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Ecotoxicology and Environmental Safety 68 (2007) 326–334

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Effects of toxaphene on soil organisms

Jitka Bezchlebová, Jitka Černohlávková, Jan Lána, Ivana Sochová,
Klára Kobetičová, Jakub Hofman
RECETOX—Research Centre for Environmental Chemistry and Ecotoxicology, Faculty of Science, Masaryk University, Kamenice 126/3,
CZ-625 00 Brno, Czech Republic
Received 29 January 2007; received in revised form 12 April 2007; accepted 3 May 2007
Available online 26 June 2007

Abstract

The polychlorinated insecticide toxaphene belonged to the most used pesticides in the 20th century. Even recently, significant residues
have been found in soils at various sites in the world. However, knowledge on toxicity to soil organisms is limited. In this study, the
effects of toxaphene on soil invertebrates Folsomia candida, Eisenia fetida, Enchytraeus albidus, Enchytraeus crypticus, Caenorhabditis
elegans, and microorganisms were investigated. Among the organisms tested, F. candida was the most sensitive. The 50% effect on
survival and reproduction output (LC50 and EC50) was found at concentrations of 10.4 and 3.6 mg/kg, respectively. Sensitivity of other
organisms was significantly lower with effective concentrations at tens or hundreds of mg/kg. Our data on soil toxicity were recalculated
to soil pore-water concentrations and good accordance with available data reported for aquatic toxicity was found. Since soil
concentrations at some sites are comparable to concentrations effective in our tests, toxaphene may negatively affect soil communities at
these sites.
r 2007 Elsevier Inc. All rights reserved.

Keywords: Toxaphene; Soil pollution; Soil organisms; Pesticides

1. Introduction Toxaphene has been shown to be biomagnified in the

food chain (de Geus et al., 1999; HSDB, 2001). It was
The polychlorinated insecticide toxaphene (CAS 800-35- found to be highly toxic to aquatic organisms, especially to
2) is a complex mixture of several hundreds of congeners, fish (Saleh, 1991). This chemical proved to induce toxic
mainly polychlorinated bornanes, with average chlorine actions such as nephrotoxicity, hepatotoxicity, and repro-
content of 67–69% (de Geus et al., 1999). It belongs to the ductive effects (de Geus et al., 1999). Endocrine toxicity
group of persistent organic pollutants (POPs) listed in and mutagenicity were reported in in vitro studies (de Geus
international conventions (UN-ECE, 1998; UNEP, 2001) et al., 1999). As reviewed by Saleh (1991), the acute LD50
and was banned in many countries. Toxaphene shows of toxaphene to laboratory mammals ranged from 5 to
ubiquitous occurrence in the environment as a result of 1075 mg/kg body weights. Toxaphene was found to be
extensive usage since 1945 until the mid-1980s, when it was highly carcinogenic in rat and mice (Saleh, 1991) and
one of the most widely used insecticides. Total global use of therefore it is probably also a human carcinogen (Group
toxaphene between 1950 and 1993 was 1.3 million tons 2B; IARC, 2001).
(Voldner and Li, 1993). Toxaphene distribution is world- Toxaphene field application rates in agriculture were
wide and significant levels were also found in the arctic usually from 1 to 4 kg/ha (FAO/WHO, 1973). The
environment and biota (Bidleman et al., 1989). toxaphene content decreases with soil depth where total
toxaphene residues between 85% and 95% were found in
the upper 20 cm of soils (cultivated layer; HSDB, 2001;
Corresponding author. Fax: +420 549 494 267. ATSDR, 1998). Similar to all POPs, toxaphene is strongly
E-mail address: [email protected] (J. Hofman). sorbed to soil particles, with log Koc between 2.5 and 6

0147-6513/$ - see front matter r 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2007.05.009
 ARTICLE IN PRESS
J. Bezchlebová et al. / Ecotoxicology and Environmental Safety 68 (2007) 326–334 327

(HSDB, 2001) and water solubility is max 3.3 mg/l. Republic). The identical spiking procedure was used in all invertebrate
tests. Toxaphene was dissolved in acetone (HPLC grade; Chromservis
Transport in soil is very slow and leaching to groundwater
Inc., Czech Republic) and dilutions were prepared with the acetone
improbable. Its half-life in soils was assessed to be between solution to obtain required soil concentrations (specified for each test
100 days and 14 years depending on the soil type, climate, thereinafter). The soil surface in each test container was sprinkled with
and possible degradation capabilities of soil microorgan- acetone solution to reach the appropriate chemical concentration. In all
isms (HSDB, 2001; ATSDR, 1998). Losses of toxaphene tests, spiking ratio was kept 1 mL of acetone per 10 g of soil (dry wt) for
from soils are due to volatilization, photodecomposition, minimal effect of acetone on organisms. Pure acetone was used for control
samples. Acetone was evaporated under the fume hood overnight after
chemical, and/or microbial degradation. The potential for which each sample was thoroughly mixed and distilled H2O was added to
volatilization increases if the soil matrix has a significant adjust soil moisture to 50% WHCmax. Spiked soil samples were left for 1
sand fraction (ATSDR, 1998). Aerobic degradation of day to stabilize before organisms were introduced. The spiking procedure
toxaphene in soil is slow (Buser et al., 2000) while differed for the microbial experiment and is described thereinafter. Range-
finding tests were performed (results not shown in this paper) to select
anaerobic degradation is faster because of reductive
concentrations for final tests.
de(hydro)chlorination mediated by microorganisms (half-
life from one to several months; Fingerling et al., 1998).
In all reports and studies published so far, toxaphene soil 2.3. Eisenia fetida test
contamination was particularly considered as a possible
A permanent culture of E. fetida was maintained in the mixture (1:1) of
source for water contamination or as a possible route of horse manure and peat. Water content was approximately 80% WHCmax
human exposure. There are much data on human toxicity and the pH was adjusted to 6–7. The temperature was 2072 1C. The
or aquatic ecotoxicity, but effects on soil organisms have performance of the test was based on OECD Guideline 222 (OECD,
not been studied so far. In our study, the effects of 2004a), but it differed from that guideline in terms of number of container
toxaphene on soil invertebrates Folsomia candida, Eisenia repetitions for each exposure concentration and evaluated endpoint
(number of cocoons). Nine exposure concentrations (1, 10, 25, 50, 100,
fetida, Enchytraeus crypticus, Enchytraeus albidus, and 200, 400 and 800 mg/kg) and controls (water and solvent) were used. Test
Caenorhabditis elegans were investigated. Impact on soil containers (1 l volume) per concentration with 500 g of soil (dry wt) were
microorganisms was also investigated, based on measure- prepared and 10 adult worms with clitellum (300–400 mg) were introduced
ments of microbial biomass, respiration activity, ammoni- into each container. Food (5 g of dry ground horse manure) was added
weekly under the soil surface. Survival and reproduction (number of
fication, and nitrification. The results are discussed,
cocoons) were evaluated after 4 weeks by manual counting. The test was
compared with data reported on aquatic toxicity and with kept in 2072 1C and under 16/8 light–dark cycle.
environmental levels that have been measured recently.
2.4. Enchytraeid tests
2. Materials and methods
Permanent cultures of enchytraeids were maintained in a mixture of
2.1. Experimental soils artificial soil and commercial garden substrate (50–60% WHCmax; pH
6–7) at 1872 1C and were fed with finely ground oat flakes. Worms were
Artificial soil (OECD, 1984) was made of 70% fine quartz sand, 20% transferred to a Petri dish with water and examined under a microscope to
kaoline clay, and 10% finely ground peat proportionally mixed on a dry find adults with well-developed clitellum that were used for tests. Tests
weight basis. pHKCl was set to 6.070.5 with CaCO3 at the beginning of with both species were performed according to OECD guideline no. 220
the tests and was found to increase to 6.570.5 at the end of the tests. The (OECD, 2004b). Concentration series of 10, 25, 50, 100, 250, 500, and
water content of artificial soil was adjusted to 50% of maximal water 1000 mg/kg and 1, 10, 100, and 1000 mg/kg were prepared for E. albidus
holding capacity (WHCmax) at the beginning of the tests and evaporated and E. crypticus tests, respectively. Five replicates of test containers were
water was replenished weekly, if necessary. All tests were performed in used for each concentration and controls. Twenty (E. albidus) or 10
artificial soil except for the assay with C. elegans. This soil was found (E. crypticus) grams of soil (dry wt) were prepared and 10 adult
unsuitable for the C. elegans test because the peat floated on the surface enchytraeids were added into each test containers after spiking. Containers
during the extraction procedure and disabled counting of worms. Hence, a were covered by lids and incubated at 1872 1C. Oat flakes were added at
natural soil was used for the C. elegans tests. The same soil was used in the the beginning of the test and after that weekly. Animals were exposed for
microbial experiment. This soil was collected from the top-soil layer 42 days (E. albidus) and 28 days (E. crypticus). At the end of exposure
(0–10 cm) in pristine area of South Moravia, Czech Republic. The soil was periods, the worms were killed by 5 mL of ethanol applied to the soil and
a loamy sand cambisol with the following particle size distribution: sand dyed by Bengal red. Survival of adults and number of juveniles were
(450 mm) 64.4%, silt (2–50 mm) 29.1%, and clay (o2 mm) 6.5%. The total manually counted. Tests were kept under 16/8 light/dark cycle.
cation exchange capacity was 16.4 meq/100 g and the pHKCl was 6.5.
Organic carbon and total nitrogen contents were 1.6% and 0.13%,
respectively. Organic pollutants and heavy metals contents were compar-
2.5. Folsomia candida test
able to the background levels according to the Czech Republic guideline
(Ministry of Environment of Czech Republic, 1996). The soil was dried, Permanent culture of F. candida was maintained in plastic containers
sieved (2 mm), and then used for C. elegans tests. For microbial with a thin layer of plaster of Paris and charcoal mixture (9:1) at 2072 1C.
experiments, the fresh soil was sieved (2 mm) and stored at 4 1C in the Animals were fed with dried baker’s yeast. Age synchronized juveniles
field-moist condition (91.5% of dry matter). (10–12 days) were used for the test. The test was performed according to
ISO 11267 guideline (ISO, 1999). The test was carried out in glass
containers with 30 g of soil (dry wt). Five replicates were designed for each
2.2. Sample preparation concentration (1, 2, 4, 6, 8, 10, 20, and 40 mg/kg) and controls. Ten
synchronized organisms were introduced into each container and about
Toxaphene standard (product number PS79; ampoule of 1 g neat) was 10 mg of dried baker’s yeast were added at the beginning of test and after
produced by Supelco and received from Sigma-Aldrich Ltd. (Czech 14 days. Test containers were closed with parafilm and incubated at
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328 J. Bezchlebová et al. / Ecotoxicology and Environmental Safety 68 (2007) 326–334

2072 1C. At the end of exposure (28 days), mortality of adults and were determined as randomized difference between values obtained before
number of juveniles were determined. Organisms were extracted by and after week and/or 3 h incubations. All microbial results are expressed
floatation and animals floated on the surface were manually counted. on the basis of dry soil weight (dry wt).

2.6. Caenorhabditis elegans test

2.8. Chemical analysis
C. elegans was cultured on nematode growth agar with a bacterial lawn
Soil samples with toxaphene nominal concentrations of 10 and 100 mg/
of uracil-deficient strain of Escherichia coli as food source and was
kg were analyzed in both tested soils (natural and artificial) immediately
maintained at 2072 1C (ASTM, 2001). Age-synchronized (3–4 days old)
after spiking and after 28 days (natural soil) and 42 days (artificial soil) of
worms were used for the test. The toxicity test was carried out according
incubation. Natural and artificial soils were incubated at 22 and 20 1C,
to ASTM guideline (ASTM, 2001). Ten grams of natural soil (dry wt) was
respectively. Soil samples were extracted with a mixture of hexane–acetone
spiked with amounts of toxaphene resulting in concentrations of 50, 100,
(3:1) by Soxtec (Foss Tecator). PCB 155 (200 ng) was added as internal
250, 500, and 1000 mg/kg for each exposure level. The test was performed
standard. Extracts were cleaned up on an acidified silica/silica column.
with three replicates per concentration level by weighing 2.33 g of soil (dry
Analyses were carried out on the Varian gas chromatograph coupled to
wt) into a Petri dish. Distilled water (1.5 mL) was added, 10 worms were
triple-stage quadrupole mass spectrometer (3800GC–1200MS). DB5-MS
introduced into each container and closed dishes were incubated at
column was used for the chromatographic separation. MS was operated in
2072 1C. After 24 and 48 h, worms were extracted from the soil using
selected reaction monitoring mode with Argon in collision cell. Transition
Ludox colloidal silica suspension (Sigma-Aldrich Ltd., Czech Republic).
from m/z 125 to m/z 89 was monitored for quantification of technical
After hand-shaking and centrifugation, the top layer was decanted into a
toxaphene.
Petri dish and worms were counted under a microscope.

2.7. Microbial tests 2.9. Statistical analysis

Two kilograms of natural soil (wet wt) was adjusted to 60% WHCmax No observed effect concentration (NOEC) and the lowest effect
and was pre-incubated in closed jar at 22 1C for 1 week to allow microbial concentration (LOEC) values were determined by analysis of variance
activity to stabilize. Toxaphene standard was dissolved in acetone and (ANOVA) and Dunnett’s test (at a 5% significance level). Estimation of
serial dilutions were prepared to get soil concentrations of 1, 10, 100, and concentrations causing 50% and 10% reduction of survival (LC50 and
1000 mg/kg. Spiking was the same for all concentrations considered. LC10 values, respectively) and reproduction (EC50 and EC10 values,
Firstly, 25 g of wet soil was thoroughly mixed with 2 mL of toxaphene respectively), and their 95% confidence limits were calculated from logistic
solution, and this soil was kept under the fume hood overnight to regression model according to Haanstra et al. (1985). All statistical
evaporate acetone. After that, lucerne meal (1.25 g), water (amount analyses were done using STATISTICA 6.0 software (StatSoft, 2004).
evaporated overnight), and the rest of the soil (225 g) were added and
thoroughly mixed. Five hundred grams of soil was prepared in the same
way for water and acetone controls. Spiked soils were transferred into 3. Results
glass jars, closed with airtight lids, and incubated at 22 1C in darkness.
After 28 days of incubation, following microbial parameters were 3.1. Chemical analysis
measured: microbial biomass, basal respiration, substrate-induced respira-
tion, ammonification, arginine ammonification, and nitrification. Three
sub-samples (replicates) were prepared for each concentration and Tested concentrations of toxaphene and their temporal
controls. Microbial biomass C was determined by the fumigation–extrac- changes were verified by chemical analysis (Table 1).
tion method (ISO, 1997a). Carbon from fumigated (i.e., samples Measured concentrations after spiking (day 0) were lower
fumigated in the desiccator by CHCl3 for 20 h) and non-fumigated sub- than nominal concentrations in both tested soils (approxi-
samples (10 g) was extracted using 40 mL of 0.5 M K2SO4. After 30 min of
shaking, the extracts were filtered through Filtrak 391 paper. Organic C in
mately 70% of nominal concentrations). At the end of the
extracts was determined by potassium dichromate oxidation and back exposure periods (day 28 for natural soil and 42 for
titration with 0.1 N ammonium iron (II) sulfate-6-hydrate. Microbial artificial soil), toxaphene concentrations were also deter-
biomass C was calculated using the formula Cbio ¼ (Cfumigated mined and means of measured values (initial and final)
Cnon-fumigated)/0.38 (ISO, 1997a). Basal respiration was measured accord- were used to express the effective concentrations.
ing to (ISO, 2002). Sub-samples (10 g) were weighed into small glass
bottles with rubber caps and incubated at 22 1C. Gas (1 mL) was sampled
from the bottles after 24 h with a syringe, and CO2 was quantified by GC
with H2 mobile phase, Porapack Q stationary phase, and thermal Table 1
conductivity detector. Substrate-induced respiration was measured as Results of chemical analysis
CO2 production in the first 6 h after the addition of glucose (5 mg Cglucose/g
soil dry wt) (ISO, 1997b). Ammonification rate was determined according Soil Time (day) Nominal Measured
to Alef (1995a) as the release of NH+ 4 -N during the incubation of flooded
concentration concentration
soil sub-samples (2 g, three replicates) at 36 1C for 1 week. Arginine (mg/kg) (mg/kg)
ammonification was measured according to Alef (1995b) as the release of
NH+ Artificial soil 0 10 7.36
4 -N in the first 3 h after addition of arginine. Sub-samples were
incubated at 30 1C. Nitrification rate was measured as the release of NO 28 10 5.87
3-
N during aerobic incubation of soil sub-samples (2 g, three replicates) at 0 100 62.74
22 1C for 1 week (Alef, 1995c). Soils were extracted with 1 M KCl (1:5 w/ 28 100 61.31
v), 30 min shaken (200 rpm), and centrifuged. NH+ 4 -N content in extracts Natural soil 0 10 7.65
was measured photometrically (Berthelot reaction) by the method 28 10 6.54
according to Forster (1995) and adapted to microplate design according 0 100 71.98
to Sims et al. (1995). The NO 3 -N was determined after reduction with 28 100 66.76
Devarda’s alloy as NH+ 4 -N (Sims et al., 1995). N-transformation rates
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Table 2
Summary results for the toxicity of toxaphene to soil invertebrates: 50% and 10% effects on survival and reproduction (LC50, EC50, LC10, and EC10,
respectively), no-observed-effect and the lowest-effect concentrations (NOEC and LOEC values)

Species Test Endpoint NOEC LOEC LC50 or EC50 (95% LC10 or EC10 (95% LC50 or EC50 (95%
duration (mg/kg) (mg/kg) CI)a (mg/kg) CI)a (mg/kg) CI)a (mg/L)

Folsomia 28 d Mortality 5.0 6.2 10.4 (9.1–11.6) 5.5 (4.0–6.9) 1.0 (0.9–1.1)
candidab Reproduction 2.5 3.7 3.6 (3.1–4.2) 2.1 (1.2–3.1) 0.3 (0.3–0.4)
(juveniles)
Eisenia fetidab 28 d Mortality 248.0 496.0 ND ND ND
Reproduction 15.5 31.0 54.5 (36.4–72.6) 10.4 (2.7–18.2) 5.2 (3.5–6.9)
(cocoons)

Enchytraeus 42 d Mortality 620.0c ND ND ND ND

albidusb Reproduction 620.0c ND ND ND ND
(juveniles)
Enchytraeus 28 d Mortality 620.0c ND ND ND ND
crypticusb Reproduction 620.0c ND ND ND ND
(juveniles)
Caenorhabditis 24 h Mortality 173.0 345.0 466.0 (326.0–607.0) 86.0 (9.7–162.4) 139.0 (97.0–181.0)
elegansd 48 h Mortality 35.0 69.0 261.0 (173.0–349.0) 15.2 (1.1–29.3) 78.0 (51.0–104.0)

ND: could not be estimated.

Values for soil effects concentration are based on measured concentration (mg/kg soil dry wt). The last column shows values of equilibrium soil pore-water
concentrations estimated from soil concentrations using the Kd values as described in the text.
a
CI, confidence interval.
b
Artificial soil.
c
The highest measured concentration.
d
Loamy sand soil.

3.2. Toxicity to soil invertebrates dose–response curve could be well established for this
endpoint. Survival and reproduction (number of juveniles)
Results were valid according to criteria defined in the test of F. candida proved the greatest sensitivity to toxaphene.
guidelines. Adult mortality in both controls (water and Effective concentrations (LC10, LC50, EC10, and EC50)
solvent) was under 10% and numbers of juveniles in both were found in levels 1 order of magnitude lower than in
controls were comparable or higher than the numbers other tests. For C. elegans, shallow dose–response curves
recommended in the guidelines. Variability of results was running over three orders of magnitude were found. Thus,
acceptable. Water and solvent controls were not signifi- NOEC values were determined at low levels of toxaphene
cantly different (ANOVA; P40.05). but LC50 values occurred at relatively high concentrations.
The highest tested concentration was 1000 mg/kg (nom- All results and dose–response relationships are presented in
inal concentration) in all tests except earthworm test. It is Fig. 1 and effective concentrations are summarized in
the highest concentration recommended in test guidelines Table 2.
(OECD, 2004a, b; ISO, 1999). In earthworm test, the For organic substances with log Kowo5, the uptake from
highest tested concentration was 800 mg/kg (nominal soil pore water is supposed to be the main route of
concentration). Significant toxic effects (ANOVA; Dun- exposure to soil organisms (Sverdrup et al., 2002a) and if
nett’s; Po0.05) were found within the range of tested the equilibrium-partitioning theory is presumed, the effect
concentrations in all tests except for tests with enchy- concentration of a chemical can be recalculated to aquatic
traeids. Therefore, in most tests dose–response curves for concentrations using soil–water distribution coefficient
adult mortality and/or reproduction output could be (Ma et al., 1998). The last column in Table 2 reports
established and effect concentrations (LC10 and LC50 concentrations causing 50% effects expressed as equili-
and/or EC10 and EC50) were estimated (Table 2). brium soil pore-water concentrations. These data were
Enchytraeid species were not affected by toxaphene even derived from soil concentrations using the soil pore-water
in the highest tested concentration (ANOVA; Dunnett’s partition coefficients—Kd. Kd values were estimated from
test; P40.05). Thus, the highest tested concentration was the equation Kd ¼ Koc  foc, where foc was the fraction of
established as NOEC value for both enchytraeid endpoints organic carbon in soils, i.e., 0.05 for the artificial soil and
in this study. Adult survival of E. fetida was inhibited to 0.016 for the natural soil. Organic carbon/water partition-
30–38% at the highest tested concentration what did not ing coefficient (Koc) value was obtained from databases and
allow the estimation of LC50. Reproduction of E. fetida literature and ranged from 103 to 106 (HSDB, 2001;
(number of cocoons) was a more sensitive endpoint and the ATSDR, 1998; FAO, 2000). Finally, the value of 210 000
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330 J. Bezchlebová et al. / Ecotoxicology and Environmental Safety 68 (2007) 326–334

Fig. 1. Effects of toxaphene on survial and reproduction (number of juveniles/cocoons) of F. candida, E. fetida, E. albidus, E. crypticus, and C. elegans.
The boxes show mean7standard errors (n ¼ 5 for F. candida, E. albidus, and E. crypticus, and n ¼ 3 for E. fetida and C. elegans). The lines indicate the
logistic regression lines fitted to data with the least-squares methods.

was used because it was most often cited for toxaphene concentrations tested. Substrate-induced respiration in-
technical mixture. creased significantly at 1 mg/kg; however, this effect
declined with increasing toxaphene concentrations. No
3.3. Effects on soil microorganisms inhibition was observed even at the greatest concentration
tested. For ammonification and short arginine ammonifi-
Results of microbial tests showed a low sensitivity of cation test, significant inhibition (ANOVA; Dunnett’s test;
indigenous microbial community to toxaphene. Nominal Po0.05) was revealed at the highest tested concentrations.
concentration of 1000 mg/kg caused 35% reduction in Nitrification was not negatively affected at any of
microbial biomass (ANOVA; Dunnett’s test; Po0.05). concentrations tested. For determination of NOEC values,
Basal respiration was not negatively affected at any of the only negative effects were considered (i.e., statistically
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J. Bezchlebová et al. / Ecotoxicology and Environmental Safety 68 (2007) 326–334 331

Substrate - induced
Microbial biomass C Basal respiration respiration
(µg . g-1) (µg CO2-C . g-1 . h-1) (µg CO2-C . g-1 . h-1)
600 1.6 16

450 1.2 12

300 0.8 8

150 0.4 4

0 0.0 0
0 1 10 100 1000 0 1 10 100 1000 0 1 10 100 1000
Toxaphene concentration (mg/kg) Toxaphene concentration (mg/kg) Toxaphene concentration (mg/kg)

Ammonification Nitrification Arginine ammonification

(µg NH4-N . g-1 . d-1) (µg NO3-N . g-1 . d-1) (µg NH4-N . g-1 . h-1)
5 2.5 4

4 2.0
3

3 1.5
2
2 2.0

1
1 0.5

0 0.0 0
0 1 10 100 1000 0 1 10 100 1000 0 1 10 100 1000
Toxaphene concentration (mg/kg) Toxaphene concentration (mg/kg) Toxaphene concentration (mg/kg)

Fig. 2. Effects of toxaphene on biomass, respiration (basal and substrate-induced), ammonification, and nitrification of indigenous microorganisms. The
boxes show mean7standard errors (n ¼ 3).

significant inhibition in comparison to the control). NOEC High sensitivity of F. candida to toxaphene in compar-
values were thus set to 100 mg/kg for microbial biomass ison with other tested species could be attributed to the
and nitrogen mineralization parameters and 1000 mg/kg specific mode of action. Vaal et al. (1997) studied the
for respirations (nominal concentrations). Results of relationship between mode of toxic action and different
microbial tests are presented in Fig. 2. sensitivity of aquatic species. In that study, toxic effects of
chemicals with non-specific mode of action showed lower
4. Discussion variations among species response than those with a
specific mode of action. Toxaphene is an insecticide with
4.1. Comparison of species sensitivity the neurotoxic action and this can explain high variation in
species response to toxic effects in our study. Another
F. candida appeared to be more sensitive than other explanation for the observed variations in sensitivity
tested species. LC50 and EC50 values for this organism were to toxaphene could be different routes of exposure.
by more than one order of magnitude lower when Whereas the main exposure route of worms is skin
compared to E. fetida and C. elegans (Table 2). Both penetration and the exposure by food is negligible for
enchytraeid species were rather insensitive. Both endpoints chemicals with log Kowo5 (Ma et al., 1998), collembolans
are sufficiently sensitive in the test with collembolans while are exposed mainly by food. However, Pedersen et al.
only the reproduction is sensitive in the earthworm test and (2000) showed that exposure through food needs higher
the adult survival is rather robust. Both endpoints (lethal total metal concentrations to exert a toxic effect than soil
and sublethal) of enchytraeids were not affected by exposure. Further, different routes of exposure do not
toxaphene in whole tested concentration scale, which explain differences in sensitivity of E. fetida and enchy-
showed to insensitivity and robustness of these organisms traeids. Sverdrup et al. (2002a) reported similar observa-
in this study. tions regarding sensitivity of enchytraeids and earthworms
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332 J. Bezchlebová et al. / Ecotoxicology and Environmental Safety 68 (2007) 326–334

in their work, where chemicals with non-polar narcosis 4.4. Comparison to soil residues levels
as a mode of action and/or heavy metals were tested.
However, other studies reported comparable sensitivity of In the past, toxaphene at pesticide disposal sites was
these species (Römbke and Moser, 2002). This shows that determined in levels over 100 mg/kg (Seiber et al., 1979)
it is not possible to compare the sensitivity of species and at some sites up to 1550 mg/kg (Troxler et al., 1993). In
without considering the type of chemical and organism the 1970s, average toxaphene levels found in the USA soils
responses to mode of toxic action as well as the biology of ranged from 0.1 to 3.0 mg/kg (Li et al., 2001). Only limited
organisms. information on toxaphene residues in soils is available
from recent years. Kannan et al. (2003) reported levels
3.3–2500 mg/kg found in South Carolina and Georgia and
4.2. Effects on soil microorganisms
levels from o3 to 2420 mg/kg were detected in Alabama
soils (Harner et al., 1999). High levels of toxaphene (50 mg/
Parameters characterizing the whole microbial commu-
kg with maximum of 3000 mg/kg) were found in soils
nity (microbial biomass content and respiration) and/or
surrounding the former production plant (Falberg-List,
associated with nitrogen cycle (ammonification, nitrifica-
Magdeburg, Germany; Fingerling, 1998) and levels up to
tion) were used to evaluate toxaphene effects on micro-
44 mg/kg were detected in soils from the coastal environ-
organisms. Microbial biomass content, ammonification,
ment of Nicaragua (Carvalho et al., 2003). Comparison of
and arginine ammonification were the only parameters
our LC50/EC10 values (Table 2) with levels reported shows
negatively affected by toxaphene at the greatest concentra-
that soil invertebrates could be under serious risk at some
tion tested (1000 mg/kg). Indistinct results that could
sites with high toxaphene concentrations.
not be satisfactorily explained were found for nitrification
Pilot screening of toxaphene residues in Czech Republic
(Fig. 2). Respiration parameters were not negatively
soils (Kosubova et al., 2003) showed no presence of
affected at any of the tested concentrations. Nevertheless,
toxaphene residues (limit of detection was 1.6 mg/kg) in
a stimulation effect at a low toxaphene concentration
matrices with low organic matter content such as arable
was observed for substrate-induced respiration, which was
and grassland soils. On the other hand, toxaphene residues
also confirmed by another study (Elmholt, 1992). In
were detected in levels from 9.1 to 60.7 mg/kg in forest soils
conclusion, our results showed difficulties in the evaluation
in mountain regions that was attributed to the contamina-
of effects of organic chemicals to an indigenous microbial
tion by long-range atmospheric transport (Kosubova et al.,
community. Microbial biomass content and respiration
2005). These levels indicate that the risk for soil organisms
activity describe the whole soil microbial community as a
in Czech Republic can be neglected.
‘‘black box’’ and changes in the structure of the microbial
community might not be observed. However, also para-
meters associated with the nitrogen cycle proved the low 5. Conclusion
sensitivity to toxaphene toxicity in our study although
some of them have been used as sensitive parameters in Toxaphene proved different toxicity among the test
toxicity tests (e.g., Sverdrup et al., 2002b; van Beelen and organisms. Folsomia candida appeared to be the most
Doelman, 1997). sensitive organisms with effective concentrations at 10 mg/
kg. These values were at least one order of magnitude lower
than effective concentrations for other tested organisms
4.3. Comparison with available studies
(earthworms, nematodes) where the effective concentra-
tions appeared at tens and hundreds of mg/kg. Enchy-
Whereas the knowledge on toxicity of toxaphene to soil
traeids were not even affected at concentration of 1000 mg/
organisms is limited, information on the toxicity to aquatic
kg. Results of toxaphene toxicity were recalculated
organisms is available. Saleh (1991) reported LC50 of
according to equilibrium partitioning theory and the
toxaphene (acute toxicity) for the most aquatic organisms
obtained effective pore-water concentrations were similar
in a range from 1 to 40 mg/L. Some organisms as freshwater
to those reported for aquatic organisms in available
molluscs are relatively tolerant to toxaphene where
literature. Effective concentrations were also compared to
effective concentration (LC50 96 h) was determined as
toxaphene levels in soils suggesting possible and adverse
0.75 mg/L (Keller, 1993). On the other hand, sublethal
effects on soil biota at some contaminated sites with high
effects on aquatic crustaceans were detected in concentra-
toxaphene levels.
tions below 0.1 mg/L (de Geus et al., 1999). Aquatic
unicellular algae and protozoa were less sensitive to
toxaphene with an effective concentration of 380 mg/L Acknowledgment
(IARC, 2001), which corresponds to our study where
microorganisms were found also rather insensitive. After This research was supported by Grant Agency of Czech
recalculation of our results to equilibrium pore-water Republic projects GACR 525/04/P159 and GACR 525/03/
concentrations, found values (Table 2) were relatively 0367 and Ministry of Education of the Czech Republic,
similar to mentioned aquatic data. Project No. MSM 0021622412 INCHEMBIOL.
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