potential risk of thesechemicals to aquatic ecosystems. A bioassay using Chir-

onomus riparius larvae and employing biological responses(development, growth) as endpoints was deployed ina rice field and in the adjacent wetland channel duringthe period of pesticide spraying. Third instar larvaewere deployed in rice fields 48 h after each pesticidetreatment (endosulfan, molinate, propanil). Bioassaysusing contaminated water from the rice fields and condi-tioned sediments were also conducted under laboratoryconditions, at the same time as the in situ bioassay, toassess whether in situ and laboratory tests demonstratedcomparable results.

Chironomidae larvae have been recorded as pests of ricegrowing in many temperate countries (Surakarn and Yano,1995). The larvae either attack the seed itself, or feed on theroots or shoots of young seedlings (Helliwell and Stevens,2000; Stevens et al., 2000). Damage can arise through theirtunnelling activity in the sediment which can destabilise theroot systems of young plants and increase turbidity,reducing photosynthesis and slowing the growth ofsubmerged seedlings (Helliwell and Stevens, 2000). ButChironomids are also important macroinvertebrates in theecology of the aquatic ecosystems. They are the mostwidespread and often abundant group of insects infreshwater environments (Pinder, 1995). They provide animportant link between different trophic levels, and play akey role in recycling organic matter (Prat and Rieradevall,1995; Garcıa-Berthou, 1999).

2. Material and methods

2.1. Study sites and pesticide treatment

In situ bioassays with C. riparius were carried out in rice fields within

the Natural Reserve of Arzila Marsh, a protected wetland located near the

Mondego river in Central Portugal (Fig. 1). The site designated as highly

contaminated was located inside a rice field, representing one of the several

rice fields that were treated with pesticides (Fig. 1). The control and

low contamination sites were located in a channel bordering the rice fields

(Fig. 1). The control site was located upstream and the low contamination

site was located downstream from the point where the channel received

water from the (highly contaminated) rice fields.

Molinate (C9H17NOS) is a selective herbicide which belongs to the

thiocarbamate class. It is used to control grassy weeds in rice fields

(Hartley and Kidd, 1983; Meister, 1991) and is of low persistence in the

soil environment, with a field half-life of 5–21 days (Wauchope et al.,

1992). It was the first pesticide to be introduced in the rice field in the study

site. Because it rapidly volatilizes if the soil is wet (Brooks, 1980), it was

ploughed into the dry soil before the water was allowed to inundate the

site. After 3 weeks, endosulfan (C9H6Cl6O3S) was applied. Endosulfan is

an organochlorine insecticide of the cyclodiene subgroup which acts as a

contact poison for a wide variety of insects and is highly persistent in the

environment (Helliwell and Stevens, 2000). This insecticide is frequently

used, associated with deltamethrin, by farmers in Central Portugal to

control chironomid larvae and crayfish Procambarus clarkii infestation in

rice fields. Approximately 1 month after endosulfan application, the

herbicide propanil (C9H9Cl2NO) was applied. Propanil is an acetanilide

post-emergence herbicide used to control numerous weeds that occur in

rice and potato fields. This herbicide is soluble in water but adsorbs

weakly to soil particles (Wauchope et al., 1992; Weed Science Society of

America, 1994). It rapidly breaks down in water due to microbial activity

and has a low persistence in soil, with a field half-life of 1–3 days

(Wauchope et al., 1992; Weed Science Society of America, 1994).

2.2. Test organisms: culture conditions

C. riparius larvae used in the bioassays were obtained from laboratory

cultures established at the Biology Department, University of Aveiro.

The culture unit was an enclosed transparent acrylic box (120 cm�

60 cm� 40 cm), containing all the conditions necessary to complete the

whole life cycle of the chironomids and large enough to allow swarming

and copulation of emerged adults (OECD, 2000). Cultures were

maintained at 2072 1C, with a 14 h light: 10 h dark photoperiod. At the

start of a new culture �120 newborn larvae were introduced into plastic

beakers (27 cm� 13 cm� 11.5 cm) containing a 2 cm layer of organic

matter free natural sediment (ignited in a muffle furnace for 6 h at 450 1C)

from the Bestanc-a river (north Portugal) and ASTM reconstituted hard

water (ASTM, 2000). This sediment was considered unpolluted because it

came from a river segment known to be free from pollution (Faria et al.,

2006). A suspension of ground TetraMins (Tetrawerke, Germany) of

1mg larvae�1 day�1 was added as the food source (Naylor and Rodrigues,

1995) and each beaker was gently aerated. Seven days later, larvae were

transferred to new culture beakers with fresh media, sediment and food

(60 larvae per beaker) until emergence.

2.3. In situ bioassays

Twenty four hours after each pesticide treatment in rice fields, five

chambers, with a layer of 5 cm natural sediment previously collected from

the Bestanc-a river, were placed in each site, to condition the sediment for

in situ and laboratory bioassays. Two additional chambers were deployed,

to determine pesticide concentrations in the sediment on day 0 and on day

6 of the in situ bioassay. Six additional chambers were placed in each site

to condition sediment during 24 h for laboratory tests. All chambers where

placed in sites using a holding structure, constructed with 2 baskets

(10� 21� 25 cm) made of plastic coated metal wire. In situ chambers were

modified from a design by Soares et al. (2005) consisting of PVC tube

(20 cm high and 4.5 cm inner diameter) with four openings, two laterals

(�10 cm� 7 cm), one at the top and one at the bottom. The openings were

covered with 200-mm nylon mesh to allow contact with the sediment and

water flow through the chambers.

ARTICLE IN PRESSM.S. Faria et al. / Ecotoxicology and Environmental Safety 67 (2007) 218–226220

On the first day of the bioassays (day 0), 2 days after pesticide

treatment, five third instar larvae of C. riparius were introduced into each

assay chamber using a plastic pipette via a plastic tube (tube size depended

on water depth) glued to the 200-mm nylon mesh that covered the assay

chamber (glued with white thermal glue, Elis-Taiwan, TN122/WS,

Taiwan, which has been shown to be nontoxic to cladocerans; Pereira

et al., 1999), to avoid sediment disturbance. TetraMins (�30mg in

suspension) was added to each of these chambers. The body length

and head capsule width of 30 additional larvae were measured to

determine initial body length and development stage of larvae. The

conditioned sediment of the additional five chambers was collected for

use in the laboratory tests and the sediment from the other chambers,

previously added were collected for pesticide analysis. Water was

also collected at each site to use in laboratory tests and for pesticide

analysis.

At the end of the experiment (day 6) in each site, the surviving larvae

were collected from the five chambers, killed and preserved in Von Torne

conservant (Gama, 1964) for later measurement. Sediment from the sixth

chamber was collected for pesticide analysis. Water samples from each site

were also collected for pesticide analysis. Larval length and developmental

stage were determined by measuring body length and head capsule width

of larvae, respectively, using a stereomicroscope (Leica MS5, Leica

Microsystems AG, Germany) fitted with a calibrated eye-piece micro-

meter. Growth (body length increase) of larvae was calculated by

subtracting the average initial length from each individual final length.

C. riparius larvae instar was determined according to Watts and Pascoe

(2000).

2.4. Laboratory bioassays

In all treatments, on day 0, field water (300ml) was introduced into five

chambers (glass flasks) with a conditioned sediment layer of 5 cm depth.

five third instar larvae were introduced into each of the chambers and

30mg of Tetramin, in suspension, was added to each chamber. The

chambers were provided with artificial aeration under a 14 h light: 10 h

dark photoperiod with a temperature of �20 1C. At the end of the

experiment (day 6), the surviving larvae were collected from the chambers,

killed and preserved in Von Torne conservant (Gama, 1964) for later

measurement. Measurement procedures were as described for in situ

bioassays. At the same time, another chamber was also included in each

treatment without the addition of larvae and its water and sediment were

collected for pesticide analysis at the end of the experiment. The

treatments used were: (1) water and conditioned sediment brought from

each site and (2) ASTM hard water (ASTM, 2000), control water, and

natural sediment from the Bestanc-a river, used as a control treatment.

2.5. Physical and chemical analysis

In field and laboratory tests, hand-held field meters were used to

measure water pH (pH 330/SEF-2, Weilheim, Germany), conductivity

(Cond 330i/SET, Weilheim, Germany) and dissolved oxygen (DO)

(Oxi 330/SET, Weilheim, Germany) and a maximum–minimum thermo-

meter was used to determine the minimum and maximum temperature

of water (880/847881, Electronic Temperature Instruments Ltd, UK)

for day 0 and day 6. Water samples were collected on day 0 and on day 6

to determine nitrate, nitrite, ammonia and phosphate concentrations

of the water with a direct reading spectrophometer (DR/2000, Hach

company, USA).

The sediment and water samples collected for pesticide analysis were

placed in dark glass containers and preserved at 4 1C in the dark. The

analysis of endosulfan and molinate were carried out by organic solvent

extraction with cartridges containing Oasiss Hydrophilic–Lipophilic

Balance Sorbent (HLB), a reversed-phase sorbent for all compounds,

followed by a concentration determination using liquid chromatography-

mass spectrometry (LC-MS). Propanil analysis was carried out by solid

phase extraction (SPE), and concentrations were determination by liquid

chromatography-mass spectrometry (LC-MS). Since endosulfan is fre-

quently used associated with deltamethrin by farmers in Central Portugal,

water and sediment samples collected to determine endosulfan concentra-

tions were also used to determined deltamethrin concentrations, but the

results of deltamethrin analysis were below the detection limits

(0.002mgL�1). All pesticide concentrations were determined by the

accredited laboratory Innova-lab, Spain.

2.6. Statistical analysis

The statistical analysis comprised one-way analysis of variance

(ANOVA) followed, when necessary, by Tukey HSD multiple comparison

tests to test for significant differences in responses of biological endpoints

(development and growth) between treatments and between laboratory

and field exposures (Zar, 1996). One-way analysis of variance (ANOVA)

followed by Dunnett tests was used to compare responses of biological

endpoints between reference and contaminated (low- and high-) treat-

ments (Zar, 1996). Responses were tested for normality using the

Kolmogorov–Smirnov test. Linear regression analyses were used to

investigate significant relationships between biological responses and

minimum and maximum water temperature. Statistical analyses were

performed using MinitabTM Release 14.

3. Results

3.1. Physical and chemical parameters

Physical and chemical conditions were more variable inthe field than in the laboratory (Tables 1 and 2). Physicaland chemical parameters of water quality in the in situbioassays were similar in reference and low contaminatedsites but differed between these sites and the highlycontaminated sites for the three pesticides (Table 1).Higher values of maximum temperature were observed inthe highly contaminated site during the exposure period.Dissolved oxygen and pH values were also higher in thehighly contaminated site than in reference and lowcontamination sites, during all pesticide treatments. Inthe laboratory bioassays, physical and chemical parametersof water samples were similar in all pesticide treatments(Table 2).

3.2. Pesticide concentrations

Molinate and propanil concentrations and total endo-sulfan concentrations, the sum of a and b isomers andendosulfan sulphate, in water and sediment during fieldand laboratory exposure are presented in Table 3. On day0, the endosulfan concentration in water varied from0.91–2.78 mgL�1. In the sediment of the rice field this was0.62 mg kg�1, while at the sites in the wetland channelendosulfan was not detected in the sediment. On day 0, themolinate concentration in the water column varied from4.63 to 0.31 mgL�1 and in sediment it ranged from 1.43 to1.09 mgL�1. On day 6, the endosulfan and molinateconcentrations in both water and sediment were higher infield than in laboratory. Propanil was not detected insediment samples, however, it was detected in the waterfrom the rice field on day 0 (2.58 mgL�1) and in thelaboratory mesocosms on day 6 (0.14 mgL�1).

ARTICLE IN PRESS

Table 1

Physical and chemical parameters of water in day 6 and day 0 (in parenthesis) of in situ bioassays

Treatmenta Tempmin Tempmax pH DO Cond Nitrate Nitrite Amonia Phosphate Hardness

(1C) (1C) (mgL�1) (ms cm�1) (mgL�1) (mgL�1) (mgL�1) (mgL�1) (mgCaCO3L�1)

Endosulfan

Ref 15.0 26.0 7.9 (7.9) 7.0 (6.8) 790 (730) 2.20 (2.13) 0.10 (0.05) 0.26 (0.59) 0.57 (0.37) 26.3 (34.5)

Low 14.0 29.0 7.8 (7.9) 7.3 (6.9) 810 (733) 2.12 (2.23) 0.12 (0.05) 0.27 (0.50) 0.50 (0.42) 25.9 (40.9)

High 12.5 40.0 8.8 (8.0) 9.7 (10.7) 667 (507) 0.95 (1.7) 0.02 (0.07) 0.20 (0.28) 0.01 (0.04) 15.8 (18.6)

Molinate

Ref 12.0 20.0 7.7 (6.8) 8.7 (8.5) 315 (389) 1.14 (1.32) 0.02 (0.02) 0.40 (1.14) 0.24 (0.15) 47.5 (44.0)

Low 13.0 29.0 7.8 (6.5) 9.2 (8.6) 442 (352) 0.72 (1.54) 0.005 (0.01) 0.93 (1.10) 0.04 (0.26) 23.86 (32.8)

High 7.0 32.0 7.2 (8.3) 10.2 (10.7) 397 (386) 0.43 (1.51) 0.002 (0.02) 0.38 (0.92) 0.02 (0.08) 16.2 (20.8)

Propanil

Ref 12.0 30.0 8.4 (7.9) 7.4 (7.8) 716 (751) 2. 03 (2.20) 0.02 (0.03) 0.28 (0.49) 0.11 (0.08) 22.6 (27.2)

Low 17.0 31.0 8.2 (7.9) (7.3) 8.02 713 (781) 1.93 (2.11) 0.02 (0.04) 0.26 (0.45) 0.10 (0.07) 27.0 (27.7)

High 18.0 47.0 8.9 (8.0) 10.5 (10.7) 636 (831) 0.12 (0.43) 0.02 (0.04) 0.51 (0.22) 0.03 (0.02) 15.1 (24.3)

Abbreviations: Temp: temperature; Cond: conductivity; DO: dissolved oxygen.aRef ¼ water from reference site and conditioned natural sediment, Low ¼ water from low contaminated site and conditioned natural sediment,

High ¼ water from high contaminated site and conditioned natural sediment.

Table 2

Physical and chemical parameters of water determined in day 6 and day 0 (in parenthesis) of laboratory bioassays

Treatmenta Temp min (1C) Temp max (1C) pH DO (mgL�1) Cond (ms cm�1)

Endosulfan

Control 17.8 18.0 7.6 (7.7) 7.0 (7.5) 666 (630)

Ref 17.3 18.8 8.0 (7.7) 7.5 (7.2) 766 (737)

Low 17.3 19.0 8.2 (7.6) 7.3 (7.7) 801 (830)

High 17.4 18.7 8.3 (7.9) 7.2 (8.0) 714 (660)

Molinate

Control 19.1 20.4 8.4 (7.6) 8.1 (8.3) 624 (595)

Ref 19.3 20.3 8.3 (7.8) 8.3 (8.9) 590 (423)

Low 19.0 19.4 8.2 (7.9) 8.0 (8.9) 459 (390)

High 19.0 19.4 8.2 (7.6) 8.5 (8.7) 490 (397)

Propanil

Control 19.8 20.5 8.0 (7.7) 7.5 (8.0) 667 (615)

Ref 19.6 20.3 8.1 (7.9) 7.5 (7.3) 710 (733)

Low 19.4 19.8 8.2 (8.0) 7.2 (8.0) 704 (730)

High 19.7 20.0 8.0 (7.9) 7.9 (8.2) 675 (798)

Abbreviations: Temp: temperature; Cond: conductivity; DO: dissolved oxygen.aControl ¼ ASTM and not conditioned natural sediment, Ref ¼ water from reference site and conditioned natural sediment, Low ¼ water from low

contaminated site and conditioned natural sediment, High ¼ water from high contaminated site and conditioned natural sediment.

M.S. Faria et al. / Ecotoxicology and Environmental Safety 67 (2007) 218–226 221

3.3. Biological responses

The herbicides molinate and propanil did not causebiological responses of C. riparius larvae (Fig. 2). Nosignificant (P40.05) differences in endpoints between treat-ments were observed in laboratory and field exposures toherbicides, although a trend of decreasing larval growth wasobserved from the highest to the lowest (reference)contaminated sites during molinate exposure in field (Fig. 2).

The insecticide endosulfan had a negative effect on larvalgrowth under field and laboratory exposure, but did notaffect larval development. During endosulfan exposure, all

larvae changed from the third to the fourth instar and nosignificant (P40.05) differences were observed in headcapsule width of larvae between treatments, although lowervalues were observed in the rice field channel (Fig. 3).In situ, larval growth was significantly lower in the rice

field than in the reference and low contaminated sites in thewetland channel (Fig. 3), whilst no significant differences inlarval growth were observed between reference and lowcontaminated sites (P40.05).In the laboratory differences in larval growth were

smaller but similarly significant between treatments, withlower growth observed in the treatment with water and

ARTICLE IN PRESS

Table 3

Pesticide concentrations in water (mgL�1) and sediment (mgKg�1) treatments during in situ and laboratory bioassays

Treatment (d.l.)a Water (mgL�1) Sediment (mg kg�1)

day 0 day 6 (field) day 6 (lab) day 0 day 6 (field) day 6 (lab)

Endosulfan (0.002mgL�1)Reference 0.91 0.62 0.19 n.d. n.d. n.d.

Low contaminated 1.18 0.81 0.33 n.d. n.d. n.d.

High contaminated 2.78 1.82 0.94 0.62 0.11 0.10

Molinate (0.03mgL�1)Reference 0.31 0.81 0.21 1.10 1.09 0.10

Low contaminated 1.48 0.97 0.60 1.28 1.14 0.57

High contaminated 4.63 1.31 1.03 2.64 1.43 0.99

Propanil (0.03mgL�1)Reference n.d. n.d. n.d. n.d. n.d. n.d.

Low contaminated n.d. n.d. n.d. n.d. n.d. n.d.

High contaminated 2.58 n.d. 0.14 n.d. n.d. n.d.

Abbreviations: d.l.: detection limit, lab: laboratory, n.d.: not detected.

Molinate - Development

Head

cap

su

le w

idth

(m

m)

+ S

E

0.0

0.2

0.4

0.6

0.8

1.0Control

Reference

Low contaminated

High contaminated

Molinate - Growth

Laboratory In situ

Bo

dy len

gth

in

cre

ase (

mm

) +

SE

0

2

4

6

8

Propanil - Development Control

Reference

Low contaminated

High contaminated

Propanil - Growth

Laboratory In situ

Fig. 2. Mean values and standard errors of biological responses, development (head capsule width) and growth (body length increase) of C. riparius larvae

exposed to molinate and propanil treatments (control, reference low and high contamination; see Table 2), in the laboratory and in the in situ bioassays.

M.S. Faria et al. / Ecotoxicology and Environmental Safety 67 (2007) 218–226222

conditioned sediment from the rice field in comparison totreatments with water and conditioned sediment fromreference and low contamination sites (Fig. 3). As wasobserved in situ, laboratory bioassay found no significantdifferences in growth between the treatments with waterand conditioned sediment from reference and low con-

tamination sites (P40.05). Comparison with the controltreatment revealed an inhibition of larval growth in high,low and reference treatments of the insecticide (Fig. 3).Regression analyses indicated that larval development wassignificantly negatively related (r2 ¼ 0.72, Po0.01) tomaximum temperature during in situ exposure to pesticides

ARTICLE IN PRESS

Growth

Laboratory In situ

Bo

dy

le

ng

th i

nc

rea

se

(m

m)

+ S

E

0

1

2

3

4

5

6

7

Development

He

ad

ca

ps

ule

wid

th (

mm

) +

SE

0.0

0.2

0.4

0.6

0.8

1.0

Control

Reference

Low contaminated

High contaminated

*

***

*

# # #

##

Fig. 3. Mean values and standard errors of biological responses,

development (head capsule width) and growth (body length increase) of

C. riparius larvae exposed to endosulfan treatments (control, reference,

low and high contaminated; see Table 2), in the laboratory and in the in

situ bioassays. Asterisks indicate significant differences between treat-

ments (* ¼ Po0.05; ** ¼ Po0.01, Tukey tests); hash marks indicate

significant differences between treatments and the control (] ¼ Po0.05;

]]] ¼ Po0.0001, Dunnett tests).

M.S. Faria et al. / Ecotoxicology and Environmental Safety 67 (2007) 218–226 223

(head capsule width ¼ 0.623–0.00325 maximum tempera-ture), but it was not related to maximum temperatureduring laboratory exposure (P40.05). Regression analysesdid not reveal significant relationships (P40.05) betweenhead capsule width and minimum temperature or betweenlarval growth and temperature during in situ andlaboratory exposure to pesticides.

Overall growth was generally greater in laboratorycompared to in situ bioassays for the same pesticidestreatments (Fig. 3). Significant differences (Po0.05) in thebiological responses of C. riparius larvae, exposedto most all pesticide treatments between in situ andlaboratory bioassays were observed. The exceptions(P40.05) were found for growth of larvae exposed toendosulfan in the reference treatment and in larvae exposedto propanil in the low contaminated treatment, and, also,for development of larvae exposed to molinate in alltreatments.

4. Discussion

4.1. Pesticide concentrations

On day 0 of the in situ bioassay, 2 days after endosulfanand molinate spraying of rice fields, the wetland channelwas contaminated due the spray drift and/or runoff. Thehighest pesticide concentrations were found in the rice field,as expected, since this site was subject to direct application(Table 3). The lowest pesticide concentrations were foundin the reference site, located in the wetland channel.Although pesticide concentrations in the reference site werelower than in the low contamination site, the differences inconcentration between the two sites were lower than weexpected, since the reference site and the low contamina-tion site were located upstream and downstream, respec-tively, from the contaminated rice field effluent. This mightbe explained by the occurrence of a reflux of water betweenthe two sites, which were in close proximity to each other inthe same channel. This effect may be greater when waterfrom the rice fields is discharged into the channel.Differences in the molinate and endosulfan concentrationsin water and conditioned sediment between field andlaboratory treatments on day 6 may have been caused bythe input of additional endosulfan during in situ exposureperiod or they may have resulted from the storage andtransport of water and sediment from field to thelaboratory.

4.2. Biological responses

C. riparius larvae were not affected by herbicides,suggesting that this species is not a suitable to assessherbicide contamination.The decreasing trend of thegrowth response to molinate from the highest to the lowest(reference) levels of contamination observed in the in situbioassay (Fig. 2) might be explained by differences inminimum and maximum temperature between treatments(Table 1). Although no significant relationships betweenlarval growth and temperature were observed in this study,Pery and Garric (2006) found high significant relationshipsbetween the growth of C. riparius larvae not exposed tocontamination and temperature.In contrast to the herbicides, the insecticide endosulfan

negatively affected growth of C. riparius larvae, conform-ing to expectations given that chironomids represent atarget group of insecticide application. Growth wasnegatively affected by endosulfan contamination in bothlaboratory and in situ bioassays (Fig. 3). However, whilethe inhibition of growth in laboratory can be related to thepresence of endosulfan concentrations, since physical andchemical conditions were similar between treatments,inhibition of growth in the rice field during in situ exposuremay also have been influenced by differences in physicaland chemical conditions, in particular the high valuesrecorded for maximum temperature. This may have causedan additional stress to C. riparius larvae. Landrum et al.

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(1999) verified that toxicity of several organophosphateinsecticides (e.g., azinphos methyl, disulfon, fensulfothion,terbufos) to C. riparius larvae varied with pH andtemperature. Similarly, Lydy et al. (1999) found a positivecorrelation between temperature and toxicity of theorganophosphate insecticides, chlorpyrifos and m-para-thion to Chironomus tentans.

Contrary to growth, larval development was not affectedby the insecticide. No significant differences were observedin larval head capsule width between treatments inlaboratory or between different sites in the in situexposures. The lowest head capsule widths were observedin the rice field, the highly contaminated site (Fig. 3), andalso for chironomids subject to in situ exposure to propanil(Fig. 2) and appear to be related to higher maximumtemperature at that site (40 and 47 1C) compared to thereference (26 and 30 1C) and the low contamination sites(29 and 31 1C) (Table 1), with regression analysis indicatinga negative relationship between head capsule width andmaximum temperature. Frouz et al.’s (2002) detailed studyof the effects of temperature on the developmental rate ofChironomus crassicaudatus demonstrated that larval devel-opment increased rapidly with increasing temperature upto 20 1C, slowed between 20 and 27.5 1C, and decreased attemperatures higher than 27.5 1C.

4.3. Relevance of C. riparius larvae bioassay to assess

ecological impact of pesticides

Head capsule with of C. riparius larvae appeared to bemore influenced by physical and chemical conditions ofexposure, especially by temperature, than by the insecticideendosulfan, suggesting that development is not a suitablemeasurement to assess endosulfan contamination. Incontrast, larval growth was highly affected by endosulfancontamination for both in situ and laboratory exposures,indicating that bioassays with C. riparius larvae usinggrowth as an endpoint is sensitive and a suitable tool toassess the impact of endosulfan contamination in fresh-water ecosystems.

Growth impairment in chironomid larvae may lead toreduced fitness with a subsequent decrease in populationabundance (Liber et al., 1996; Sibley et al., 1997). Becausechironomids are typically one of the predominant macro-invertebrates in freshwater ecosystems (Vos, 2001) and areimportant prey items for birds and fish (Van de Bund,1994; Prat and Rieradevall, 1995; Garcıa-Berthou, 1999),the detrimental effects on the growth of chironomids larvaecaused by a contaminant may have ramifying effectsthroughout the ecosystems. Organochlorine insecticides,such as endosulfan, are lipid-soluble and tend to accumu-late in the fatty tissues of organisms. This problem can befurther compounded by biomagnification, which results inincreased concentrations of contaminants in higher trophiclevels. Although endosulfan in the water column can betoxic to fish (Maier-Bode, 1968; Jonsson et al., 1993) andaquatic birds (National Library of Medicine, 1987),

endosulfan entering organism through food ingestionmay increase its toxic effects. Nagel and Loskill (1991)found that where endosulfan had been taken up by the preyitems of small fish, the resultant body load in fish was fiveorders of magnitude higher than the concentrations in theambient water. Bahner et al. (1977) similarly observed thatsignificant quantities of the organochlorine insecticidechlordecone (kepone) were transferred from preys topredatory fish. In aquatic birds, concentrations of organo-chlorine contaminants can be up to 100 times greater inbody tissue than in the surrounding water (Anderson andHickey 1976; Norstrom et al., 1976).The sublethal effects (growth inhibition) on C. riparius

larvae caused by endosulfan contamination in the wetlandchannel of the Natural Reserve of Arzila Marsh, resultingfrom pesticide treatment, may be indicative of ecologicalimpairment in this protected wetland channels andsimilarly indicate the ecological risk that the endosulfanspray drift and runoff from rice fields constitute tofreshwater ecosystems.

4.4. Laboratory versus in situ bioassays

Higher growth of C. riparius larvae found duringlaboratory bioassays compared to in situ bioassays, hasalso been documented by other authors (e.g., Castro et al.,2003) and maybe be explained by the occurrence of hightemperatures and the increased variability in physical andchemical conditions under condition of in situ exposureNevertheless, a similar biological response to pesticides,principally inhibition on larval growth, was found underboth laboratory and field exposures to endosulfan,providing evidence for comparability of the respectiveexperimental designs. Tucker and Burton (1999) also foundsignificantly higher lethal effects on C. tentans larvaeexposed to contaminated rivers surrounded by an agricul-tural area compared to larvae exposed to water andsediment from the same rivers but under laboratoryconditions. These authors similarly attributed this differ-ence to the variation in the parameters of physical andchemical water-quality, highlighting the potential for biasin extrapolating laboratory results to field-based scenarios.In the laboratory bioassay it was also possible to detect

toxicity effects in larval growth resulting from endosulfancontamination of the wetland channel, by comparing thegrowth of larvae in treatments with water and conditionedsediment from the wetland channel with the controltreatment, while this was not possible in the in situexposure due to absence of a control. Although, in situbioassays have several advantages: (1) they eliminate biasesassociated with laboratory-to-field extrapolation; (2) theyreduce sampling-related artefacts; (3) they allow stressorconcentrations to fluctuate naturally (Tucker and Burton,1999), laboratory bioassays can be important for theassessment of low levels of contamination by enabling thecomparison under controlled conditions. This was the caseof the study area because all wetland channels within the

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limits of Natural Reserve of Arzila Marsh receive inputsagriculture fields. Furthermore, laboratory bioassays withwater and sediment collected in situ provide an effectiveway to discriminate effects caused by stress contaminationfrom effects caused by stress of other physical and chemicalconditions. Thus, when in situ toxicity testing is used incombination with laboratory toxicity testing and physico-chemical characterization, a more realistic assessment ofpesticide effects to freshwater ecosystems can be made.

5. Conclusions

Impairment of larvae growth by endosulfan contamina-tion in the wetland channel adjacent to rice fields, suggeststhat insecticide spray in rice fields may cause ecologicalimpairment in adjacent freshwater ecosystems. Theseresults also lends support to the use of bioassay based onC. riparius larvae using growth as the endpoint to assessecological impairment caused by insecticide applications torice fields. The study also indicates that bioassays with C.

riparius larvae conducted in the laboratory provide avaluable auxiliary tool to the interpretion of in situbioassays based on this species.

Acknowledgments

The authors would like to thank Anabela Simoes and the‘‘ICN—Instituto da Conservac- ao da Natureza’’ for allow-ing the execution of this study in the Natural Reserve ofArzila Marsh, Reinaldo Maia for consenting to conductthis study in his rice field during the pesticide spray period,Joao Malcato for help with nutrient analysis, Pedro Reisfor help in the field, Ricardo Lopes, Philippe Ross andKieran Monaghan for revising and comments on themanuscript. The authors also thank ‘‘Fundac- ao para aCiencia e a Tecnologia’’, for providing a Ph.D. grant toMafalda S. Faria (SFRH/BD/2935/2000).

Funding sources: This research was supported by theFundac- ao para a Ciencia e a Tecnologia (FCT) via aPh.D. studentship grant to M.S. Faria (SFRH/BD/2935/2000).

This study involved experimental animals that wereconducted in accordance with national and institutionalguidelines for the protection of human subjects and animalwelfare.

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