REPRODUCTIVE EFFECTS AND BIOACCUMULATION OF CHLORDANE INDAPHNIA MAGNA

RACHID MANAR,{{ HLIMA BESSI,{ and PAULE VASSEUR*{{Lab Interaction, Ecotoxicologie, Biodiversite, Ecosystemes, CNRS UMR 7146, Universite de Metz, rue du General Delestraint,

57070 Metz, France{Lab d’Ecotoxicologie et Microbiologie pour l’Environnement, UFR Environnement et Sante, Faculte des Sciences et Techniques,

Universite Hassan II, BP 146 Mohammedia, Morocco

(Received 6 November 2008; Accepted 23 April 2009)

Abstract—Acute and chronic toxicity of high-grade chlordane (98%) and bioaccumulation were investigated in Daphnia magna atwater soluble concentrations obtained without cosolvent. The measured effective concentrations immobilizing 50% of themicrocrustacea (95% confidence interval) were 22.6 (19.7–26.1) mg/L at 24 h and 13.4 (11.3–15.8) mg/L at 48 h. This indicated anincrease of chlordane toxicity with time of exposure as confirmed in chronic studies. After 21 d of exposure, significant effects onsurvival were recorded at a chlordane concentration greater than 2.9 mg/L, whereas reproduction (number of offspring per adult, broodsize) and length of adults decreased at 0.7 mg/L or more in a concentration- and time-dependent manner. The production of maleoffspring and developmental abnormalities, consisting of underdeveloped second antennae and shell spines in live neonates, wererecorded. The chlordane concentration tested with no significant adverse effect (NOEC) on reproduction of daphnids after 21 dcompared with controls was 0.18 mg/L. The bioaccumulation factor of chlordane by daphnids exposed at a level of concentration closeto the 21-d NOEC reached 10,600, wet weight, and 244,000, dry weight, after 40 d. The trans-chlordane bioaccumulated to a greaterextent than the cis isomer in daphnids, whereas the cis isomer was predominant in the test medium. The results suggest a crucial role ofthe invertebrates in transfer of chlordane in aquatic food webs and can be used to derive a freshwater guideline for environmentalprotection accounting for bioaccumulation.

Keywords—Chlordane Daphnia magna Reproduction Embryotoxicity Bioaccumulation

INTRODUCTION

Chlordane is an organochlorine insecticide introduced in

the 1940s and widely produced for agricultural and residential

uses and for termite control. The term chlordane refers to a

mixture of two isomers, cis(alpha)- and trans(gamma)-chlor-

dane, present to a certain extent in commercial products.

Technical chlordane can contain between 40 and 75% of cis-

and trans-chlordane, associated with a number of other

chlorinated hydrocarbons, including heptachlor, trans- and

cis-nonachlor, and chlordene isomers [1].

Chlordane, like other chlorinated insecticides from the first

generation, is stable, highly lipophilic, and persistent, as

illustrated by a biological half-life of several years in soils

and sediments [2]. These properties give chlordane a high

bioaccumulation and biomagnification potential in biota. This

pesticide is also suspected of having carcinogenic and

endocrine disruptive effects [3]. Chlordane was forbidden for

phytosanitary treatments in Europe in the late seventies. All

uses in the United States, except termite control, were banned

in 1978, and its use as a termiticide was voluntary suspended in

1988 [4]. Despite this ban, chlordane is still present in soils and

in hydrosystems [5–8]. Chlordane was listed as a pollutant of

concern in the U.S. Environmental Protection Agency (U.S.

EPA) Great Water Program in 2000 [5]. The Stockholm

convention in 2001 targeted chlordane among the 12 persistent

organic pollutants (POPs) that should be totally banned from

the environment. Indeed, exemptions to the convention are

possible and new production of chlordane according to

permitted uses as a termiticide are allowed [9]. A recent

NATO workshop on the fate of POPs in the environment

underlined that chlordane is still produced in countries such as

China and Botswana, and because of long-range transport, it

remains a serious problem around the world [7]. It was recently

identified as a contaminant of concern in arctic human

communities as a result of consumption of local food, heavily

contaminated with chlordane residues through the food chain

[10]. According to Seemamahannop et al. [11] approximately

20% of the 70,000 tons of technical chlordane manufactured

since 1946 still exist unaltered in the environment. As a result,

environmental risk assessment is being carried out in many

countries to establish standards for protection of wildlife and

communities at risk. Chronic data is necessary that is more

appropriate than the acute toxicity information used so far to

define water quality standards. Indeed, little is known about

the chronic effects of chlordane and its bioaccumulation at

lower trophic levels of food webs.

Despite its widespread occurrence and environmental

persistence, chronic toxicity of chlordane to aquatic species is

not well documented. Most studies on chlordane effects have

focused on acute toxicity. The median lethal effect concentra-

tion values (L[E]C50) on freshwater and marine species range

from 0.5 to 115 mg/L for fish and from 0.4 to 63 mg/L for

crustaceans [1,12]. Chronic data have dealt with only a few

species, especially fish, although invertebrates are said to be

more sensitive to its effects than vertebrates. Toxicity has been

mostly assessed with technical chlordane, including a number of

components whose effects could not be discriminated from

chlordane itself. Toxicity of the hundred impurities of technical

* To whom correspondence may be addressed(vasseur@univ-metz.fr).

Published on the Web 7/9/2009.

Environmental Toxicology and Chemistry, Vol. 28, No. 10, pp. 2150–2159, 2009’ 2009 SETAC

Printed in the USA0730-7268/09 $12.00 + .00

2150

chlordane is not known, with the exception of nonachlor and

heptachlor, which were shown to be more toxic than chlordane

itself [10]. Unfortunately, toxicity of chlordane with a high

degree of purity has been insufficiently investigated.

In addition, chronic results based on measured concentra-

tions are scarce. Chronic values published so far generally have

been expressed as nominal concentrations and have been

obtained from experiments performed with a carrier solvent to

counteract the low water solubility of chlordane (0.1 mg/L at

22uC). Yet, effects of solvents routinely employed in ecotox-

icology for testing hydrophobic or ‘‘difficult substances’’

cannot be excluded, as recently reviewed by Hutchinson et

al. [13]. These authors gave evidence that some solvents might

affect the reproduction of aquatic species and affect biomark-

ers of endocrine disruption. They recommended avoiding the

use of carrier solvents wherever possible in chronic ecotoxicity

testing and reproduction studies and, where possible, through

the use of saturation systems [14]. Effects of modulation of

enzyme activities involved in the metabolism of endogenous

signaling molecules were found with solvents such as

dimethylsulfoxide (DMSO) [15], methanol, acetone, dimethyl-

formamide, and isopropanol [16]. Solvent effects can, in turn,

confound interpretation of data from chronic studies, leading

to problems in establishing values for the lowest observed

effect concentration (LOEC) and no observed effect concen-

tration (NOEC).

This study has been designed to fill in the lack of data

regarding long-term effects of chlordane on freshwater inver-

tebrates. Here, we aim to study the chronic toxicity of chlordane

of high-grade purity on invertebrates at concentrations below

water solubility, prepared without any cosolvent, and checked

analytically. The purpose of these experimental conditions was

to ensure environmentally relevant conditions of exposure to get

a better knowledge of chlordane effects in hydrosystems in the

long term and to produce results that can be used to derive

freshwater quality standards for environmental protection.

Chlordane toxicity and bioaccumulation were investigated

on Daphnia magna, a cladoceran representative of freshwater

crustacean species and zooplankton. Survival, growth, and

fecundity of D. magna were measured in the long term, as well

as bioaccumulation of the cis- and trans-chlordane isomers.

The Daphnia model is recommended by standard methods for

aquatic ecotoxicity assessment and is required by many

international regulations because of its sensitivity to chemical

environmental stressors. The genus Daphnia holds a central

position in aquatic food webs and is an intermediate between

primary producers and fish. Daphnids are the most significant

herbivores among invertebrates and are considered an

important source of food for fish [17]. Zooplankton has a

role not only in the transfer of energy, but also in

contamination of the trophic chains. Chlordane concentra-

tions in plankton [6] and trophic transfer in wildlife from fish

to mammals have been reported [18]. Uptake, elimination, and

residues have been studied in fish, but bioaccumulation by

zooplankton has not been investigated in the long run despite

the critical position of invertebrates in food webs. We

measured bioaccumulation after 25 and 40 d of exposure at

concentrations considered safe to daphnids under a low or a

normal feeding regime to explore the capacity of pollutant

transfer on this cladoceran.

In this study, we report data on freshwater invertebrates

that will make it possible to calculate freshwater quality

standards from chronic values instead of calculating them

from the acute data that have been used so far. This data will

allow the U.S. EPA to refine the acute chronic ratio (ACR)

used to derive a final chronic value from acute data. Here, we

emphasize bioaccumulation of chlordane by daphnids at

concentrations said to be safe for these invertebrates. It

stresses that pollutant transfer from zooplankton to fish

should be taken into account to protect ecosystems and species

at the top of food chains. The results of this study are intended

to be the basis for further environmental risk assessment.

MATERIALS AND METHODS

Chemical testing and preparation of test media

Chlordane (Chemical Abstracts Service: 12789-03-06,

pestanal quality, high-performance liquid chromatography

grade, purity 98.4%) was purchased from Sigma-Aldrich.

A saturated solution of chlordane in pure water was

prepared without the use of any cosolvent, and this solution

was diluted with the Daphnia medium for the preparation of

test solutions. The saturated solution was prepared by stirring

glass microspheres, impregnated with the chemical in the test

medium, in a dark space for 20 h at 20uC. The procedure was

as follows: 2 g of 1-mm glass microspheres were impregnated

with the chemical with a solution of 0.2 g chlordane in a liter of

acetone, which would be eliminated by using a rotary

evaporator. Then, microspheres (2 g) were introduced into a

small basket plunged into 0.1 L of the Daphnia medium and

stirred in the dark for 20 h. Thereafter, the saturated solution

was filtered on a paper disk (1.2 mm porosity) and diluted with

the Daphnia medium to obtain a range of decreasing

concentrations of the test chemical. Fresh saturated solutions

and the corresponding dilutions were prepared every 2 d

before each renewal of the test media. The chemical

concentrations in the test dilutions of chlordane were analyzed

once a week in the test media (800 ml) freshly prepared.

Chlordane analyses

Chlordane analyses were carried out according to standard

method NF EN ISO6468 [19].

Chlordane was measured by gas chromatography (GC)

with electron capture detection. The GC analyses were carried

out on a Varian 3400 chromatograph with the use of a JW

DB5 column (30 m 3 0.32 mm diameter and a 0.25-mm film of

5% phenyl–95% dimethyl polysiloxane; Alltech). The initial

temperature (80uC for 2 min) was increased to 180uC (an

increase of 15uC/min and a plateau for 6 min), then to 220uC(an increase of 4uC/min and a plateau for 2 min), then to 275uC(an increase of 5uC/min and a plateau for 13 min). The

temperature was 275uC for the injector and 320uC for the

detector. The quantification limits were 10 ng/L for cis- and

trans-chlordane, and the detection limits were 3 ng/L.

Extraction procedures

Chlordane was extracted from the Daphnia test media with

a 50:50 (v/v) mixture of hexane/dichloromethane. The extrac-

tion solvent was dried on anhydrous sodium sulfate then

evaporated and adjusted to 1 ml, from which microliters were

used for GC analysis. A 91% 6 4% recovery was achieved by

the extraction procedure because of losses by adsorption and

possibly by volatilization, although the vapor pressure of

chlordane is quite low (1.3 3 10–3 Pa at 25uC). The

concentrated extract was diluted if necessary to fulfill

conditions of linearity between signals and concentrations.

Chronic toxicity of chlordane to Daphnia magna Environ. Toxicol. Chem. 28, 2009 2151

Chlordane accumulated by daphnids was extracted with

hexane (1 ml). Daphnids submitted to analysis were unfed for

2 d in the contaminated medium before extraction to facilitate

emptying the gut within 24 h. To this end, pools of 30 to 60

daphnids of each batch corresponding to each condition of

exposure were collected and separated from the test medium

by gentle centrifugation then extracted with the use of hexane.

The hexane extract was separated by centrifugation before use

for analysis.

Test organism

Daphnia magna was obtained from continuous culture

maintained in our laboratory in 2-L aquaria at 20uC in a

synthetic medium of Lefevre–Czarda (LC) medium:Volvic

mineral water (20:80, v/v) with a 16:8 h light:dark photoperiod

and at a density below 40 animals per liter. The medium was

supplemented with a mixture of vitamins (0.1 ml/L) containing

thiamine-HCl (750 mg/L), vitamin B12 (10 mg/L), and Biotine

(7.5 mg/L) and was renewed three times weekly. Daphnids

were fed daily with a mixture of three algal species (5 3 106

Pseudokirchneriella subcapitata, 2.5 3 106 Scenedesmus sub-

spicatus, and 2.5 3 106 Chlorella vulgaris/daphnia per day).

The algae were cultivated continuously in the laboratory with

LC medium according to Graff et al. [20]. The offspring

produced were discarded every day. Brood daphnids were

discarded after 1 mo in culture and replaced with neonate

organisms. These culture conditions maintained the daphnids

in the parthenogenetic reproductive stage.

Acute toxicity

The acute toxicity of technical chlordane to neonate

daphnids was determined during 48 h of exposure (,24 h

old at the onset of the test). All experiments were performed

according to the International Organization for Standardiza-

tion procedure 6341.2 [21] for the determination of inhibition

of mobility of D. magna. Preliminary experiments were

conducted. The definitive test was carried out at the measured

chlordane concentrations of 4.5, 6.3, 9.2, 13.5, 19.8, 26, and 45

mg/L in parallel with the blank control for 48 h.

Four replicates of five neonates (,24 h old) from a

designated brood were placed in a 30-ml glass beaker

containing 10 ml for each test concentration and control. Test

organisms were not fed during the testing period. Observations

were made at 24 and 48 h, and results were recorded. The

endpoint examined was immobilization, wherein a daphnia

was considered to be immobile if it did not move after 15 s of

gentle agitation. The effective concentrations immobilizing

50% of the daphnids tested after 24 and 48 h of exposure (24-h

EC50 and 48-h EC50) were determined.

Chronic toxicity

In the chronic toxicity test, daphnids (,24 h old) were

exposed for 21 d to measured concentrations of chlordane

(mean 6 standard deviation) of 0, 0.18 6 0.05, 0.73 6 0.15,

1.82 6 0.16, 2.9 6 0.5, and 7.0 6 3.5 mg/L according to the

Daphnia magna reproduction test of the Organization for

Economic Cooperation and Development Guideline 211 [22].

Daphnids were raised individually in 50-ml glass beakers

containing 40 ml of test solution, which was composed of LC–

Volvic culture medium with food and pesticide at a desired

concentration. The alga P. subcapitata (at a density of 2.5 3

105 algal cells/ml, i.e., 107 algal cells/Daphnia per day) was used

as food. A total of 10 replicates for each treatment was

performed. The incubation temperature was controlled at 20 6

1uC and a 16:8 h light:dark photoperiod was maintained. The

test solution was renewed every 2 d.

The endpoints examined were longevity, size (body length),

days to first brood, total number of neonates per female, molt

rate (number of molts), number of broods, brood size, and sex

ratio. Digital image processing equipment was used to record

individual body lengths. The equipment consisted of a video

camera mounted on the ocular lens of a stereomicroscope that

was connected to a monitor and a computer. Pictures of living

specimens were recorded on hard disk or on videotape for

measurement. Body lengths (from the top of the head to the

base of the tail spine) were measured with the image analysis

software Motic Image Plus 2.0 (Motic China Group LTD).

Neonates were counted daily and discarded. The sex and

morphology of neonates were observed and counted with the

use of a dissecting microscope. Male daphnids were identified

by the presence of large, prominent first antennules. The sex

ratio was determined as the total number of males divided by

the total number of neonates.

The population growth rate (r) was calculated from the

integration of age-specific data on survival and fecundity

probabilities.

The intrinsic rate of population increase (r) was estimated

according to Stearns [23] from the Euler–Lotka equation (g

lxmxe–rx 5 1) with the equations

r& lnX

lxmxð Þ=Tand

T~X

x lxmx

.Xlxmx

where lx is the proportion of individuals surviving to age x, mx

is the age-specific fecundity (number of neonates produced per

surviving female at age x), and x is days.

Bioaccumulation test

The uptake of chlordane by daphnids during 25 and 40 d

was measured at two concentrations about the two lowest

concentrations of chlordane tested in the chronic test (i.e., 0.18

and 0.73 mg/L). Two separate experiments were conducted

with daphnids (,24 h old at the beginning of the test).

In the first experiment (25 d), 40 daphnids were exposed in

2-L glass beakers to the test solution (1 L) comprising LC–

Volvic culture medium with food and chlordane at a desired

concentration. Mean measured chlordane concentrations for

this test period were 0.15 6 0.03 and 0.65 6 0.19 mg/L. The

alga P. subcapitata was used as food and provided daily at a

low rate (106 algae/daphnia per day). Two replicates for each

treatment were performed. The incubation temperature was

controlled at 20 6 1uC and a 16:8 h light:dark photoperiod

was maintained. The test solution was renewed every 2 d. The

neonates were discarded daily. At the end of the experiment,

live daphnids were collected and pooled. After the determina-

tion of the wet weight, the daphnids were homogenized in a

glass Potter–Elvehjem tissue grinder with 1 ml of hexane

(analytical grade).

The second experiment was performed in the same

conditions as the first with the exception of the duration of

exposure (40 d), the number of daphnids (30) per modality, the

medium renewal (every 3 d), and the amount of food (supplied

at a higher level, with an increase during the last 10 d of the

test). The feeding schedule was as follows: 107 algae/daphnia

2152 Environ. Toxicol. Chem. 28, 2009 R. Manar et al.

per day up to day 29, then 2 3 107 algae/daphnia per day from

day 30 to day 40 to allow daphnia growth. The measured

chlordane concentrations over this second experiment were

0.21 6 0.03 and 0.84 6 0.07 mg/L.

Because daphnids were fed with algae (added daily in the

test medium), uptake of chlordane by daphnids would be

achieved not only by absorption from water, but also from the

algal food. Indeed, algae will adsorb a part of the chlordane

from the contaminated medium. Therefore, the term bioaccu-

mulation, as defined by Gobas and Morrison [24] and used by

Mackay and Fraser [25], can be used here to describe the

increased chlordane concentration in daphnids compared with

that in water. Bioaccumulation was measured as the ratio of

chlordane concentration accumulated by daphnids (mg/kg) to

the chlordane concentration in the test medium (mg/L). The

bioaccumulation factor (BAF) for chlordane concentration in

daphnids was expressed on a wet weight or dry weight basis.

Statistical analyses

The EC50s for acute toxicity were calculated by probit

analysis (Probit Ver 1.5, U.S. EPA). All chronic data, except

the percentage of abnormal neonates and of males, were tested

for statistical significance by single-factor one-way analysis of

variance followed by Duncan’s multiple range post hoc test.

Significant differences were established at p , 0.05. Homoge-

neity of variances and normality among replicates were

determined by Bartlett’s test and Kolmogorov–Smirnov test,

respectively. For cases in which the latter criterion was not

met, nonparametric methods (Kruskal–Wallis analysis of

variance followed by Mann–Whitney tests for pairwise

comparisons) were applied.

The LOEC used in this study was defined as the lowest

concentration to produce a significant effect of the parameter

studied compared with controls.

Toxicity endpoints, such as effective concentration values

(EC50 and EC10) that were used for D. magna, were estimated

with the bootstrap method in the REGTOX Excel macro (E.

Vindimian, French Ministry of Ecology and Sustainable

Development, http://eric.vindimian.9online.fr/en_index.html),

which models the data set according to the Hill model. The

software estimates the parameters of the model by means of

nonlinear regression (confidence intervals are estimated by a

bootstrap simulation). The results are expressed as ECx values

with their confidence intervals. All statistical analyses were

performed with Statistica for WindowsH ( p , 0.05; Statistica

Ver 5.1 for Windows, Statsoft).

RESULTS

Acute test result

The acute toxicity of chlordane on D. magna was evaluated

for 24 and 48 h. The percentage of neonate immobilization

increased with the time of exposure in the range of 4.5 to 45 mg/L.

The measured chlordane EC50s with confident intervals were

22.6 (19.7–26.1) mg/L after 24 h and 13.4 (11.3–15.8) mg/L after 48

h. The 48-h EC10 was 10.4 (6.4–11.2) mg/L.

Chronic test result

The survival and reproduction of D. magna exposed to

chlordane for 21 d are shown in Figure 1a and b, respectively,

and the sublethal effects on growth and fecundity registered at

the end of the exposure time are described in Table 1.

Mortality remained below 10% after 21 d at concentrations

of 1.82 mg/L or less, but survival was significantly affected at

concentrations of 2.9 mg/L chlordane or more. Survival

decreased with increased chlordane concentrations and time

of exposure at the two highest concentrations tested of 2.9 and

7 mg/L (Fig. 1a). At 2.9 mg/L, the mothers died massively

during the last days of exposure, and survival dropped from

100% at day 17 to 30% at day 21. At 7 mg/L chlordane,

mortality increased along the test period from day 3 to day 21,

and survival was 20% at the end of the exposure time.

Reproduction was affected at concentrations below those

permitting survival, and the decrease of the mean number of

offspring per daphnid was dose- and time-dependent (Fig. 1b).

A significant decrease was observed during the second week of

Fig. 1. Effects of chlordane on survival (a) and reproduction (b) of Daphnia magna during 21 d of exposure.

Chronic toxicity of chlordane to Daphnia magna Environ. Toxicol. Chem. 28, 2009 2153

exposure at 2.9 and 7 mg/L, whereas it appeared in the third

week at 0.73 and 1.82 mg/L.

Reproduction parameters, such as the number of offspring

per adult, brood size, and body length of adults, were

significantly reduced at chlordane concentrations of 0.73 mg/L

or more, with no significant effect being registered at 0.18 mg/L

after 21 d (Table 1). The number of neonates declined in a dose-

dependent manner from 116 in the controls to 73 at 0.73 mg/L, 33

at 2.9 mg/L, and 19 at 7 mg/L, which corresponded to a decrease of

37, 45, 72, and 84%, respectively. Likewise, the mean brood size

was reduced from 22 in the controls to 16 at 0.73 mg/L and 6 at 7

mg/L. The body length of adults was significantly lower than in

controls at chlordane concentrations of 0.73 mg/L or more and

was reduced by 24% at the highest tested concentration (7 mg/L).

The number of broods per adult decreased at chlordane

concentrations of 2.9 mg/L or more ( p , 0.05), and it was

reduced by 55% at 7 mg/L.

The first brood of D. magna was delayed in the presence of

chlordane, but only at 7 mg/L, wherein the first brood occurred

around the ninth day compared with the seventh day in

controls. The molt frequency and longevity of D. magna were

also reduced at this concentration, as well as the intrinsic rate

(r) of natural increase, which lessened to 0.2 compared with 0.3

in the controls.

Embryotoxicity and production of male offspring

Embryotoxicity in daphnids was observed at the highest

concentrations of chlordane tested during the 21 d of the

experiment (Table 2). Developmental abnormalities consisted

of curved or unextended shell spines and underdeveloped first

antennae (Fig. 2). The neonate deformities affected 2 and 4%

of the total offspring at 2.9 and 7 mg/L of chlordane,

respectively. No effect was observed in the control group

and at chlordane concentrations less than 2.9 mg/L.

Chlordane increased the incidence of males in D. magna at

the 1.82 mg/L and more (Table 2). The percentage of males

increased with the concentration of chlordane and reached 8%

at 7 mg/L. In the control group and at low concentrations (0.18

and 0.73 mg/L), only female neonates were produced.

The LOEC on the most sensitive reproduction parameters,

the number of offspring per female and brood size, and on

body length was 0.73 6 0.15 mg/L after 21 d of exposure

(Table 1). The 21-d EC10 values with 95% confidence interval

for these parameters were 0.15 (0.05–0.34) mg/L for brood size

and 0.17 (0.06–0.34) mg/L for number of offspring (Table 3).

Bioaccumulation

At the end of the first experiment conducted during 25 d

with chlordane concentrations of 0.15 6 0.03 and 0.65 6 0.19

mg/L, no significant difference in mean wet weight (3.7 mg/

daphnid) was noted between daphnids of the control group

and those exposed to 0.15 mg/L chlordane. On the other hand,

a decrease in the wet weight (22%) and in the activity of

daphnids exposed to 0.65 mg/L chlordane compared with the

controls was registered. The BAF on a wet weight basis was

6,340 at 0.15 mg/L, two times higher than at the concentration

of 0.65 mg/L (2,800). On a dry weight basis, the BAF in

daphnids was 145,800 at 0.15 mg/L chlordane and 64,500 at

0.65 mg/L in the test medium (Fig. 3).

At the end of the second experiment conducted for 40 d

with chlordane concentrations of 0.21 6 0.03 and 0.84 6 0.07

mg/L, no significant difference in mean wet weight (9 mg/

daphnid) appeared between the control group and the group

exposed to 0.21 mg/L chlordane. On the other side, a 21%

decrease was registered in the mean wet weight of daphnids

exposed to 0.84 mg/L chlordane compared with the control

group. The BAF on a wet weight basis was 10,600 at 0.21 mg/L,

2.7 times higher than at 0.84 mg/L (3,900). On a dry weight

basis, the BAF in daphnids was 244,000 at 0.21 mg/L chlordane

and 90,000 at 0.84 mg/L (Fig. 3c).

In both experiments, chlordane residues measured in

daphnids increased with chlordane concentrations, and the

BAF was much higher in daphnids exposed to the lowest tested

concentrations of chlordane (Fig. 3b). The trans isomer of

chlordane accumulated more heavily in daphnids than the cis

isomer. Whereas trans:cis averaged 0.5 in the test media and

1.0 in daphnids, trans:cis of the BAFs in daphnids ranged

between 1.2 and 2.0 for all the modalities tested (Fig. 3a).

DISCUSSION

In this study, we investigated acute and chronic aquatic

toxicity of high-grade chlordane (98% purity) at water soluble

concentrations and without the use of cosolvent to eliminate

any possible interference of carrier and impurities. Semistatic

conditions of exposure were used, with test media renewed

every 2 d in chronic bioassays. To this end, saturated aqueous

solutions of chlordane were prepared according to a well-

defined protocol, diluted just before medium renewal, and

measured once a week.

Table 1. Longevity, size, molting, reproduction, and population growth rate (r) of Daphnia magna exposed to measured concentrations of chlordanein a 21-d life study (values are means 6 standard deviation; * p , 0.05)

Chlordane(mg/L)

Longevity(d)

Length(mm)

Days tofirst brood

No. of neonatesper adult Brood size

Cumulativemolts

No. ofbroods r

Control 21 6 0 4,207 6 90 7.3 6 0.48 116 6 10.3 22 6 1.8 10.8 6 0.6 5.2 6 0.4 0.305 6 0.010.18 6 0.05 20.7 6 0.9 4,108 6 67 7.2 6 0.42 103.2 6 21 19.5 6 3 10.5 6 0.9 5.5 6 0.5 0.31 6 0.020.73 6 0.15 19.9 6 3.5 4,054 6 114* 7 6 0 73 6 24* 16.2 6 3.1* 10.2 6 1.8 4.6 6 0.5 0.31 6 0.021.82 6 0.16 20.4 6 1.9 3,894 6 64* 7.2 6 0.63 64 6 15* 14 6 2.2* 9.9 6 1.2 4.7 6 1.2 0.32 6 0.022.9 6 0.48 19.8 6 1 3,232 6 33* 8 6 0.94 33 6 8* 8.1 6 1.7* 8.9 6 1.2 4.1 6 0.5* 0.29 6 0.047 6 3.5 14.8 6 5.9* 3,202 6 159* 9.22 6 1.09* 19 6 11* 6.1 6 1.5* 7 6 2.8* 2.3 6 1.3* 0.22 6 0.08*

Table 2. Embryotoxicity and percentage of males in the offspring ofdaphnids during a 21-d exposure to different chlordane concentrations

Chlordaneconcentration (mg/L)

Developmentallyabnormal neonates (%)

Male(%)

Control 0 00.18 6 0.05 0 00.73 6 0.15 0 01.82 6 0.16 0 2.92.9 6 0.48 2.1 6.87 6 3.5 4.2 8.4

2154 Environ. Toxicol. Chem. 28, 2009 R. Manar et al.

The pattern of response from daphnids was typical of the

persistence and the cumulative properties of the insecticide,

whose killing effects accentuated with time of exposure. This

was reflected by the EC50 values, which decreased from 22.6

mg/L after 24 h to 13.4 mg/L after 48 h, and by the dose–effect–

time relationships obtained in the chronic test, indicating a

steady state had not been reached at the two highest

concentrations tested of 2.9 and 7 mg/L.

The acute toxicity values of chlordane measured in this

study were in the lower range of the limited data already

published on daphnids [1,26]. An EC50 value at 48 h of 98 mg/L

for D. magna, on the basis of nominal concentrations of

chlordane (44% purity), was reported by Moore et al. [26].

Cardwell et al. [27] measured acute toxicity in two bioassays

carried out independently with technical chlordane (43%

chlordane and 40% mixture of chlordenes) and found EC50

values of 28 and 35 mg/L for D. magna. Lower nominal acute

concentration values were published on the freshwater shrimp

Neocaridina denticulata collected in the field, but experimental

conditions and chlordane grade were undefined, leading to

disputable results [28]. Saltwater invertebrates appeared

quite sensitive to chlordane, in that EC50 values at 96 h of 0.4

and 4 mg/L were reported for Penaeus duorarum and Palaeomo-

netes pugio, respectively, when tested in flow-through conditions

with chlordane of 99% purity [29]. All the studies published so far

have been conducted with the use of a cosolvent to solubilize

Fig. 2. Developmental abnormalities elicited by chlordane exposure. (A) Normal neonatal daphnid. (B) Neonatal daphnid with curved shell spine.(C) Neonatal daphnid with underdeveloped first antennae and curved shell spine (male, presence of first antennules). (D) Neonatal daphnid withunderdeveloped first antennae and curved shell spine (female).

Chronic toxicity of chlordane to Daphnia magna Environ. Toxicol. Chem. 28, 2009 2155

chlordane, and the work here is the first carried out without any

carrier.

Our study on chronic toxicity showed that reproduction

was a more sensitive index of toxicity than survival for

chlordane. Detrimental effects on reproduction were recorded

at chlordane concentrations of 0.7 mg/L or more, and survival

was affected at concentrations greater than 2.9 mg/L. Male

offspring and embryo abnormalities were observed from

chlordane concentrations of 1.8 and 2.9 mg/L respectively.

No significant effects were observed on growth, reproduction,

and survival at the lowest chlordane concentration tested, and

the 21-d EC10 value on D. magna brood size was 0.15 (0.05–

0.36) mg/L.

The few data available on chronic effects of chlordane to

aquatic invertebrates and vertebrates showed that daphnids

were one of the most sensitive species tested. Growth and

survival of the amphipod Hyalella azteca were affected at

measured concentrations of technical chlordane between 5.3

and 11.5 mg/L [27]. A significant reduction in the size of the

amphipods, as we noted in daphnids, was reported at these

concentrations, but reproduction was not studied. Adverse

chronic effects on reproduction and hatching success of the

freshwater fish Lepomis macrochirus (bluegill) were found at

2.2 mg/L of technical chlordane [27], and the concentration

inducing no adverse effects was estimated to be 1.6 mg/L [1].

The saltwater fish Cyprinodon variegatus was quite sensitive to

chronic toxicity of chlordane, and the no-effect chronic value

was 0.63 mg/L for this species [1]. The 21-d EC10 value of 0.15

mg/L on brood size of D. magna found here is the lowest of the

chronic data published so far. From our acute and chronic

results, an ACR of 89 (13.4:0.15) was calculated on D. magna.

This ACR is the first one based on effects of measured

concentrations of pure chlordane on a freshwater invertebrate.

This value can be used to recalculate the mean ACR allowing

the U.S. EPA to derive a freshwater final chronic value from a

freshwater final acute value. So far, the mean ACR used is 14

[1]. Integrating this new ACR of 89 in the mean, will allow

U.S. EPA to improve the environmental quality standards

derived from the final chronic value.

In addition, the work here offers the first chronic value for

high-grade chlordane on a freshwater species that can be used

in the on-going environmental risk assessment set by the

European Union (EU). According to the EU guidelines [30],

an assessment factor of 100 is assigned to the chronic NOEC

obtained from a single trophic level to extrapolate from single-

species laboratory data to a multispecies ecosystem. The

assessment factor can be decreased to 50 or 10 when chronic

data exist from two or three species at different trophic levels.

Chronic data for three chronic levels (algae, daphnids, and

fish) are available for technical chlordane, so an assessment

factor of 10 applied to the lowest NOEC corresponding to the

freshwater invertebrate will lead to a predictive no-effect

concentration (PNEC) of 18 ng/L (0.18 mg/L/10). For high-

grade chlordane, a single chronic NOEC is available, and a

PNEC of 1.8 ng/L (0.18 mg/L/100) can be calculated. A better

approach, recommended by the EU, is to derive the lowest

confidence interval limit of the EC10 value obtained from

modeling (0.15 6 0.05–0.34 mg/L). This will result in a more

protective guideline value of 0.5 ng/L (0.05 mg/L/100) for high-

grade chlordane in freshwater systems.

Bioaccumulation of chlordane by daphnids was shown to

reach 10,600 on a wet weight basis after 40 d of exposure to a

chlordane concentration close to the NOEC value estimated at

0.18 mg/L in the reproduction test. After 25 d of exposure

under a restricted feeding regime, the BAF value was 6,340 at

the same level of concentration. The first experiment used

Table 3. Concentrations of chlordane corresponding to the effective concentrations (EC) immobilizing 10 and 50% (EC10 and EC50, respectively) ofDaphnia magna after 21 d of exposure. (The EC values are given with 95% confidence intervals)

Parameter

EC10 EC50

Chlordane (mg/L) Confidence interval Chlordane (mg/L) Confidence interval

No. of offspring 0.17 0.06–0.34 1.54 1.1–2.05Brood size 0.15 0.05–0.34 2.65 2.02–3.68Longevity 3.37 1.2–5.9 9.72 7.5–16.5No. of broods per female 1.49 0.6–2.98 6.35 4.85–9.01Cumulative molt 1.79 0.55–3.63 11.86 7.88–2.95Population growth rate 4.09 2.18–6.28 10.1 7.57–18.8

Fig. 3. Concentrations of the cis (&) and trans (&) isomers and total(h) chlordane in (a) daphnids (ng/g dry wt) and (b) test medium (mg/L)during 25 and 40 d of exposure, and the resulting bioaccumulationfactor (c), expressed as dry weight of daphnids.

2156 Environ. Toxicol. Chem. 28, 2009 R. Manar et al.

restricted food supply, whereas the second was designed to

allow daphnia growth on longer exposure time. In the second

experiment, bioaccumulation by daphnids was about two

times higher than in the first experiment as a result of both an

extended period of exposure, an increased algal food supply,

and a better health status of daphnids, as attested to by their

higher weight. Technical chlordane had no toxic effects on

populations of microalgae at the concentrations we tested, but

it can be adsorbed on the cells, as indicated by bioconcentra-

tion values of 6,000 or more [31], leading to daphnid

contamination through water and food. Our experiments were

designed to mimic environmental contamination of the water

compartment for measuring invertebrate contamination and

not to determine the fraction of chlordane taken up from water

or food by daphnids, although it could be of interest. Indeed,

the information regarding partitioning of chlordane between

the medium and the uncontaminated food given to the tested

species has not been given in any chronic study reported in the

literature dealing with chlordane bioaccumulation by inverte-

brates and vertebrates. In the case here, some inference can be

made regarding the contribution of water and food to daphnid

bioaccumulation. Although bioaccumulation studies with P.

subcapitata have not been conducted, a bioconcentration

factor for green algae within the same taxonomic group

(Scenedesmus quadricauda) has been reported, at 6,000 (wet wt)

for 1 d of exposure at 0.4 mg/L [31]. Given the static renewal

design, it is probable that algae used for feeding accumulated

chlordane to similar levels. If algae accumulated chlordane at

similar efficiencies, it is possible to calculate the factor of

bioaccumulation by daphnids from food from the ratio of

daphnids concentrations to algal concentrations (wet weight).

The estimation gives a value averaging 1.5, suggesting that

primary producers have a crucial role in the process of food

chain contamination.

Compared with other invertebrates, the bioaccumulation of

chlordane by daphnids in our experiments appears higher than

bioaccumulation by H. azteca, P. duorarum, and P. pugio, with

BAF values in the range 1,900 to 6,000 on a wet weight basis in

the latter species compared with 2,800 to 10,600 in D. magna in

this study (Table 4). According to the literature, bioaccumu-

lation had not been measured in zooplankton during long

periods of time. Most experiments have been conducted over a

few days. Yet, a steady-state equilibrium requires longer time

to be reached. In this study, the BAF values we measured in

daphnids after 40 d of exposure were twice as high as those

found after 7 d [1]. Results from this study also showed that

trans-chlordane bioaccumulated more extensively than the cis

isomer in daphnids, despite a concentration of cis isomer twice

the trans concentration in the test medium. Moore et al. [32]

also found that absorption of trans-chlordane was 30% higher

than the cis isomer by daphnids.

The isomer metabolism somewhat differs in fish. Indeed, in

most field studies already published, the residues of the cis

isomer in fish were found at greater concentrations than the

trans isomer whatever the site—estuaries, coastal areas [7], and

rivers [33]. Because the cis and trans isomer have similar

lipophilic properties with a partition coefficient between water

and octanol (KOW) of 6.1 6 0.1 for cis and 6.2 6 0.1 for trans,

Table 4. Bioaccumulation factor of chlordane in aquatic organisms expressed as dry weight or wet weight

Species ChemicalaDurationin days

Bioaccumulation factor

ReferenceDry wt Wet wt

Alga Ankistrodesmus amalloides trans-Chlor 1 2,000–5,500 Moore et al. [32]Scenedesmus quadricauda Tech chlor 1 6,000–15,000 Glooschenko et al. [31]

5 6,700–10,300Invertebrate Hyalella azteca Tech chlor 65 5,200 Cardwell et al. [27]

Daphnia magna Tech chlor 7 3,800 Cardwell et al. [27]D. magna cis:trans 1.8:1 25 64,500–145,800 2,800–6,340 This study

cis-Chlor 48,767–135,162trans-Chlor 95,472–170,742cis:trans 1.8:1 40 90,000–244,000 3,900–10,600cis-Chlor 61,047–222,036trans-Chlor 108,697–420,362

Daphnia pulex cis:trans 1:1 1 16,000–24,000 Moore et al. [32]cis-Chlor 1 10,000–16,040trans-Chlor 1 13,333–20,130cis:trans 1:1 3 7,460–9,850

Penaeus duorarum Chlor 99.9% 4 4,000–6,000 Parrish et al. [29]Palaemonetes pugio 4 1,900–2,300

Mollusc Crassostrea virginica Chlor 99.9% 4 3,200–8,300 Parrish et al. [29]Fish Cyprinodon variegatus Chlor 99.9% 4 12,600–18,700 Parrish et al. [29]

C. variegatus (juvenile) 28 8,500–12,300Lagodon rhomboıdes 4 3,000–7,500C. variegatus (juvenile) Chlor 99.9% 28 15,300 U.S. EPA [1]C. variegatus Chlor 99.9% 189 16,000 U.S. EPA [1]C. variegatus (juvenile) trans-Chlor 28 6,600 U.S. EPA [1]C. variegatus Tech chlor 4 12,900 U.S. EPA [1]Pimephelas promelas Tech chlor 32 37,800 U.S. EPA [1]Lelostomus xanthurus Tech chlor 4 9,250 U.S. EPA [1]L. xanthurus Tech chlor 3 4,600 U.S. EPA [1]Carassius auratus cis:trans 1:1 4 67–162 Moore et al. [32]Cyprinus carpio Tech chlor 3 . 200 Seemamahannop et al. [11]

cis-Chlor 3 162trans-Chlor 3 312

a Chlor 5 chlordane; Tech chlor 5 technical chlordane; cis-Chlor 5 cis-chlordane; trans-Chlor 5 trans-chlordane.

Chronic toxicity of chlordane to Daphnia magna Environ. Toxicol. Chem. 28, 2009 2157

lipophilicity cannot explain the different fate of two isomers in

aquatic species [34]. Rather, differences in metabolism and

excretion might explain the different patterns observed, as

suspected by Seemamahannop et al. [11] and Murphy and

Gooch [35].

Our investigation showed that chlordane bioaccumulation

by daphnids increased at low concentrations of exposure.

Indeed, the BAF values were two or three times higher at the

lowest chlordane concentrations tested (0.15 and 0.21 mg/L)

than at the highest (0.65 and 0.84 mg/L), whatever the duration

of exposure (25 or 40 d).

From an ecological point of view, these results mean that

transfer of chlordane in the food chain will be favored at very

low concentrations of the pollutant in environmental com-

partments. Therefore, low concentrations of chlordane with-

out apparent effects on specific trophic levels at the bottom of

food webs can yet be detrimental in the long term on top

predators because of transfer and biomagnification, as shown

in seabirds and mammals [18,36–38]. On the basis of

bioaccumulation results, the concentrations considered to be

safe for the cladoceran population cannot be certified as safe

for their predators and the ecosystem. This justified the use of

an assessment factor of 100, taking into account the pollutant

transfer to higher trophic levels to derive a concentration

protecting the whole ecosystem from a chronic EC10 or

NOEC value on daphnids. It could be of interest to confirm

that the PNEC of 0.5 ng/L is protecting fish from secondary

poisoning by pollutant transfer from zooplankton in the long

term. To this end, the study of fish fed from a contaminated

diet made of precontaminated zooplankton at this chlordane

concentration could be proposed.

Chlordane concentrations in surface waters up to 39 ng/L

were recorded in China, where chlordane is still used as a

termiticide [39]. This concentration is approximately four

times lower than the lowest concentration of 0.18 mg/L tested

here. Yet, it is above the PNEC values evaluated for the high-

grade and technical chlordanes, indicating some concerns for

aquatic species in the long term. In other countries in which the

insecticide has been banned, contamination by chlordane and

other POPs was found to decline over the last decade in all

aquatic compartments [38]. Concentrations found in San

Francisco Bay (California, USA) were less than 140 pg/L [8],

and they were less than 4 pg/L in the Great Lakes (USA and

Canada) [40]. In the latter case, the range of concentrations in

freshwater is two orders of magnitude below the lowest PNEC

values established. Yet chlordane remains a pollutant of

potential human health concern in these countries as the result

of food chain transfers. The work here provides information to

refine water quality standards, taking into account bioaccu-

mulation at lower levels of food webs.

Chlordane is known to be neurotoxic and to exert its

insecticide effects via antagonism of the GABA (c-aminobu-

tyric acid) receptor–chloride channel complex and inhibition

of Ca/Mg adenosine triphosphatase [41]. Chlordane has the

same mode of action as aldrin, dieldrin, heptachlor, and

related compounds [42]. The endpoints measured here do not

allow us to determine whether chronic toxicity is a conse-

quence of neurotoxicity, endocrine effects, or both.

Production of male offspring and deformities in daphnids

can be produced by various mechanisms like unfavorable

environmental conditions [43]. Such conditions cannot be

retained in our experiments because no food deprivation,

crowding, or photoperiod changes occurred that could explain

the production of males in the treated invertebrates. Broods of

female offspring were exclusively produced in controls; males

were found only in daphnids exposed to chlordane. Other

authors have reported male production after treatment with

the crustacean juvenoid hormone methyl farnesoate and its

insecticidal analogs, methoprene and pyriproxyfen [44,45].

Developmental abnormalities have been triggered by ecdyster-

oid antagonism and deprivation. The similarity to results

obtained in the present study does not prove an endocrine

disrupting potential of chlordane, but suggests that such a

mechanism deserves to be addressed.

CONCLUSION

This study highlights the sensitivity of the freshwater

cladoceran D. magna to the acute and chronic effects of

chlordane. The dose–effect–time relationship registered are

typical of the persistence of this chlorinated compound, whose

toxicity threshold lessens with length of exposure. Further

investigations would be required to clarify mechanisms of male

production and embryotoxicity. The high bioaccumulation of

chlordane by daphnids confirms the role of invertebrates as

important links for transfer of the chlorinated molecule in

aquatic food webs.

Although trends in regression of contamination level have

been described over the last decade, concentrations found

recently in species at the top of food chains attest that wildlife

could be endangered through transfer and biomagnification in

trophic webs. On the basis of high potential of bioaccumula-

tion of the pesticide by aquatic species and long half-life, the

survey of chlordane and its metabolites in the environment

remains necessary to identify the level of contamination at risk

and to prevent long-term disorders for aquatic biota and

human population.

Acknowledgement—The authors gratefully acknowledge EGIDE, theFrench Leading Agency for International Mobility, for the grantattributed to this research within the exchange research program AIMA/04/105F. They also thank the Ministry of Research and theRegion Lorraine in France for financial support. The authors warmlythank A. Laalou for linguistic proofreading of this paper.

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