Journal of Environmental Science and Health, Part B (2015) 50, 266–274 Copyright © Taylor & Francis Group, LLC ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601234.2015.999599

Bioavailability and influence of 14C-carbofuran on Eisenia andrei avoidance, growth and reproduction in treated natural tropical soils  1 REGINA C. B. FERREIRA1, SOLANGE PAPINI2 and MARA M. DE ANDREA 1 2

Laboratory of Agrochemical Ecology, Biological Institute, S~ ao Paulo, Brazil Environmental Surveillance of the City of S~ ao Paulo, S~ ao Paulo, Brazil

The bioavailability of carbofuran to the compost worms Eisenia andrei and the influence of its residual amounts on the avoidance, reproduction and growth of this species were studied in two natural tropical soils: a Typic Humaquept (GM) and a Typic Hapludox (LVD), as indicated by the Brazilian environmental authorities for ecotoxicological tests. The worms avoided the soil LVD treated with different doses of carbofuran. The pesticide also affected the production of juvenile specimens in both soils, but cocoon production was reduced only in the GM soil. The earthworms’ growth and weight loss were affected by carbofuran (2,2-dimethyl2,3-dihydro-1-1-benzofuran-7-yl methylcarbamate. CAS number 1563-66-2) only in the LVD and the mortality detected at 56 days of contact with the treated soils was not statistically significant in both of them. Fourteen days after the soil treatment with14 ccarbofuran, most residues detected in the soils were bound residues (approximately 36% and 30% in the GM and LVD, respectively) and neither mortality nor bioaccumulation was detected in the earthworms, even with absorptions of 13% and 43%, respectively. The LVD soil has lower organic matter content, and the effects of carbofuran on different aspects of the earthworms’ life were more pronounced in this soil, most likely due to the higher bioavailability of the pesticide in the soil solution. The results for carbofuran clearly demonstrate that even small quantities of residues do not assure lack of toxicity. They also make evident the necessity of studying the effects of pesticides in natural agricultural soils. Furthermore, as the bound residues and the earthworm contamination are not detected by conventional techniques, they are not taken into account and may be underestimated on environmental risk assessments. Keywords: Ecotoxicology, bioavailability, exposure time, residues, Typic Humaquept soil, Typic Hapludox soil.

Introduction Fertilizers and pesticides may generate harmful residues in the soil environment, contaminate water, food and organisms, and affect the health of the population, mainly rural workers who use these chemicals.[1,2] Prolonged exposure to contaminants may increase their negative impacts on the ecosystem due to the higher risks to organisms in the soil and the possible effects on their growth, reproduction and survival.[3,4] In addition, the contaminants may be absorbed by the soil organisms and be biomagnified in the food webs.[5,6] Among the soil organisms, earthworms are very important because they are the first soil colonizers;[7] are a route

Address correspondence to Mara M. de Andrea, Laboratory of Agrochemical Ecology, Biological Institute, Av. Cons. Rodrigues Alves 1252, S~ ao Paulo-SP CEP 04014-002, Brazil; Email: [email protected] Received August 15, 2014.

for the transfer of contaminants to terrestrial food chains;[8] have a role in the maintenance of soil structure, aggregation of soil particles, water and air infiltration and work on biological pest control.[9] They also contribute to organic matter degradation and influence the cycling and distribution of nutrients in the soil.[10] Earthworms of the Eisenia andrei or Eisenia fetida species have been chosen as bioindicators in tests of ecotoxicity and reproduction[11] because they are easily reared and have high reproduction rates, facilitating the international comparison of results.[5,12] Additionally, the presence of soil contaminants may be detected by morphological and behavioral changes, mortality[5,13,14] and variations on the reproduction rate of these earthworms.[15] The toxicity and the effects of several pesticides on earthworm reproduction have been demonstrated in several studies. Yasmin and D’Souza[16] observed that the presence of carbendazim, dimethoate and glyphosate influenced the weight gain and reproduction of E. fetida. Zhou et al.[17] detected significant reductions in the reproduction of both E. fetida and E. andrei in soil treated with

Bioavailability and influence of 14C-carbofuran on Eisenia andrei in natural soils cypermethrin at 20 mg kg¡1 of soil. The tropical earthworms Perionyx excavatus and Pontoscolex corethrurus are more sensitive to chlorpyrifos, carbofuran, mancozeb[18] and glyphosate[19] than E. fetida and E. andrei. Milanovic et al.[4] demonstrated that even small amounts of different pesticides affected the growth of E. fetida, mainly in longer contacts with contaminated soils. Among the pesticides most widely used in Brazil, the methylcarbamate carbofuran (2,2-dimethyl-2,3-dihydro-11-benzofuran-7-yl methylcarbamate. CAS number 156366-2) is an acaricide, termiticide, insecticide and nematicide that is directly applied or incorporated in the soil at doses of 5 to 80 kg ha¡1 for different types of food crops. It is highly toxic and very harmful to the environment,[20] with high water solubility (700 mg L¡1) and poor degradation by soil microorganisms. It can, therefore, be transported by the soil solution and reached underground water.[21] According to some manufacturers, besides its mobility, carbofuran is moderately persistent in the soil.[22] Its degradation depends on chemical and environmental conditions, such as moisture and temperature. It is stable under neutral or acidic conditions but degrades in alkaline media.[23] While carbofuran can be used to control soil pests, it also affects beneficial organisms. Carbofuran has been shown to be toxic to different organisms, including some in the aquatic ecosystems.[24–26] Moreover, it is very toxic to earthworms of different species.[18,27–29] According to one manufacturer, the lethal concentration for 50% of earthworms (LC50) is 1.46 mg kg¡1 soil.[22] Based on the hypothesis that the soil type may influence the effects of carbofuran, the worms E. andrei were placed in two carbofuran-treated natural soils with different physical and chemical characteristics. The treatment doses were below the recommended for agricultural practices, in order to detect the bioavailability of carbofuran and possible effects of its residues, that may remain in soil after agricultural applications, on different aspects of the earthworms’ life, such as growth, avoidance and reproduction.

Materials and methods Earthworm Adult earthworms Eisenia andrei (>300 mg, with clitellum) were taken from the rearing boxes maintained by the

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Laboratory of Agrochemical Ecology (Instituto Biol ogico - Secretaria de Agricultura e Abastecimento do Estado de S~ao Paulo, SP - Brazil). Before their placement in soil, they were washed and kept on wet filter paper for at least 3 h to ensure their guts are empty. For the avoidance study, the worms were acclimatized for 24 h in the untreated test soils before being placed in the containers. Soils Two natural soils with different physical and chemical characteristics were utilized: a Typic Humaquept (GM) and a Typic Hapludox (LVD), which were collected and characterized by the Soil Science Department of the Escola Superior de Agricultura of S~ao Paulo University (ESALQ/ USP), according to the Brazilian Institute of Environment and Renewable Natural Resources recommended parameters (Table 1) for some ecotoxicological studies.[30] Treatments Avoidance. To obtain bioactive soils, replicates of the needed amount of each soil type for each test were previously moistened to 60% of the maximum water holding capacity (MWHC) with deionized water, 1 week before the study. As the recommended dose range for application of carbofuran in agriculture is large (0.7 to 105 kg ha¡1)[20] and may result in residual amounts, for the avoidance test, the soils were treated with technical grade carbofuran (Noragro, 95% purity) solution in methanol at 0.5, 1.0, and 2.5 mg kg¡1 carbofuran soil. Reproduction, growth and mortality. Because carbofuran is highly toxic to worms, to investigate the effect of a sublethal dose on reproduction, growth and mortality, (4 £) 500 g samples of each soil type were treated with 1.0 mg carbofuran kg¡1 soil. The control samples (4 £ 500 g of each soil type) received the same volume (250 mL) of methanol used as vehicle for the treatment with carbofuran. The samples were placed in 550 mL glass jars and exposed to a stream of air for 1 hour, so as to evaporate the solvent, before placing the worms. Bioaccumulation. Replicates of (4 £) 350 g of GM and (4 £) 200 g of LVD dry weight of soils equivalent were,

Table 1. Main physical and chemical characteristics of the soils.

Soil

pH (H2O)

OM* (g kg¡1)

Clay

Silt

Sand

MWHC**

Typic Humaquept (GM) Typic Hapludox (LVD)

4.6 4.6

163.4 58.5

576 610

366 140

58 250

1.43 0.62

OM D organic matter content. MWHC D maximum water holding capacity.

*

**

268 respectively, treated with 0.28 e 0.25 mg g¡1 of carbofuran (technical product with 95% purity) and 0.27 e 0.18 kBq g¡1 of 14 C-carbofuran (Institute of Isotopes, specific activity of 2.17 MBq mg¡1 and 98.1% of radiochemical purity). The duplicate control samples of each soil received just the same maximum volume (280 mL) of methanol used for treatments. All the glass jars (550 mL capacity) were also exposed to a stream of air for 1 hour, so as to evaporate the solvent, before placing the worms.

Experimental setup Avoidance. The earthworms’ avoidance behavior to the carbofuran-treated soils followed, with limited modifications, the Brazilian Association for Technical Standards— ABNT NBR ISO 17512-1 guidelines,[31] using (3 £) samples of 100 g (dry weight equivalent) of each soil type and of each treatment (0.5; 1.0, and 2.5 mg carbofuran kg¡1 soil). The samples were placed in plastic chambers (11.5 cm wide £ 11.5 cm long £ 4.5 cm high) divided in two halves by a resistant plastic divider. One compartment was filled with 100 g of soil treated with a dose of carbofuran (T) and the other was filled with untreated control soil (CO). The experiment was also performed with (3 £) double control chambers (CO–CO). After 1 hour of exposure to a stream of air, to evaporate the solvent, the plastic divider was removed, and six worms were placed together in the groove between the chambers. The size of the chambers and the proportion of soil/earthworms were nearly identical to that used by Das Gupta et al.[1] for an acute toxicity test in a 4 day study. The outside of the chambers was covered with a dark plastic sheet to block the light, and the surfaces were closed with perforated plastic film to permit air circulation and to hinder the escape of the worms. The systems were maintained at 25 C and cycles of 12 h light/dark for 48 h. At the end of the test period, the plastic divider was reinserted between the chambers, and the worms on each side were counted. According to ABNT,[31] the amount of earthworms was converted into percentage of avoidance by the following equation: R(%) D [(C ¡ T)/N] c_ 100, where R D avoidance; C D number of worms in the control (CO) condition; T D number of worms in each dose in the same soil; N D total number of worms. The results were statistically analyzed by comparing the differences between medium values using the t distribution test with a  0.05. Reproduction, growth and mortality. The determinations of the mortality, growth and reproduction rates of the E. andrei earthworms followed, with limited modifications, the guideline OECD no. 222.[11] Groups of 10 selected adult earthworms were previously weighed and kept for 24 h in the same untreated soil samples in 550 mL glass

Ferreira et al. jars, to acclimatize. They were then placed in the treated soils (1.0 mg carbofuran kg¡1 soil) and, during the first 28 days, fed weekly with 5.0 g of dry horse manure, while the soil moisture was assured by adding 2.0 mL of deionized water. The bottles were covered with perforated plastic film and a porous tissue to permit air circulation and prevent the escape of the worms. All systems were maintained at 25 C and 12 h light/dark. After the first 28 days, mortality was checked. The live organisms were washed with tap water, dried with a filter paper and weighed to determine the effect of carbofuran present in the soil on the worms’ growth. The worms were returned to the bottles after a final addition of horse manure and deionized water, and the bottles were returned to the chamber with controlled light and temperature. The effect of carbofuran on the earthworms’ mortality and growth was again checked by counting and weighing the live organisms at the end of a 56 day period. The weight of the worms was compared by the t distribution test with a  0.05. The effect of carbofuran on worms’ reproduction was determined by counting the cocoons and juveniles present in each replicate. The glass bottles were incubated in a water bath at an initial temperature of 40 C, followed by an increase to 60 C over 20 min.[11] During this period, the juvenile specimens were counted after rising to the soil surface. The number of cocoons was determined manually after carefully spreading the soil on trays. The numbers of cocoons and juvenile specimens present in the control and carbofuran-treated samples were compared by the t distribution test with a  0.05. The mortality rate was compared between the soils by the X2 test (a D 0.05). Bioavailability and bioaccumulation. The test to determine the bioavailability of carbofuran to the earthworms was based on Vampre et al.[32] and the OECD protocol no. 317.[33] Groups of five earthworms were weighed and placed in each jar with carbofuran-treated and untreated soil type. The containers were also covered with perforated plastic film to allow gas exchange. During the 14 days of test, no food was given and the flasks were maintained at 25 C and 12 h periods of darkness. Every 2 days, the moisture content was weighed to check the need of water addition. After 14 days, the worms were removed, washed with tap water and dried in filter paper; the biomass was evaluated by weighing the earthworms’ groups. The organisms were then frozen (¡16 C) till the analysis of 14 C-carbofuran residues by combustion. The bioaccumulation factor (BAF) was determined as the ratio between the radiocarbon amounts in the organisms (wet weight) and in the soil samples, according to Andrea and Papini.[34] The BAF values were compared by the t distribution test with a  0.05.

Bioavailability and influence of 14C-carbofuran on Eisenia andrei in natural soils Determination of the 14 C-carbofuran extractable residues in the soils The extractable residues in the soils were determined just after the treatment and after the 14 day test by extraction of (3 £) 5.0 g (dry weight equivalent) of soil of each flask with 10 mL of methanol and 3 h mechanical shaking. The extracts were filtered through filter paper and the radiocarbon in duplicates of 2.0 mL samples was determined by LSC, according to Andrea et al.[35] The extracts were also analyzed by high performance liquid chromatography (HPLC) of 1.0 mL aliquots after filtering through 0.45 mm membranes for detection and quantification of carbofuran and its metabolites. The analyses were first done in a Shimadzu LC 10 UV/Vis (Shimadzu, Japan); mobile phase: acetonitrile:water (50:50) at 1.5 mL min¡1 flux; column C18; pump DAD 10; oven temperature: 40 C; wavelength of 270 nm, where carbofuran had a retention time of 4.8 min. Confirmation was done in Shimadzu - DGU-20A UV/Vis; mobile phase: acetonitrile:water (30:70) at 1.0 mL min¡1 flux; column C18; pump LC 20 AT; oven temperature: 40 C; wavelength of 270 nm, where the retention times of carbofuran and its hydroxy- and keto-carbofuran metabolites were, respectively, 10.5, 3.0, and 4.7 min.

Determination of 14 C-residues in earthworms and bound residues in soils The amount of 14 C-carbofuran actually applied to soils and its remaining residues after 14 days were determined by combustion of (3 £) 0.5 g soil of each flask, using a Biological Oxidizer (Harvey OX-600, Harvey, Tappan, NY, USA; 4 min at 900 C).[35] The results were corrected to dry weight equivalent. Triplicates of 0.5 g subsamples of the extracted soil samples had the 14C-bound residues also determined by combustion after drying for a week. The amount of 14Cresidues in the worms was determined by combustion of (3 £) 0.5 g (wet weight) of the homogenized pieces of each group of worms. Results of the differences between 14Cextractable and -bound residues of both soils were also compared by the t distribution test with a  0.05.

Results and discussion Avoidance The LVD soil was avoided in all treatments (Table 2, R D 11.2%, 44.4% and 11.1%, respectively, for 0.5, 1.0 and 2.5 mg kg¡1), and these differences were statistically significant (P D 0.012). Inexplicably, GM soil was avoided (R D 11.1%, P D 0.301) only when treated with the lower dose of carbofuran, i.e., 0.5 mg kg¡1. Buch et al.[19] also detected avoidance of artificial soil treated with carbofuran by E. andrei and Pontoscolex corethrurus earthworms, although

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Table 2. Avoidance of Eisenia andrei of Typic Humaquept (GM) and Typic Hapludox (LVD) soils treated with different doses of carbofuran. Doses(mg carbofuran kg¡1 of Dry Soil Equivalent)

GM Avoidance(%)

LVD

0.5 1.0 2.5

11.1 0 0

11.2 44.4 11.1

only with treatments of, respectively, 5.0 and 2.5 mg kg¡1 of artificial soil. There was no statistically significant difference between the earthworms’ avoidance of control GM soil and carbofuran-treated GM soil (P D 0.301). However, the difference between the avoidance of the carbofuran-treated LVD soil and its corresponding control was significant (P D 0.012). Therefore, the carbofuran-treated LVD soil was more harmful to the earthworms than the GM soil. The greater sensitivity of E. andrei in LVD soil, rather than in GM soil, indicates that the effects of pesticides on natural soils should as well be evaluated, in order to obtain a more accurate assessment of the effects of contaminants closer to the reality of soil ecosystems.

Mortality, growth and reproduction Upon initial contact with the treated soils, even with carbofuran concentrations below those recommended in agricultural practice, earthworm sensitivity was observed in both GM and LVD soils. The earthworms curled themselves up and were less sensitive to touch, as observed previously by Santos et al.[36] for a mixture of acaricide spirodiclofeninsecticide dimethoate. The worms also took longer to bury in the treated soils, as observed by Ellis et al.[37] with carbendazim. E. andrei earthworms are known to be less sensitive to pesticides, including carbofuran, than the native endogeic species in soil.[19] Thus, if this bioindicator species exhibits behavioral changes, then endogeic worms that feed on the soil as well as other beneficial soil organisms may also be affected. As a result, the processes of soil maintenance, including soil restoration [7] and acceleration of organic matter degradation, which these organisms perform,[38] may suffer the negative consequences of soil environmental contamination with carbofuran. In contrast, neither mortality nor morphological change was detected in either carbofuran-treated soil types until 28 days. A zero mortality rate in the first 28 days validated the study according to the protocol of OECD no. 222,[11] which states mortality  10% of the organisms in each control sample in the first 28 days as a criterion for validating the study. Despite the small dose, compared with other studies,[19,29] and the lack of difference between the

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Ferreira et al. of undesirable consequences for all beneficial activities performed by earthworms in the soils. The pesticide affected worm growth again only in the LVD soil (Fig. 2). Even with doses that may be considered residues, the effects of the pesticide increased as the contact time of the earthworms with the carbofuran-treated LVD increased (P D 0.011 and 0.002, respectively, at 28 and 56 days of contact), but not with the treated GM (P D 0.067 and 0.070, at 28 and 56 days). Results again indicate the harm of persistent residues in soils with small OM to these organisms.

Bioavailability of 14C-carbofuran to the worms Fig. 1. Reproduction of Eisenia andrei after 56 days of contact with Typic Humaquept (GM) and Typic Hapludox (LVD) soils treated with 1.0 mg carbofuran kg¡1 soil. *Statistically significant effect (P < 0.05).

soils in the studied period (P D 0.073), carbofuran caused some mortality in the GM (20%) and LVD (50%) natural soils at 56 days. This indicates the influence on the results of exposure time of the organisms to the pesticide residues. According to the OECD guideline no. 222[11] on earthworm reproduction, the amount of specimen juveniles produced in the control samples must be 3 juveniles earthworm¡1 added to validate the study. At the end of this study, the results were validated because the mean of the total number of juveniles detected was 30 and 38 specimens, respectively, in the GM and LVD control samples, thus meeting or surpassing the required criterion. Nevertheless, a marked reduction in the number of juveniles (Fig. 1) was observed in both natural soils treated with carbofuran (P D 0.000, for both), and the number of cocoons in the carbofuran-treated soil was statistically smaller than in the control only in the GM soil (P D 0.04). Although no cocoon was detected in the LVD soil treated with carbofuran (Fig. 1), the difference between the control and insecticide-treated samples was not significant (P D 0.07). The difference between the number of juveniles produced in the control samples in both soil types was not significant (P D 0.5), but the number of cocoons differed significantly (P D 0.02). The same pattern occurred in the carbofuran-treated samples, i.e., there was no difference in the number of juveniles between the soils (P D 0.137), but the number of cocoons was different (P D 0.017). The effect of carbofuran on the earthworms’ reproduction was therefore similar in both soils, indicating that reproduction might not be the most recommended study parameter for natural soils. However, the effects on reproduction clearly indicate that the harm to the worms’ reproduction may be underestimated and these effects show the possibility

Again, neither mortality nor morphological change was detected, but a reduction on sensitivity to touch was observed in the worms that remained 14 days in the 14Ccarbofuran-treated soils. As the recommended doses are from 1.4 mg kg¡1 to 210 mg kg¡1,[20] and recent studies found a LC50 of 13.5 mg kg¡1 in artificial tropical soil,[19] no mortality was expected in the residual doses here used (0.28 mg carbofuran and 0.27 kBq 14C-carbofuran g¡1 GM and 0.25 mg carbofuran and 0.18 kBq 14C-carbofuran g¡1 LVD). Nevertheless, the low doses caused, again, a significant loss of biomass (P D 0.01) in the short period of 14 days, but only in the LVD. The 14C-carbofuran residues recovery rate in the soils was significantly different, being higher (P D 0.002) in the LVD than in the GM soil, which is richer in organic content (Table 3, Fig. 3). On the other hand, the short period of 14 days was long enough to produce bound residues in both soils, but in larger quantities (P D 0.123) in GM, and also larger than the extractable or bioavailable residues (Table 3). The higher OM in GM probably provided more sites for chemical reactions and adsorption of the pesticide, which contributed to the formation of carbofuran-bound residues in significantly larger quantities (Fig. 3). Carbofuran was also identified and quantified by HPLC, being 20.1 § 2.6% and 25.1 § 5.9%, respectively, recovered in GM and LVD, which mean 0.056 mg kg¡1 and 0.063 mg kg¡1 of the applied quantities. These values are lower than the ones recovered by the radiometric technique, which is known for its lower detection limits. But, the chromatographic analysis did not confirm the degradation of carbofuran in both soils in the time interval of the study (Fig. 4). Thus, the influence of carbofuran on avoidance and reproduction was clearly larger in the soil with lower OM (Table 2 and Fig. 1), as indicated by others.[28,29] Previous studies[34,39] also demonstrated larger bioaccumulation of 14 C-pesticides in earthworms in soils and substrates with lower organic matter content. De Silva et al.[28] ascribed the higher toxicity of carbofuran to earthworms to the lower adsorption of the pesticide in the soil particles and

Bioavailability and influence of 14C-carbofuran on Eisenia andrei in natural soils

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Table 3. Bioconcentration factor (BAF) and recovery of 14 C-residues in Typic Humaquept (GM), Typic Hapludox (LVD) soils treated with 14 C-carbofuran, and in earthworms Eisenia andrei, after 14 days into the soils (% of the treatments). 14

C-soil

Soil Type**

14

GM LVD

23.4 § 1.4 35.1 § 2.1

C-extracted %

14

14

14

BFA**

36.6 § 1.7 30.4 § 3.6

12.9 § 8.9 43.2 § 10.8

73.0 § 7.3 108.7 § 4.9

0.2 0.7

C-bound

C-earthworms

C-total Recovered

*

GM: Typic Humaquept; LVD: Typic Hapludox. FBA: bioaccumulation factor (14C-earthworms/14C-soil).

**

the greater bioavailability of carbofuran in soils with lower OM, similar to the LVD soil used in the present study. The effect of OM does not seem to be exclusive to pesticides because an indirect relationship between organic content and bioavailability to E. fetida was also observed for phenanthrene[40] and metals.[41] The effects of carbofuran on the mobility, curling, avoidance and growth of E. andrei earthworms, used as bioindicators in natural soils, indicate that carbofuran can also affect other species and soil food webs, i.e., ecosystem

Fig. 2. Biomass of the earthworms Eisenia andrei after contact with Typic Humaquept (GM) and Typic Hapludox (LVD) soils treated with carbofuran. *Statistically significant effect (P < 0.05).

functioning and structure,[12] particularly in light of the reduced sensitivity of this species.[42] But, despite the fact the doses of carbofuran used in this study were not bioaccumulated (Table 3), the earthworms in both soils absorbed some, being the absorption significantly higher (P D 0.03) from the LVD, where more extractable or bioavailable residues were detected, what probably caused greater weight loss (Table 3 and Fig. 3). Larger quantities of bound residues were formed in GM; however, as these residues are not promptly available, the earthworms in this soil did not have significant weight loss. The 14C-residues found in worms and the 14C-soil bound residues are important because they are not detectable by conventional analytical techniques and thus are underestimated in the biomonitoring programs of soil contamination. The bound residues cause limited availability of pesticides, what may demand the reduction of time intervals between applications. Finally, as the earthworms absorbed from 13% to 43% of the small amount of carbofuran present in the soil, the residues, after agricultural applications, may not be environmentally safe to the soil food webs because they do not guarantee the lack of longterm toxicity in the soil and along the trophic chains of soil environment where earthworms belong.

Fig. 3. Organic matter contents and 14 C-residues in earthworms E. andrei, 14 C-extractable and bound to soils Typic Humaquept (GM) and Typic Hapludox (LVD) treated with 14 C-carbofuran, 14 days after the treatment (%).

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Ferreira et al.

Fig. 4. Chromatograms of (a) carbofuran-treated Typic Humaquept (GM); (b) carbofuran-treated Typic Hapludox (LVD); (c) carbofuran; (d) hydroxy-carbofuran and (e) keto-carbofuran.

Conclusion E. andrei earthworms were sensitive bioindicators of carbofuran in natural soils. They presented clear negative reactions, such as avoidance, behavioral changes, decreased reproduction, weight loss and reduced survival rates in carbofuran-treated soils. Although the worms did not bioaccumulate carbofuran, they absorbed up to 43% of the small applied amount of the pesticide, depending on the soil. The effects were larger and long-lasting in the soil (LVD) with lower OM, where adsorption is probably reduced and carbofuran is more bioavailable in the soil

solution, what strongly influence survival, avoidance behavior, growth, absorption and reproduction rates. On the other hand, the higher OM of the Typic Humaquept (GM) induced higher production of soil bound residues, which are slowly released from the soil particles and which, together with the residues in the worms, are underestimated in biomonitoring programs. So, the results clearly indicated the influence of soil type on absorption of residues by earthworms and the soil bound residues formation. Furthermore, the results demonstrated that the effects of the pesticide rose with longer contact time of the worms

Bioavailability and influence of 14C-carbofuran on Eisenia andrei in natural soils with carbofuran-contaminated soils, suggesting that even small amounts of persistent residues may continue to affect the organisms and consequently the food webs that originate in the soil.

Funding The authors thank the International Atomic Energy Agency (IAEA) for the financial support to this study as part of the project BRAZIL IAEA-CRP-14018.

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Bioavailability and influence of ¹⁴C-carbofuran on Eisenia andrei avoidance, growth and reproduction in treated natural tropical soils.

The bioavailability of carbofuran to the compost worms Eisenia andrei and the influence of its residual amounts on the avoidance, reproduction and gro...
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