**Sorption of Terbuthylazine in Organic Matter Amended Soils: Effects on Eisenia Fetida and Lumbricus Terrestris**

Manuel Tejada, Marina del Toro, Paloma Osta, Juan Parrado and Isidoro Gómez

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54948

### **1. Introduction**

Pesticides are important tools in agriculture that help to minimize economic losses caused by weeds, insects, and pathogens. Although their use has helped to increase crop yields and value, they may also contribute to environmental degradation [1].

Pesticides are recognized as a source of potential adverse environmental impacts and their persistent in surface and ground waters has grown considerably [2]. Some soil applied herbicides reach surface and ground waters by the losses associated with runoff and leaching processes [3]. These losses are attenuated by the natural process of sorption, since degradation, transport, and biological activity of pesticides are greatly influenced by sorption on soil constituents [2].

In order to compensate for the losses caused by transport and degradation processes, some pesticides applicators are exceeding limits on labels which greatly exceed those required for control of the target organisms, and the excessive quantities added increase the environmental impact of these compounds [4, 5]. Because of this, public concern over the residues of pesticides in environment, food and related commodities has increased over the last decades. In Europe, pesticides are considered hazardous substances in accordance with current legislation regarding water [6, 7].

Traditional agricultural requires the use of herbicides, and prevention of ground water pollution is much cheaper than restoring polluted aquifers. Thus, it is of maximum interest that development of agricultural strategies continues to be directed to the decrease in pesticide movement [8].

© 2013 Tejada et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Tejada et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Triazine herbicides have been largely used in agriculture worldwide for selective and nonselective control of broadleaf and small seeded grass weeds in diverse crops, such as cotton (*Gossypium hirsutum*, L.), maize (*Zea mays,* L.), soybean (*Glycine max*, L.), groundnut (*Arachis hypogea*, L.) and vineyards (*Vitis vinifera*, L.). However, due to their physicochemical properties (in particular, their relatively long persistence) there have been numerous reports of their presence in surface or ground waters [3, 9]. Several bioremediation strategies have been proposed to reduce the presence of pesticides in soil from which they can reach groundwater, such as remediation by enhancing the microbial population able to degrade specifically the target compounds. This strategy has been approached by addition of organic exogenous matter of different origin [9, 10, 11].

**2. Material and methods**

CO3 2- (g kg-1) 203 ± 12 Fine sand (g kg-1) 142 ± 35 Coarse sand (g kg-1) 387 ± 26 Silt (g kg-1) 242 ± 19 Clay (g kg-1) 229 ± 10

shown in Table 1.

Clay types

of four samples.

**2.1. Soil, organic amendment and herbicide characteristics**

The soil used in this experiment is a Plagic Antrosol [24]. The main soil characteristics are

Sorption of Terbuthylazine in Organic Matter Amended Soils: Effects on Eisenia Fetida and Lumbricus Terrestris

pH (H2O) 8.6 ± 0.2 7.1 ± 0.3 6.2 ± 0.3 8.3 ± 0.2

Organic matter (g kg-1) 1.1 ± 0.2 614 ± 26 469 ± 15 764 ± 29 Humic acid-C (mg kg-1) 18.5 ± 2.4 672 ± 1.4 1030 ± 17 461 ± 13 Fulvic acid-C (mg kg-1) 9.8 ± 1.1 715 ± 10 711 ± 10 631 ± 24 Total N (g kg-1) 0.4 ± 0.1 38.8 ± 2.9 17.3 ± 1.3 29.2 ± 2.1 Fe (mg kg-1) 35.8 ± 3.7 180 ± 22 815 ± 38 407 ± 28 Cu (mg kg-1) 9.7 ± 1.3 1.6 ± 0.3 82.6 ± 9.8 24.2 ± 1.8 Mn (mg kg-1) 11.3 ± 2.1 4.2 ± 0.9 75.6 ± 8.1 14.1 ± 1.2 Zn (mg kg-1) 8.1 ± 1.5 3.3 ± 0.8 134 ± 13 10.3 ± 1.6 Cd (mg kg-1) 6.5 ± 1.2 0.35 ± 0.07 1.1 ± 0.3 0.28 ± 0.09 Pb (mg kg-1) 0.36 ± 0.11 0.94 ± 0.12 82.4 ± 3.6 5.3 ± 0.8 Ni (mg kg-1) 2.9 ± 0.7 1.3 ± 0.2 13.6 ± 1.5 2.4 ± 0.6 Cr (mg kg-1) 5.3 ± 0.6 0.12± 0.02 19.4 ± 1.7 0.29 ± 0.04

**Table 1.** Characteristics of the experimental soil and organic amendment (mean ± standard error). Data are the means

Soil pH was determined in distilled water with a glass electrode (soil:H2O ratio 1:2.5). Soil texture was determined by the Robinson's pipette method [25] and quantification and dominant clay types were determined by X-ray diffraction. Total carbonates were measured by estimating the quantity of the CO2 produced by HCl addition to the soil [26]. Soil organic matter was determined by the method of Yeomans and Bremner [27]. Humic and fulvic acids-

Smectite: 66% Kaolinite: 20% Illite: 14 %

**Soil PM MSW CM**

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21

However, the influence of organic matter on soil properties and sorption process depends upon the type, amount and dominant components of the added organic materials [12, 13]. This aspect is of great interest, since it supposes an important advance in the behavior of the herbicides in the soil after the addition of different sources of organic matter.

Earthworms are one of the important components in decomposer communities and contribute significantly to the organic decomposition, nutrient cycling and soil formation [14]. Continu‐ ous application of pesticides may present risks to lead to soil pollution affect soil fauna [15]. For this reason, the use of earthworms for toxicity testing is highly recommended by the European Communities [16] and are considered as preferred bioindicators for assessing the environmental health status of chemical pollution [17, 18]. Earthworm species such as *Eisenia fetida* is considered as a suitable biomonitors to determine the ecological hazard of heavy metals and chemicals contaminated soil because of its low cost, easy culturing and the standardization of the acute and sub-chronic ecotoxicological tests [14, 19].

Of the potential biomarkers, earthworm glutathione-S-transferase and cellulalse enzymes are shown to respond to toxin exposure [14]. Glutathione-S-transferase is an important detoxifi‐ cation enzyme and its activity has been used as a potential bioindicator and biomarker of earthworms for heavy metals, pesticides and PAHs exposure [14, 15, 20]. Also, cellulase activity of earthworms indicates their role in the decomposition of plant litter and other cellulosic materials. It has been used as a biomarker of a pesticide contamination on earth‐ worms [14, 20].

However, the biological relevance of *Eisenia fetida* is still open to debate [21] since they are often less susceptible to pollutants than other species [22] and rarely found in conventionally tilled agricultural soils. In this respect, Ma and Bodt [23] found and different levels of chlorpyrifos insecticide sensitivity to earthworms (*Eisenia* sp.< *Aporrectodea* sp.< *Lumbricus* sp.).

Few studies have been performed comparing different sources of organic matter types on the sorption and mobility of herbicides. For this reason, the objective of this study was to investi‐ gate the sorption and mobility of terbuthylazine herbicide in a soil amended with three organic amendment and their effects on acute toxicity and morphological alterations in two earthworm species (*Eisenia fetida* and *Lumbricus terrestris*).

### **2. Material and methods**

Triazine herbicides have been largely used in agriculture worldwide for selective and nonselective control of broadleaf and small seeded grass weeds in diverse crops, such as cotton (*Gossypium hirsutum*, L.), maize (*Zea mays,* L.), soybean (*Glycine max*, L.), groundnut (*Arachis hypogea*, L.) and vineyards (*Vitis vinifera*, L.). However, due to their physicochemical properties (in particular, their relatively long persistence) there have been numerous reports of their presence in surface or ground waters [3, 9]. Several bioremediation strategies have been proposed to reduce the presence of pesticides in soil from which they can reach groundwater, such as remediation by enhancing the microbial population able to degrade specifically the target compounds. This strategy has been approached by addition of organic exogenous matter

However, the influence of organic matter on soil properties and sorption process depends upon the type, amount and dominant components of the added organic materials [12, 13]. This aspect is of great interest, since it supposes an important advance in the behavior of the

Earthworms are one of the important components in decomposer communities and contribute significantly to the organic decomposition, nutrient cycling and soil formation [14]. Continu‐ ous application of pesticides may present risks to lead to soil pollution affect soil fauna [15]. For this reason, the use of earthworms for toxicity testing is highly recommended by the European Communities [16] and are considered as preferred bioindicators for assessing the environmental health status of chemical pollution [17, 18]. Earthworm species such as *Eisenia fetida* is considered as a suitable biomonitors to determine the ecological hazard of heavy metals and chemicals contaminated soil because of its low cost, easy culturing and the standardization

Of the potential biomarkers, earthworm glutathione-S-transferase and cellulalse enzymes are shown to respond to toxin exposure [14]. Glutathione-S-transferase is an important detoxifi‐ cation enzyme and its activity has been used as a potential bioindicator and biomarker of earthworms for heavy metals, pesticides and PAHs exposure [14, 15, 20]. Also, cellulase activity of earthworms indicates their role in the decomposition of plant litter and other cellulosic materials. It has been used as a biomarker of a pesticide contamination on earth‐

However, the biological relevance of *Eisenia fetida* is still open to debate [21] since they are often less susceptible to pollutants than other species [22] and rarely found in conventionally tilled agricultural soils. In this respect, Ma and Bodt [23] found and different levels of chlorpyrifos

Few studies have been performed comparing different sources of organic matter types on the sorption and mobility of herbicides. For this reason, the objective of this study was to investi‐ gate the sorption and mobility of terbuthylazine herbicide in a soil amended with three organic amendment and their effects on acute toxicity and morphological alterations in two earthworm

insecticide sensitivity to earthworms (*Eisenia* sp.< *Aporrectodea* sp.< *Lumbricus* sp.).

herbicides in the soil after the addition of different sources of organic matter.

of the acute and sub-chronic ecotoxicological tests [14, 19].

species (*Eisenia fetida* and *Lumbricus terrestris*).

of different origin [9, 10, 11].

20 Herbicides - Advances in Research

worms [14, 20].

### **2.1. Soil, organic amendment and herbicide characteristics**

The soil used in this experiment is a Plagic Antrosol [24]. The main soil characteristics are shown in Table 1.


**Table 1.** Characteristics of the experimental soil and organic amendment (mean ± standard error). Data are the means of four samples.

Soil pH was determined in distilled water with a glass electrode (soil:H2O ratio 1:2.5). Soil texture was determined by the Robinson's pipette method [25] and quantification and dominant clay types were determined by X-ray diffraction. Total carbonates were measured by estimating the quantity of the CO2 produced by HCl addition to the soil [26]. Soil organic matter was determined by the method of Yeomans and Bremner [27]. Humic and fulvic acidsfractions were extracted with 0.1 M sodium pyrophosphate and 0.1 M sodium hydroxide at pH 13 [28]. The supernatant was acidified to pH 2 with HCl and allowed to stand for 24 h at room temperature. To separate humic acids-fraction from fulvic acids-fraction, the solution was centrifuged and the precipitate containing humic acids-fraction was dissolved with sodium hydroxide [27]. After the removal of humic acids-fraction, the acidic filtrate containing the dissolved fulvic acid-fraction was passed through a column of XAD-8 resin. The adsorbed fulvic was then recovered by elution with 0.1 M NaOH, desalted using Amberlyst 15-cationexchange resin, and finally freeze-dried. The carbon content of humic and fulvic acids-fractions were determined by the method described. Total N was determined by the Kjeldhal method [26]. After nitric and perchloric acid digestion, total Ca, Mg, Fe, Cu, Mn, Zn, Cd, Pb, Ni and Cr concentrations were determined by atomic absorption spectrometer and K was determined by atomic emission spectrometer, according to MAPA methods [26].

**2.2. Incubation procedure**

Two kg of soil were pre-incubated at 25 ºC for 7 days at 30–40% of their water-holding capacity, according to Moreno et al. [31], prior to the treatments. After this pre-incubation period, soil samples were treated with three concentrations of terbuthylazine (1, 10 and 50 µg terbuthy‐ lazine g-1 soil) and treated with MSW at a rate of 10% or PM at a rate of 7.6% or CM at a rate of 5.8%, respectively, in order to applying the same amount of organic matter to the soil. A

Sorption of Terbuthylazine in Organic Matter Amended Soils: Effects on Eisenia Fetida and Lumbricus Terrestris

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23

non-mended treated as well as a amended non-treated soil were used as controls.

**2.** C2, soil treated with 1 µg terbuthylazine g-1 soil and non-organic amended

**3.** C3, soil treated with 10 µg terbuthylazine g-1 soil and non-organic amended

**4.** C4, soil treated with 50 µg terbuthylazine g-1 soil and non-organic amended

**6.** MSW2, soil treated with 1 µg terbuthylazine g-1 soil and amended with MSW

**7.** MSW3, soil treated with 10 µg terbuthylazine g-1 soil and amended with MSW

**8.** MSW4, soil treated with 50 µg terbuthylazine g-1 soil and amended with MSW

**10.** PM2, soil treated with 1 µg terbuthylazine g-1 soil and amended with PM

**11.** PM3, soil treated with 10 µg terbuthylazine g-1 soil and amended with PM

**12.** PM4, soil treated with 50 µg terbuthylazine g-1 soil and amended with PM

**14.** CM2, soil treated with 1 µg terbuthylazine g-1 soil and amended with CM

**15.** CM3, soil treated with 10 µg terbuthylazine g-1 soil and amended with CM

**16.** CM4, soil treated with 50 µg terbuthylazine g-1 soil and amended with CM

cultures on organic amendment materials, vermicomposts principally.

Triplicate treatments were kept in semi-closed microcosms at 25 ºC for 3, 15, 45 and 90 days,

Twenty two earthworms of the species *Eisenia fetida* (approximately 210 mg fresh weight) and *Lumbricus terrestris* (approximately 190 mg fresh weight) were included in each microcosm. Each microcosm was covered with fine nylon mesh to prevent the soil loss and to keep earthworms from escaping. *Lumbricus terrestris* were collected in the field, in an area that has not been treated with pesticides for 20 years, whereas *Eisenia fetida* were bred in laboratory

The incubation treatments are detailed as follows:

**5.** MSW1, soil non- treated and amended with MSW

**9.** PM1, soil non- treated and amended with PM

**13.** CM1, soil non- treated and amended with CM

respectively.

**1.** C1, control soil, soil non-polluted and non-organic amended

The organic amendment applied were the organic fraction of a municipal solid waste (MSW), poultry manure (PM) and cow manure (CM). The general properties of the organic amendment are shown in Table 1. Organic matter was determined by dry combustion, according to the official methods of the Spanish Ministry of Agriculture [26]. Humic and fulvic acids-fraction were extracted, separated and determined by the methods previously described. Total N was determined by the Kjeldhal method [26]. After nitric and perchloric acid digestion, total Ca, Mg, Fe, Cu, Mn, Zn, Cd, Pb, Ni and Cr concentrations were determined by atomic absorption spectrometer and K was determined by atomic emission spectrometer, according to MAPA methods [26].

Table 2 shows the acidic functional group contents of humic acids isolated from both organic amendment. The carboxyl group content was estimated by direct potentiometric titration at pH 8, the phenolic hydroxyl group content was estimated as two times the change in charge between pH 8 and pH 10, and the total acidity was calculated by addition [29].


**Table 2.** Acidic functional group contents (mean ± standard errors) of humic acids isolated from PM, MSW and CM

The herbicide used in this experiment was the terbuthylazine. Terbuthylazine (N2-tert-butyl-6 chloro-N4-ethyl-1,3,5-triazine-2,4-diamine) is a selective herbicide for the control of broadleaf and grass weeds in forestry, lucerne (*Medicago sativa*, L.), maize (*Zea mays,* L.), sweetcorn (*Zea mays,* L. var. *rugosa*), peas (*Pisum sativum*, L.), orchard and non-cropland, with a water solubility of 8.5 mg l−1 at 20 ºC. It is absorbed by roots and inhibits Hill reaction and CO2 sorption in the chlorophyllic function [30].

### **2.2. Incubation procedure**

fractions were extracted with 0.1 M sodium pyrophosphate and 0.1 M sodium hydroxide at pH 13 [28]. The supernatant was acidified to pH 2 with HCl and allowed to stand for 24 h at room temperature. To separate humic acids-fraction from fulvic acids-fraction, the solution was centrifuged and the precipitate containing humic acids-fraction was dissolved with sodium hydroxide [27]. After the removal of humic acids-fraction, the acidic filtrate containing the dissolved fulvic acid-fraction was passed through a column of XAD-8 resin. The adsorbed fulvic was then recovered by elution with 0.1 M NaOH, desalted using Amberlyst 15-cationexchange resin, and finally freeze-dried. The carbon content of humic and fulvic acids-fractions were determined by the method described. Total N was determined by the Kjeldhal method [26]. After nitric and perchloric acid digestion, total Ca, Mg, Fe, Cu, Mn, Zn, Cd, Pb, Ni and Cr concentrations were determined by atomic absorption spectrometer and K was determined

The organic amendment applied were the organic fraction of a municipal solid waste (MSW), poultry manure (PM) and cow manure (CM). The general properties of the organic amendment are shown in Table 1. Organic matter was determined by dry combustion, according to the official methods of the Spanish Ministry of Agriculture [26]. Humic and fulvic acids-fraction were extracted, separated and determined by the methods previously described. Total N was determined by the Kjeldhal method [26]. After nitric and perchloric acid digestion, total Ca, Mg, Fe, Cu, Mn, Zn, Cd, Pb, Ni and Cr concentrations were determined by atomic absorption spectrometer and K was determined by atomic emission spectrometer, according to MAPA

Table 2 shows the acidic functional group contents of humic acids isolated from both organic amendment. The carboxyl group content was estimated by direct potentiometric titration at pH 8, the phenolic hydroxyl group content was estimated as two times the change in charge

**Table 2.** Acidic functional group contents (mean ± standard errors) of humic acids isolated from PM, MSW and CM

The herbicide used in this experiment was the terbuthylazine. Terbuthylazine (N2-tert-butyl-6 chloro-N4-ethyl-1,3,5-triazine-2,4-diamine) is a selective herbicide for the control of broadleaf and grass weeds in forestry, lucerne (*Medicago sativa*, L.), maize (*Zea mays,* L.), sweetcorn (*Zea mays,* L. var. *rugosa*), peas (*Pisum sativum*, L.), orchard and non-cropland, with a water solubility of 8.5 mg l−1 at 20 ºC. It is absorbed by roots and inhibits Hill reaction and CO2 sorption in the

**Total acidity (mol kg-1) COOH Phenolic OH**

between pH 8 and pH 10, and the total acidity was calculated by addition [29].

PM 3.99 ± 0.13 2.99 ± 0.09 0.99 ± 0.05 MSV 4.29 ± 0.04 3.19 ± 0.03 1.10 ± 0.03 CM 2.81 ± 0.02 2.00 ± 0.03 0.80 ± 0.01

by atomic emission spectrometer, according to MAPA methods [26].

methods [26].

22 Herbicides - Advances in Research

chlorophyllic function [30].

Two kg of soil were pre-incubated at 25 ºC for 7 days at 30–40% of their water-holding capacity, according to Moreno et al. [31], prior to the treatments. After this pre-incubation period, soil samples were treated with three concentrations of terbuthylazine (1, 10 and 50 µg terbuthy‐ lazine g-1 soil) and treated with MSW at a rate of 10% or PM at a rate of 7.6% or CM at a rate of 5.8%, respectively, in order to applying the same amount of organic matter to the soil. A non-mended treated as well as a amended non-treated soil were used as controls.

The incubation treatments are detailed as follows:


Triplicate treatments were kept in semi-closed microcosms at 25 ºC for 3, 15, 45 and 90 days, respectively.

Twenty two earthworms of the species *Eisenia fetida* (approximately 210 mg fresh weight) and *Lumbricus terrestris* (approximately 190 mg fresh weight) were included in each microcosm. Each microcosm was covered with fine nylon mesh to prevent the soil loss and to keep earthworms from escaping. *Lumbricus terrestris* were collected in the field, in an area that has not been treated with pesticides for 20 years, whereas *Eisenia fetida* were bred in laboratory cultures on organic amendment materials, vermicomposts principally.

### **2.3. Adsorption studies**

For adsorption studies the treatments used were:


Terbuthylazine sorption was determined according to Cabrera et al. [32] criteria. Triplicate samples (5 g) of the non-amended and organic amended soil (S, S+CM, S+PM, S+MSW) were treated with 10 ml of terbuthylazine (50%:50%, v/v) solution (initial concentrations, Ci, ranging from 5 to 50 µM in 0.01 CaCl2). Previously, it was determined that equilibrium was reached in less than 24 h, and that no measurable degradation occurred during this period. Equilibrium concentrations (Ce) in the supernatants were determined by HPLC. Sorption isotherms were fitted to Freundlich equation (Cs=Kf x Ce1/nf) and sorption coefficients Kf and 1/nf were calculated.

**2.6. Statistical analysis**

**3. Results**

**3.1. Sorption studies**

0

20

40

60

Concentration in soil (µg kg-1)

80

100

120

Two-ways analysis of variance (ANOVA) was performed for all parameters, considering two variables involved (incubation time and the terbuthylazine concentration applied to the soil) using the Statgraphics v. 5.0 software package [38]. The means were separated by the Tukey's test, considering a significance level of *P*<0.05 throughout the study. For the ANOVA, triplicate

Sorption of Terbuthylazine in Organic Matter Amended Soils: Effects on Eisenia Fetida and Lumbricus Terrestris

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25

Sorption isotherms of terbuthylazine on soil, soil+CM, soil+PM and soil+MSW are shown in Figure 1. The results indicated that sorption of terbuthylazine on organic amended soils significantly increased compared to non-organic amended soil. For each organic amended soil,

0 1 2 3 4 5 6

Concentration in soil solution (µg ml-1) S S+MSW S+PM S+CM

**Figure 1.** Terbuthylazine sorption isotherms in non-amended and organic amended soils. Symbols are experimental

Sorption isotherms were fit to the Freundlich equation and sorption coefficients Kf and 1/nf were calculated (Table 3). The results indicated that Kf values significantly increased in organic amended soils than for non-organic amended soils. However, terbuthylazine sorption increased by a factor of 4.5 upon amendment with MSW, whereas for PM and CM, the factor increased 4 and 3.4, respectively. Again, the results indicate significant differences between S

data points, whereas lines are the Freundlich-fit sorption isotherms.

data were used for each treatment and every incubation day.

the herbicide sorption with MSW was higher than with PM and CM.

### **2.4. Herbicide analysis**

Herbicide was extracted twice with methanol (Merck, Darmstadt, Germany) at 1:2 soil/solution ratio for 15 min. Extracts were mixed and rotary-vacuum evaporated almost to dryness at 40 ºC. The residue was dissolved in 2 ml of methanol and analyzed by HPLC [30]. Terbuthylazine was analyzed using a Beckman, System Gold, Autosampler 508 HPLC chromatograph coupled to a Waters 2996 diode-array detector. The analytical conditions were: Nova-Pack C18 column (159 mm length X 3.9 mm internal diameter), eluent mixture, 50:50 acetonitrile/water at a flow rate of 1 ml min-1, 25 µl injection volume, and UV detection at 220 nm [33]. External calibration curves with four standard solutions between 0.2 and 26 µM were used in the calculations.

### **2.5. Earthworm analysis**

Earthworm cocoon production was determined after 30 days of exposure. Cocoons were collected by hand sorting and weighed, and then incubated for four additional weeks as described by Maboeta et al. [34]. Cocoons were cultured in Petri dishes at 25±1 ºC covered with three moist filter papers. According to Xiao et al. [14], the filter papers in these dishes were changed every three days to prevent bacterial growth. At the end of the experiment (30 days), the weight of per cocoon and number of juveniles per cocoon were determined.

After 3, 15, 45 and 90 incubation days for each treatment, three worms were selected and placed on wet filter paper in Petri dishes for 24 h to clear gut contents, and their weights were recorded after blotting them dry on paper towels. Earthworms were digested in the 1:1 nitric-perchloric extract after digestion at 450 ºC for 6 h. The terbuthylazine was measured by the method previously mentioned. Cellulase activity was measured as described by Mishra and Dash [35], and glutathione-S-transferase activity was measured according to the method described by Habig et al. [36] and Saint-Denis et al. [37].

### **2.6. Statistical analysis**

**2.3. Adsorption studies**

24 Herbicides - Advances in Research

calculated.

**2.4. Herbicide analysis**

**2.5. Earthworm analysis**

Habig et al. [36] and Saint-Denis et al. [37].

For adsorption studies the treatments used were:

**1.** S, non-organic amended control soil (10 g of soil)

**2.** S+CM, soil amended with CM at rate of 10% (10 g of soil + 1 g of CM)

**3.** S+PM, soil amended with PM at a rate of 12.4% (10 g of soil + 1.24 g of PM)

**4.** S+MSW, soil amended with MSW at a rate of 16.3% (10 g of soil + 1.63 g of MSW)

Terbuthylazine sorption was determined according to Cabrera et al. [32] criteria. Triplicate samples (5 g) of the non-amended and organic amended soil (S, S+CM, S+PM, S+MSW) were treated with 10 ml of terbuthylazine (50%:50%, v/v) solution (initial concentrations, Ci, ranging from 5 to 50 µM in 0.01 CaCl2). Previously, it was determined that equilibrium was reached in less than 24 h, and that no measurable degradation occurred during this period. Equilibrium concentrations (Ce) in the supernatants were determined by HPLC. Sorption isotherms were fitted to Freundlich equation (Cs=Kf x Ce1/nf) and sorption coefficients Kf and 1/nf were

Herbicide was extracted twice with methanol (Merck, Darmstadt, Germany) at 1:2 soil/solution ratio for 15 min. Extracts were mixed and rotary-vacuum evaporated almost to dryness at 40 ºC. The residue was dissolved in 2 ml of methanol and analyzed by HPLC [30]. Terbuthylazine was analyzed using a Beckman, System Gold, Autosampler 508 HPLC chromatograph coupled to a Waters 2996 diode-array detector. The analytical conditions were: Nova-Pack C18 column (159 mm length X 3.9 mm internal diameter), eluent mixture, 50:50 acetonitrile/water at a flow rate of 1 ml min-1, 25 µl injection volume, and UV detection at 220 nm [33]. External calibration curves with four standard solutions between 0.2 and 26 µM were used in the calculations.

Earthworm cocoon production was determined after 30 days of exposure. Cocoons were collected by hand sorting and weighed, and then incubated for four additional weeks as described by Maboeta et al. [34]. Cocoons were cultured in Petri dishes at 25±1 ºC covered with three moist filter papers. According to Xiao et al. [14], the filter papers in these dishes were changed every three days to prevent bacterial growth. At the end of the experiment (30 days),

After 3, 15, 45 and 90 incubation days for each treatment, three worms were selected and placed on wet filter paper in Petri dishes for 24 h to clear gut contents, and their weights were recorded after blotting them dry on paper towels. Earthworms were digested in the 1:1 nitric-perchloric extract after digestion at 450 ºC for 6 h. The terbuthylazine was measured by the method previously mentioned. Cellulase activity was measured as described by Mishra and Dash [35], and glutathione-S-transferase activity was measured according to the method described by

the weight of per cocoon and number of juveniles per cocoon were determined.

Two-ways analysis of variance (ANOVA) was performed for all parameters, considering two variables involved (incubation time and the terbuthylazine concentration applied to the soil) using the Statgraphics v. 5.0 software package [38]. The means were separated by the Tukey's test, considering a significance level of *P*<0.05 throughout the study. For the ANOVA, triplicate data were used for each treatment and every incubation day.

### **3. Results**

### **3.1. Sorption studies**

Sorption isotherms of terbuthylazine on soil, soil+CM, soil+PM and soil+MSW are shown in Figure 1. The results indicated that sorption of terbuthylazine on organic amended soils significantly increased compared to non-organic amended soil. For each organic amended soil, the herbicide sorption with MSW was higher than with PM and CM.

**Figure 1.** Terbuthylazine sorption isotherms in non-amended and organic amended soils. Symbols are experimental data points, whereas lines are the Freundlich-fit sorption isotherms.

Sorption isotherms were fit to the Freundlich equation and sorption coefficients Kf and 1/nf were calculated (Table 3). The results indicated that Kf values significantly increased in organic amended soils than for non-organic amended soils. However, terbuthylazine sorption increased by a factor of 4.5 upon amendment with MSW, whereas for PM and CM, the factor increased 4 and 3.4, respectively. Again, the results indicate significant differences between S +MSW and S+CM treatments. Also, the 1/nf coefficients significantly decreased in organic amended soils than for non-organic amended soil. For organic amended soils, the 1/nf coefficient was higher in the soil amended with MSW, followed by PM and CM, respectively.

*Eisenia fetida Lumbricus terrestris* **Incubation days Incubation days 3 15 45 90 3 15 45 90**

Sorption of Terbuthylazine in Organic Matter Amended Soils: Effects on Eisenia Fetida and Lumbricus Terrestris

C1 208ab ± 12 211b ± 14 211b ± 9 212b ± 10 192ab ± 5 190ab ± 8 195ab ± 7 199ab ± 10 C2 206ab ± 15 190a ± 20 173a ± 15 162a ± 13 188ab ± 10 172a ± 10 160a ± 8 148a ± 14 C3 201ab ± 17 180a ± 11 164a ± 16 145a ± 18 180a ± 11 163a ± 15 150a ± 10 132a ± 13 C4 197ab ± 19 164a ± 23 140a ± 12 129a ± 16 170a ± 13 156a ± 11 134a ± 8 116a ± 12 MSW1 216b ± 13 233b ± 16 244b ± 19 255b ± 14 203ab ± 11 215b ± 15 222b ± 12 236b ± 17 MSW2 215b ± 12 217b ± 18 213b ± 18 220b ± 17 198ab ± 13 195ab ± 12 197ab ± 10 201ab ± 13 MSW3 214b ± 10 215b ± 13 209ab ± 10 205ab ± 11 194ab ± 10 190ab ± 11 186ab ± 13 182a ± 14 MSW4 213b ± 13 211b ± 10 204ab ± 13 197ab ± 13 190ab ± 14 188ab ± 10 182a ± 11 179a ± 12 PM1 215b ± 14 229b ± 17 233b ± 12 244b ± 11 198ab ± 12 206b ± 15 219b ± 12 227b ± 14 PM2 214b ± 12 212b ± 13 209ab ± 18 207ab ± 18 195ab ± 10 194ab ± 12 194ab ± 8 192a ± 11 PM3 213b ± 11 210ab ± 15 199ab ± 13 189a ± 15 193ab ± 11 188ab ± 15 180a ± 16 172a ± 15 PM4 212b ± 15 205ab ± 12 190a ± 11 180a ± 11 190ab ± 12 181a ± 10 175ab ± 12 168a ± 13 CM1 213b ± 17 220b ± 15 225b ± 14 236b ± 18 196ab ± 11 202ab ± 13 210b ± 10 219b ± 12 CM2 221b ± 15 201ab ± 10 195ab ± 15 193ab ± 14 193ab ± 10 193ab ± 9 180a ± 12 186ab ± 11 CM3 210ab ± 11 195ab ± 12 180a ± 10 174a ± 11 190ab ± 8 185ab ± 10 170a ± 13 162a ± 13 CM4 209ab ± 13 190a ± 17 173a ± 12 165a ± 13 188ab ± 6 179a ± 11 164a ± 11 157a ± 14

**Table 4.** Changes in weight (mean ± standard error) (mg) of *Eisenia fetida* and *Lumbricus terrestris* exposed to different concentrations of terbuthylazine herbicide. Column (mean ± standard errors) followed by the same letter(s)

> **Number of juveniles per cocoon**

C1 2.93ab ± 0.35 8.76b ± 0.47 3.09b ± 0.36 2.63b ± 0.22 8.15b ± 0.31 2.81ab ± 0.17 C2 2.05a ± 0.22 6.64ab ± 0.24 2.49ab ± 0.17 1.78a ± 0.18 6.06ab ± 0.23 2.21a ± 0.14 C3 1.74a ± 0.15 5.68a ± 0.31 2.34a ± 0.21 1.54a ± 0.24 5.20a ± 0.16 2.13a ± 0.10 C4 1.18a ± 0.17 4.88a ± 0.25 1.98a ± 0.13 1.03a ± 0.10 4.33a ± 0.13 1.77a ± 0.15 MSW1 3.42b ± 0.22 8.95b ± 0.39 3.37b ± 0.28 3.03b ± 0.34 8.31b ± 0.24 3.03b ± 0.17 MSW2 2.90ab ± 0.17 7.50b ± 0.25 2.94b ± 0.17 2.51b ± 0.19 6.76ab ± 0.17 2.60ab ± 0.13 MSW3 2.69ab ± 0.13 7.12ab ± 0.20 2.87b ± 0.13 2.33b ± 0.15 6.43ab ± 0.22 2.54ab ± 0.19 MSW4 2.37ab ± 0.13 6.97ab ± 0.22 2.55ab ± 0.11 2.06ab ± 0.17 6.24ab ± 0.25 2.22a ± 0.16

*Eisenia fetida Lumbricus terrestris*

**Cocoon numbers**

**Average weigh of per cocoon (mg)**

**Number of juveniles per cocoon**

http://dx.doi.org/10.5772/54948

27

are not significantly different (p<0.05)

**Cocoon numbers** **Average weigh of per cocoon (mg)**


**Table 3.** Freundlich sorption coefficients Kf and 1/nf and standard error for terbuthylazine in non-amended and organic amended soils. Column (mean ± standard errors) followed by the same letter(s) are not significantly different (p<0.05)

### **3.2. Effect of terbuthylazine on weight on earthworms**

In treated and non-organic amended soils, the *Eisenia fetida* weight decreased during the experimental period and when increased the terbuthylazine concentration in soil (Table 4). At the end of the experiment, the worm weight decreased 23.6%, 31.4% and 39.1% in soil treated with 1, 10 and 50 µg terbuthylazine g-1 soil, respectively. For *Lumbricus terrestris*, the weight decreased 25.6%, 33.6% and 41.8% in soil treated with 1, 10 and 50 µg terbuthylazine g-1 soil, respectively.

The application of organic matter to non-treated soil increased the worm weight. However, this increased depended of the organic matter type. At the end of the experiment, *Eisenia fetida* weight increased 20.3%, 15.1% and 11.3% in MSW, PM and CM-amended soils, compared to control soil, whereas *Lumbricus. terrestris* weight increased 18.6%, 13.9% and 10.1% in MSW, PM and CM-amended soils, compared to control soil. However, the ANOVA analysis indicated no significant differences between these treatments.

In treated and organic amended soils, the both worms weight increased respect to the treated and non-organic amended soils. This increase was higher for MSW, followed by PM and CMamended soils.

The non-treated and organic amended soils have the highest mean cocoon numbers (Table 5). For both worms, the cocoon numbers were highest in MSW followed by PM and CMamended soils. Terbuthylazine treatments in organic amended soils decreased the cocoon numbers. However, this decrease was lowest in MSW followed by PM and CM-amended soils.

The average weight per cocoon was also higher in non-polluted and organic amended soils, compared to control soil. Again, the average weight per cocoon in terbuthylazine treated and organic amended soils were highest in MSW followed by PM and CM-amended soils.

Sorption of Terbuthylazine in Organic Matter Amended Soils: Effects on Eisenia Fetida and Lumbricus Terrestris http://dx.doi.org/10.5772/54948 27

+MSW and S+CM treatments. Also, the 1/nf coefficients significantly decreased in organic amended soils than for non-organic amended soil. For organic amended soils, the 1/nf coefficient was higher in the soil amended with MSW, followed by PM and CM, respectively.

S 6.93a ± 0.95 0.92a ± 0.05 0.963 S+MSW 31.22c ± 2.46 0.80c ± 0.06 0.943 S+PM 28.03bc ± 2.03 0.83bc ± 0.05 0.958 S+CM 23.48b ± 1.99 0.86b ± 0.04 0.977

**Table 3.** Freundlich sorption coefficients Kf and 1/nf and standard error for terbuthylazine in non-amended and organic amended soils. Column (mean ± standard errors) followed by the same letter(s) are not significantly different

In treated and non-organic amended soils, the *Eisenia fetida* weight decreased during the experimental period and when increased the terbuthylazine concentration in soil (Table 4). At the end of the experiment, the worm weight decreased 23.6%, 31.4% and 39.1% in soil treated with 1, 10 and 50 µg terbuthylazine g-1 soil, respectively. For *Lumbricus terrestris*, the weight decreased 25.6%, 33.6% and 41.8% in soil treated with 1, 10 and 50 µg terbuthylazine g-1 soil,

The application of organic matter to non-treated soil increased the worm weight. However, this increased depended of the organic matter type. At the end of the experiment, *Eisenia fetida* weight increased 20.3%, 15.1% and 11.3% in MSW, PM and CM-amended soils, compared to control soil, whereas *Lumbricus. terrestris* weight increased 18.6%, 13.9% and 10.1% in MSW, PM and CM-amended soils, compared to control soil. However, the ANOVA analysis

In treated and organic amended soils, the both worms weight increased respect to the treated and non-organic amended soils. This increase was higher for MSW, followed by PM and CM-

The non-treated and organic amended soils have the highest mean cocoon numbers (Table 5). For both worms, the cocoon numbers were highest in MSW followed by PM and CMamended soils. Terbuthylazine treatments in organic amended soils decreased the cocoon numbers. However, this decrease was lowest in MSW followed by PM and CM-amended soils.

The average weight per cocoon was also higher in non-polluted and organic amended soils, compared to control soil. Again, the average weight per cocoon in terbuthylazine treated and

organic amended soils were highest in MSW followed by PM and CM-amended soils.

**3.2. Effect of terbuthylazine on weight on earthworms**

indicated no significant differences between these treatments.

(p<0.05)

26 Herbicides - Advances in Research

respectively.

amended soils.

**Kf 1/nf R2**


**Table 4.** Changes in weight (mean ± standard error) (mg) of *Eisenia fetida* and *Lumbricus terrestris* exposed to different concentrations of terbuthylazine herbicide. Column (mean ± standard errors) followed by the same letter(s) are not significantly different (p<0.05)



**4. Discussion**

*andrei* over a period of three generations.

with herbicides and heavy metals [40].

Our results indicated that terbuthylazine induced negative effects on weight, reproductive and enzymatic activities on the both earthworms. These negative effects increased with increasing herbicide concentration and/or exposure time. These results are in accordance with Brunninger et al. [39] who studied the toxicity of terbuthylazine on the growth and reproduction of *Eisenia*

Sorption of Terbuthylazine in Organic Matter Amended Soils: Effects on Eisenia Fetida and Lumbricus Terrestris

http://dx.doi.org/10.5772/54948

29

The weight loss may indicate a feeding inhibition situation, with the earthworms regulating the intake of the terbuthylazine by reducing consumption rate and thus affecting their subsequent growth rate. This strategy is commonly used by earthworms to avoid poisoning

The decrease of earthworm cellulase and gluthatione-S-transferase activities possibly is due to a physiological adaptability to compensate for pesticide stress. To overcome the stress situation, animals require high energy, and this energy demand may have led to protein catabolism [41]. Furthermore, this decrease in protein content might be a result of mechanical lipoprotein formation, which is used to repair damaged cells, tissues, and organs [40].

C1 556b ± 22 624bc ± 28 610bc ± 19 590b ± 20 527b ± 19 542b ± 15 575bc ± 18 558bc ± 13 C2 550b ± 19 543b ± 18 538b ± 17 529ab ± 22 520b ± 15 508b ± 12 496b ± 17 489b ± 17 C3 544b ± 20 529ab ± 20 510ab ± 19 491ab ± 17 511b ± 17 492b ± 18 470ab ± 14 450ab ± 15 C4 530ab ± 17 498ab ± 15 456a ± 21 416a ± 13 510b ± 11 479b ± 13 425a ± 18 384a ± 12 MSW1 563b ± 26 679c ± 25 710c ± 19 748c ± 22 539b ± 10 568bc ± 18 602c ± 20 687c ± 18 MSW2 558b ± 19 652c ± 21 684c ± 17 703c ± 15 520b ± 15 547b ± 14 589bc ± 13 595bc ± 21 MSW3 549b ± 14 624bc ± 18 643bc ± 22 663c ± 19 511b ± 13 530b ± 11 559b ± 13 539b ± 19 MSW4 536b ± 17 560b ± 24 582b ± 26 606bc ± 20 505b ± 12 520b ± 15 528b ± 10 676c ± 17 PM1 570b ± 24 668b ± 26 700c ± 15 738c ± 21 528b ± 20 560b ± 17 608c ± 15 616c ± 18 PM2 552b ± 23 601bc ± 17 643bc ± 19 683c ± 18 515b ± 18 547b ± 19 590bc ± 22 568bc ± 13 PM3 540b ± 19 590b ± 15 628bc ± 22 642bc ± 16 504b ± 14 530b ± 12 546b ± 19 568bc ± 13 PM4 529ab ± 22 541b ± 20 561b ± 20 580b ± 13 493b ± 17 519b ± 17 522b ± 15 514b ± 16 CM1 575b ± 19 647c ± 24 680c ± 17 700c ± 22 524b ± 13 555b ± 15 586bc ± 15 650c ± 19 CM2 539b ± 21 565b ± 17 596b ± 23 642bc ± 17 510b ± 21 532b ± 12 558b ± 19 588bc ± 15 CM3 520ab ± 18 540b ± 18 573b ± 22 525ab ± 13 498b ± 19 517b ± 11 524b ± 18 483b ± 13 CM4 500ab ± 19 509ab ± 21 516ab ± 20 525ab ± 13 480b ± 18 496b ± 15 482b ± 19 476ab ± 11

**Table 6.** Cellulase activity (mean± standard error) (mg glucose mg protein hour-1) of *Eisenia fetida* and *Lumbricus terrestris* exposed to different concentrations of terbuthylazine herbicide. Column (mean ± standard errors) followed

by the same letter(s) are not significantly different (p<0.05)

*Eisenia fetida Lumbricus terrestris* **Incubation days Incubation days 3 15 45 90 3 15 45 90**

**Table 5.** Cocoon production, average weight of cocoons (mg) and number of juveniles per coccon (mean ± standard error) of *Eisenia fetida* and *Lumbricus terrestris* exposed to different concentrations of terbuthylazine herbicide. Column (mean ± standard errors) followed by the same letter(s) are not significantly different (p<0.05)

The number of juveniles per cocoon decreased when terbuthylazine concentration increased. This decrease was higher for *Lumbricus terrestris* than for *Eisenia fetida*. The application of organic matter in terbuthylazine treated soils increased this parameter. Again, this increase was higher MSW followed by PM and CM-amended soils.

### **3.3. Biochemical assay**

At the end of the experiment and for 50 µg terbuthylazine g-1 soil treatment, the cellulase activity of *Eisenia fetida* and *Lumbricus terrestris* worms was significantly recudec (29.4% and 31.1%) compared to the control soil (Table 6). The application of organic matter in herbicide treated soil increased the cellulase activity. At the end of the incubation day and for the higher concentration of terbuthylazine, the *Eisenia fetida* cellulase activity decreased 25%, 21.4% and 19% in soils amended with CM, PM and MSW, respectively, compared to organic amended and non-treated soils. For *Lumbricus terrestris*, cellulase activity decreased 26.8%, 23.9% and 21.5% in soils amended with CM, PM and MSW, respectively, compared to organic amended and non-treated soils.

Compared to the control soil, the glutathione-S-transferase activity of *Eisenia fetida* decreased 12.3%, 19.6% and 30.9% in soils treated with 1, 10 and 50 µg terbuthylazine g-1 soil, respectively, whereas for *Lumbricus terrestris* the glutathione-S-transferase activity decreased 14%, 21% and 32.4%, respectively (Table 7). At the end of the experiment, the glutathione-S-transferase activity of both worms had higher increase in MSW, PM and CM-amended soils, respectively, compared to control soil. The application of organic matter in herbicide treated soils increased the glutathione-S-transferase activity. Again, this increase was higher in MSW followed by PM and CM-amended soils.

### **4. Discussion**

*Eisenia fetida Lumbricus terrestris*

**Cocoon numbers**

**Average weigh of per cocoon (mg)**

**Number of juveniles per cocoon**

**Number of juveniles per cocoon**

PM1 3.29b ± 0.25 8.91b ± 0.45 3.29b ± 0.22 2.92b ± 0.19 8.27b ± 0.35 2.96b ± 0.19 PM2 2.68ab ± 0.20 7.39b ± 0.18 2.82b ± 0.16 2.34b ± 0.22 6.67ab ± 0.24 2.46ab ± 0.23 PM3 2.41ab ± 0.18 6.82ab ± 0.24 2.73ab ± 0.19 2.19ab ± 0.15 6.28ab ± 0.26 2.41ab ± 0.18 PM4 2.10a ± 0.15 6.38ab ± 0.21 2.39a ± 0.23 1.91ab ± 0.11 6.10ab ± 0.28 2.12a ± 0.15 CM1 3.17b ± 0.19 8.83b ± 0.53 3.21b ± 0.17 2.83b ± 0.14 8.21b ± 0.39 2.89a ± 0.29 CM2 2.47ab ± 0.10 7.09ab ± 0.21 2.68ab ± 0.19 2.16ab ± 0.12 6.35ab ± 0.22 2.38ab ± 0.21 CM3 2.19a ± 0.11 6.42ab ± 0.15 2.58ab ± 0.13 1.88ab ± 0.15 5.78a ± 0.21 2.28ab ± 0.12 CM4 1.90a ± 0.13 5.95a ± 0.13 2.23a ± 0.15 1.66a ± 0.17 5.35a ± 0.18 1.98a ± 0.16

**Table 5.** Cocoon production, average weight of cocoons (mg) and number of juveniles per coccon (mean ± standard error) of *Eisenia fetida* and *Lumbricus terrestris* exposed to different concentrations of terbuthylazine herbicide. Column (mean ± standard errors) followed by the same letter(s) are not significantly different (p<0.05)

The number of juveniles per cocoon decreased when terbuthylazine concentration increased. This decrease was higher for *Lumbricus terrestris* than for *Eisenia fetida*. The application of organic matter in terbuthylazine treated soils increased this parameter. Again, this increase

At the end of the experiment and for 50 µg terbuthylazine g-1 soil treatment, the cellulase activity of *Eisenia fetida* and *Lumbricus terrestris* worms was significantly recudec (29.4% and 31.1%) compared to the control soil (Table 6). The application of organic matter in herbicide treated soil increased the cellulase activity. At the end of the incubation day and for the higher concentration of terbuthylazine, the *Eisenia fetida* cellulase activity decreased 25%, 21.4% and 19% in soils amended with CM, PM and MSW, respectively, compared to organic amended and non-treated soils. For *Lumbricus terrestris*, cellulase activity decreased 26.8%, 23.9% and 21.5% in soils amended with CM, PM and MSW, respectively, compared to organic amended

Compared to the control soil, the glutathione-S-transferase activity of *Eisenia fetida* decreased 12.3%, 19.6% and 30.9% in soils treated with 1, 10 and 50 µg terbuthylazine g-1 soil, respectively, whereas for *Lumbricus terrestris* the glutathione-S-transferase activity decreased 14%, 21% and 32.4%, respectively (Table 7). At the end of the experiment, the glutathione-S-transferase activity of both worms had higher increase in MSW, PM and CM-amended soils, respectively, compared to control soil. The application of organic matter in herbicide treated soils increased the glutathione-S-transferase activity. Again, this increase was higher in MSW followed by PM

**Cocoon numbers**

28 Herbicides - Advances in Research

**3.3. Biochemical assay**

and non-treated soils.

and CM-amended soils.

**Average weigh of per cocoon (mg)**

was higher MSW followed by PM and CM-amended soils.

Our results indicated that terbuthylazine induced negative effects on weight, reproductive and enzymatic activities on the both earthworms. These negative effects increased with increasing herbicide concentration and/or exposure time. These results are in accordance with Brunninger et al. [39] who studied the toxicity of terbuthylazine on the growth and reproduction of *Eisenia andrei* over a period of three generations.

The weight loss may indicate a feeding inhibition situation, with the earthworms regulating the intake of the terbuthylazine by reducing consumption rate and thus affecting their subsequent growth rate. This strategy is commonly used by earthworms to avoid poisoning with herbicides and heavy metals [40].

The decrease of earthworm cellulase and gluthatione-S-transferase activities possibly is due to a physiological adaptability to compensate for pesticide stress. To overcome the stress situation, animals require high energy, and this energy demand may have led to protein catabolism [41]. Furthermore, this decrease in protein content might be a result of mechanical lipoprotein formation, which is used to repair damaged cells, tissues, and organs [40].


**Table 6.** Cellulase activity (mean± standard error) (mg glucose mg protein hour-1) of *Eisenia fetida* and *Lumbricus terrestris* exposed to different concentrations of terbuthylazine herbicide. Column (mean ± standard errors) followed by the same letter(s) are not significantly different (p<0.05)


in agricultural soils, probably as a result of the humic substances containing several major functional groups, such as carboxyl, phenolic, alcohol and carbonyl [44, 45]. However, our results also suggested that the chemical composition of the organic matter influenced in the

Sorption of Terbuthylazine in Organic Matter Amended Soils: Effects on Eisenia Fetida and Lumbricus Terrestris

Several studies of metal complexation with organic matter indicated that the sorption of heavy metals increased when the humic acid-fraction content increased in the organic matter, compared to the fulvic acid-fraction content, probably due to the humic acid-fraction possess

The terbuthylazine sorption isotherms and Freundlich sorption coefficients indicated higher herbicide sorption in MSW-amended soils, followed by PM and CM. Therefore, and similar to the heavy metals complexation, the sorption of herbicide increased with the humic acidfraction content in the organic amendment applied to the soil. The higher sorption probably caused a larger decrease of herbicide in the soil solution, and therefore, lowest availability of terbuthylazine availability for earthworms. This fact probably is the responsible of the increase

It can be concluded that the sensitivity of earthworm to pesticides differ depending on the taxonomic species, *Lumbricus terrestris* being more sensitive than *Eisenia fetida* to terbuthylazine herbicide. The application of organic matter have a positive effect on reducing the toxic effect of terbuthylazine on both *Eisenia fetida* and *Lumbricus terrestris*, which is attributable to their capability of absorbing the pesticide decreasing its concentration in soil solution. This positive effect will depend on the organic amendment characteristics, those with higher amount or

, Paloma Osta1

logía y Química Agrícola, E.U.I.T.A. Universidad de Sevilla, Sevilla, Spain

1 Grupo de investigación "Edafología ambiental". Departamento de Cristalografía, Minera‐

2 Departamento de Bioquímica y Biología Molecular. Facultad de Farmacia. Universidad de

, Juan Parrado2

and Isidoro Gómez1

http://dx.doi.org/10.5772/54948

31

a higher number of carboxylic groups than fulvic acid-fraction [12, 13].

in earthworm weight, reproductive and enzymatic activities.

reactive humic acid being the most effective.

\*Address all correspondence to: mtmoral@us.es

Manuel Tejada1\*, Marina del Toro1

terbuthylazine sorption.

**5. Conclusions**

**Author details**

Sevilla, Sevilla, Spain

**Table 7.** Glutathione-S-transferase activity (mean ± standard error) (nmol mg protein min-1) of *Eisenia fetida* and *Lumbricus terrestris* exposed to different concentrations of terbuthylazine herbicide. Column (mean ± standard errors) followed by the same letter(s) are not significantly different (p<0.05)

However, these negative effects were higher in *Lumbricus terrestris* than for *Eisenia fetida*. Therefore, the sensitivity of each worm is different to the terbuthylazine herbicide. According to Ma and Bodt [23] this can be due to some physiological property of the worms or to factors governing the exposure to herbicide. Studies on the effect of benomyl and carbofuran on earthworms have similarly shown that the toxicity is much greater to *Lumbricus terrestris* than for *Eisenia fetida* when tested under standardized conditions in soil substrates [42].

The addition of organic matter to the herbicide treated soil increased the earthworm weight, reproductive and enzymatic activities probably due to the sorption of terbuthylazine with the organic matter. These results are in agreement with Dolaptsoglou et al. [43] and Cabrera et al. [30, 32], who found a decrease of terbuthylazine in the soil solution after the addition of organic matter to soil due to the herbicide sorption.

The terbuthylazine sorption isotherms and Freundlich sorption coefficients obtained in this study, suggested that organic matter play a fundamental role in the sorption of the herbicide in agricultural soils, probably as a result of the humic substances containing several major functional groups, such as carboxyl, phenolic, alcohol and carbonyl [44, 45]. However, our results also suggested that the chemical composition of the organic matter influenced in the terbuthylazine sorption.

Several studies of metal complexation with organic matter indicated that the sorption of heavy metals increased when the humic acid-fraction content increased in the organic matter, compared to the fulvic acid-fraction content, probably due to the humic acid-fraction possess a higher number of carboxylic groups than fulvic acid-fraction [12, 13].

The terbuthylazine sorption isotherms and Freundlich sorption coefficients indicated higher herbicide sorption in MSW-amended soils, followed by PM and CM. Therefore, and similar to the heavy metals complexation, the sorption of herbicide increased with the humic acidfraction content in the organic amendment applied to the soil. The higher sorption probably caused a larger decrease of herbicide in the soil solution, and therefore, lowest availability of terbuthylazine availability for earthworms. This fact probably is the responsible of the increase in earthworm weight, reproductive and enzymatic activities.

### **5. Conclusions**

*Eisenia fetida Lumbricus terrestris* **Incubation days Incubation days 3 15 45 90 3 15 45 90**

C1 118ab ± 10 116ab ± 11 121ab ± 10 120ab ± 12 110ab ± 9 113ab ± 11 117ab ± 10 115ab ± 11 C2 116ab ± 9 111ab ± 13 108a ± 9 105a ± 10 110ab ± 7 106a ± 10 104a ± 11 98.9a ± 6.2 C3 113ab ± 8 108a ± 12 103a ± 11 96a ± 8 108a ± 10 104a ± 11 95.3a ± 8.6 90.9a ± 5.8 C4 110a ± 11 102a ± 9 90a ± 10 82.9a ± 6.9a 104a ± 6 99.6a ± 7.8 82.4a ± 7.2 77.7a ± 4.9 MSW1 130ab ± 12 140ab ± 14 152b ± 15 161b ± 13 123ab ± 13 131b ± 12 142b ± 10 154b ± 10 MSW2 127ab ± 10 134b ± 8 140b ± 10 148b ± 16 118ab ± 12 127b ± 11 135b ± 9 140b ± 12 MSW3 122ab ± 11 130ab ± 10 135b ± 13 141b ± 15 115ab ± 11 120ab ± 13 126b ± 11 132b ± 11 MSW4 118ab ± 12 121ab ± 11 123ab ± 11 126ab ± 11 109a ± 7 112ab ± 10 115ab ± 12 117ab ± 10 PM1 128ab ± 13 136b ± 17 147b ± 10 156b ± 11 120ab ± 10 129ab ± 11 140b ± 11 148b ± 11 PM2 124ab ± 10 130ab ± 12 135b ± 12 142b ± 13 117ab ± 10 124ab ± 12 130b ± 10 134b ± 12 PM3 120ab ± 8 125ab ± 9 129ab ± 11 134b ± 15 112ab ± 9 118ab ± 11 120ab ± 11 124ab ± 10 PM4 115ab ± 9 119ab ± 10 121ab ± 9 120ab ± 11 107a ± 9 111ab ± 9 107ab ± 9 109ab ± 9 CM1 124ab ± 12 130ab ± 15 140b ± 12 150b ± 10 117ab ± 10 125b ± 10 136b ± 12 142b ± 13 CM2 123ab ± 10 128ab ± 11 133b ± 10 135b ± 12 113ab ± 11 116ab ± 8 123ab ± 9 127b ± 11 CM3 118ab ± 11 122ab ± 10 123ab ± 12 125ab ± 13 106a ± 8 113ab ± 7 115ab ± 9 118ab ± 10 CM4 111ab ± 9 112ab ± 10 113ab ± 11 113ab ± 10 100a ± 9 104a ± 9 108a ± 8 106a ± 9

**Table 7.** Glutathione-S-transferase activity (mean ± standard error) (nmol mg protein min-1) of *Eisenia fetida* and *Lumbricus terrestris* exposed to different concentrations of terbuthylazine herbicide. Column (mean ± standard errors)

for *Eisenia fetida* when tested under standardized conditions in soil substrates [42].

However, these negative effects were higher in *Lumbricus terrestris* than for *Eisenia fetida*. Therefore, the sensitivity of each worm is different to the terbuthylazine herbicide. According to Ma and Bodt [23] this can be due to some physiological property of the worms or to factors governing the exposure to herbicide. Studies on the effect of benomyl and carbofuran on earthworms have similarly shown that the toxicity is much greater to *Lumbricus terrestris* than

The addition of organic matter to the herbicide treated soil increased the earthworm weight, reproductive and enzymatic activities probably due to the sorption of terbuthylazine with the organic matter. These results are in agreement with Dolaptsoglou et al. [43] and Cabrera et al. [30, 32], who found a decrease of terbuthylazine in the soil solution after the addition of organic

The terbuthylazine sorption isotherms and Freundlich sorption coefficients obtained in this study, suggested that organic matter play a fundamental role in the sorption of the herbicide

followed by the same letter(s) are not significantly different (p<0.05)

30 Herbicides - Advances in Research

matter to soil due to the herbicide sorption.

It can be concluded that the sensitivity of earthworm to pesticides differ depending on the taxonomic species, *Lumbricus terrestris* being more sensitive than *Eisenia fetida* to terbuthylazine herbicide. The application of organic matter have a positive effect on reducing the toxic effect of terbuthylazine on both *Eisenia fetida* and *Lumbricus terrestris*, which is attributable to their capability of absorbing the pesticide decreasing its concentration in soil solution. This positive effect will depend on the organic amendment characteristics, those with higher amount or reactive humic acid being the most effective.

### **Author details**

Manuel Tejada1\*, Marina del Toro1 , Paloma Osta1 , Juan Parrado2 and Isidoro Gómez1

\*Address all correspondence to: mtmoral@us.es

1 Grupo de investigación "Edafología ambiental". Departamento de Cristalografía, Minera‐ logía y Química Agrícola, E.U.I.T.A. Universidad de Sevilla, Sevilla, Spain

2 Departamento de Bioquímica y Biología Molecular. Facultad de Farmacia. Universidad de Sevilla, Sevilla, Spain

### **References**

[1] Farenhorst, A. Importance of soil organic matter fractions in soil-landscape and re‐ gional assessments of pesticide sorption and leaching in soil. Soil Science Society America Journal 2006; 70(3) 1005-1012.

[12] Tejada, M., Hernández, M.T., García, C. Application of two organic wastes in a soil polluted by lead: Effects on the soil enzymatic activities. Journal of Environmental

Sorption of Terbuthylazine in Organic Matter Amended Soils: Effects on Eisenia Fetida and Lumbricus Terrestris

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33

[13] Tejada, M., Moreno, J.L., Hernández, M.T., García, C. Soil amendments with organic wastes reduce the toxicity of nickel to soil enzyme activities. European Journal of Soil

[14] Xiao, N., Jing, B., Ge, F., Liu, X. The fate of herbicide acetochlor and its toxicity to *Eisenia fetida* under laboratory conditions. 2006; Chemosphere 62(8) 1366-1373.

[15] Booth, L.H., Hodge, S., O'Halloran, K. Use of cholinesterase in *Aporrectodea caliginosa* (Oligochaeta, Lumbricidae) to detect organophosphate contamination: comparison of laboratory test, mesocosms, and foeld studies. Environmental Toxicology Chemistry

[16] EC. 2004. Biological test method: tests for toxicity of contaminated soil to earthworms (*Eisenia foetida*, *Eisenia andrei*, or *Lumbricus terrestris*). Report EPS 1/RM/43, Environ‐

[17] Zhou, S.P., Duan, C.Q., Hui, F.U., Chen, Y.H., Wang, X.H., Yu, Z.F. Toxicity assess‐ ment for chlorpyrifos-contaminated soil with three different earthworm test meth‐

[18] Chakra Reddy, N., Venkateswara Rao, J. Biological response of earthworm, *Eisenia foetida* (Savigny) to an organophosphorus pesticide, profenofos. Ecotoxicology and

[19] Castellanos, L.R., Hernández, T.C.S. Earthworm biomarkers of pesticide contamina‐ tion: current status and perspectives. Journal of Pesticide Science 2007; 32(4) 360-371.

[20] Tejada, M., Masciandaro, G. Aplication of organic wastes on a benzo(a)pyrene pollut‐ ed soil. Response of soil biochemical and role of *Eisenia fetida.* Ecotoxicology and En‐

[21] Capowiez, Y., Raulth, H., Castagliola, G., Mazzia, C. Lethal and sublethal effects of imidacloprid on two earthworm species (*Aporrectodea nocturna* and *Allolobophora icter‐*

[22] Spurgeon, D.J., Weeks, J.M. Evaluation of factors influencing results from laboratory toxicity tests with earthworms. In: Sheppard, S.C., Brembridge, J.D., Holmstrup, M., Posthuma, L. (eds.) Advances in earthworm ecotoxicology. SETAC, Pensacola, pp

[23] Ma, W., Bodt, J. Differences in toxicity in the insecticide chlorpyrifos to six species of earthworms (Oligachaeta, Lumbricidae) in standardiezed soil tests. Bulletin of Envi‐

ment Canada, Environmental Technology Centre, Ottawa, ON, Canada.

ods. Journal of Environmental Science 2007; 19(7) 854-858.

Environmental Safety 2008; 71(2) 574-582.

vironmental Safety 2011; 74(4)668-674.

15-25; 1998.

*ica*). Biology and Fertility of Soils 2005; 41(3) 135-143.

ronmental Contamination and Toxicology 1993; 50(6) 864-870.

[24] FAO. Carte mondiale des sols. Légende révisée. pp. 125; 1989.

Quality 2007; 36(1) 216-225.

Biology 2008; 44(1) 129-140.

2000; 19(2) 417-422.


[12] Tejada, M., Hernández, M.T., García, C. Application of two organic wastes in a soil polluted by lead: Effects on the soil enzymatic activities. Journal of Environmental Quality 2007; 36(1) 216-225.

**References**

32 Herbicides - Advances in Research

3797-3802.

3803-3809.

1489-1495.

32(3) 389-401.

19 of December of 2000.

64, 4 of March of 2006.

Environment 65(2) 151-161.

and Food Chemistry 2007; 55(15) 6213-8.

America Journal 2006; 70(3) 1005-1012.

of America Journal 2001; 65(6) 1688-1695.

[1] Farenhorst, A. Importance of soil organic matter fractions in soil-landscape and re‐ gional assessments of pesticide sorption and leaching in soil. Soil Science Society

[2] Cox, L., Cecchi, A., Celis, R., Hermosín, M. C., Koskinen, W.C., Cornejo, J. Effect of exogenous carbon on movement of simazine and 2,4-D in soils. Soil Science Society

[3] Blanchoud, H, Moreau-Guigon, E, Farrugia, F, Chevreuil, M, Mouchel, JM. Contribu‐ tion by urban and agricultural pesticide uses to water contamination at the scale of

[4] Gerstl, Z., Nasser, A., Mingelgrin, U. Controlled release of pesticides into soils from clay-polymer formulations. Journal of Agriculture and Food Chemistry 1998a; 46(9)

[5] Gerstl, Z., Nasser, A., Mingelgrin, U. Controlled release of pesticides into water from clay-polymer formulations. Journal of Agriculture and Food Chemistry 1998b; 46(9)

[6] Directive 2000/60/EC of 23 of October of 2000, related to Communitary Marc to Ac‐ tuation about Water Politic. Diary Official of European Communities (DOCE), L 321,

[7] Directive 2006/11/EC of the European Parliament and Council of 15 of February of 2006, related to Pollution Caused by Dangerous Substances tipped in the Aquatic En‐ vironment of the Community. Diary Official of European Communities (DOCE), L

[8] Cox, L., Celis, R., Hermosin, M.C., Becker, A., Cornejo, J. 1997. Porosity and herbicide leaching in soils amended with olive-mill wastewater. Agriculture, Ecosystems and

[9] Delgado-Moreno, L., Peña, A. Compost and vermicompost of olive cake to biore‐ mediate triazines-contaminated soil. Science of Total Environment 2009; 407(5)

[10] Delgado-Moreno, L., Peña, A. Organic amendments from olive-mill wastes as a strat‐ egy to modify degradation of sulfonylurea herbicides in soil. Journal of Agriculture

[11] Abdelhafid, R., Houot, S., Barriuso, E. Dependence of atrazine degradation on C and N availability in adapted and non-adapted soils. Soil Biology and Biochemistry 2000;

the Marne watershed. Science of Total Environment 2007; 375(1-3) 168-79.


[25] Soil Survey of England and Wales. Soil Survey Laboratory methods, Tech. Monogr. vol. 6, Harpenden, UK; 1982.

[39] Brunninger, B., Viswanathan, R., Beese, F. Terbuthylazine and carbofuran effects on growth and reproduction within three generations of *Eisenia andrei* (Oligochaeta). Bi‐

Sorption of Terbuthylazine in Organic Matter Amended Soils: Effects on Eisenia Fetida and Lumbricus Terrestris

http://dx.doi.org/10.5772/54948

35

[40] Ribeiro, S., Sousa, J.P., Nogueira, A.J.A., Soares, A.M.V.M. Effect of endosulfan and parathion on energy reserves and physiological parameters of the terrestrial isopod

[41] Mosleh, Y.Y., Paris-Palacios, S., Couderchet, M., Vernet, G. Acute and sublethal ef‐ fects of two insecticides on earthworms (*Lumbricus terrestris* L.) under laboratory con‐

[42] Haque, A., Ebing, W. Toxicity determination of pesticides to earthworms in the soil substrates. Zeitschrift fuer Pflanzenkrankheiten und Pflanzenschutz 1983; 90(4)

[43] Dolaptsoglou, Ch., Karpouzas, D.G., Menkissogh-Spirondi, U., Eleftherohorinos, I., Voudrias, E.A. Influence of different organic amendments on the degradation, me‐ tabolism, and adsorption of terbuthylazine. Journal of Environmental Quality 2007;

[44] Sluzny, C., Graber, E.R., Gerstl, Z. Sorption of s-triazine herbicides in organic matter amended soils: Fresh and incubated systems. Water, Air and Soil Pollution 1999;

[45] Datta, A., Sanyal, S.K., Saha, S. 2001. A study on natural and synthetic humic acids and their complexing ability towards cadmium. Plant Soil 2001; 235(1) 115-125.

*Porcellio dilalatus*. Ecotoxicology and Environmental Safety 2001; 49 131-138.

ology and Fertility of Soils 1994 ; 18(2) 83-88.

ditions. Environmental Toxicology 2003; 18(1) 1-8.

395-408.

36(6) 1793-1802.

115(1-4) 395-410.


[39] Brunninger, B., Viswanathan, R., Beese, F. Terbuthylazine and carbofuran effects on growth and reproduction within three generations of *Eisenia andrei* (Oligochaeta). Bi‐ ology and Fertility of Soils 1994 ; 18(2) 83-88.

[25] Soil Survey of England and Wales. Soil Survey Laboratory methods, Tech. Monogr.

[26] MAPA. Métodos oficiales de análisis. Ministerio de Agricultura, Pesca y Alimenta‐

[27] Yeomans, J.C., Bremner, J.M. A rapid and precise method for routine determination of organic carbon in soil. Communications in Soil Science and Plant Analysis 1988;

[28] Kononova, M.M. Soil organic matter, (second ed.), Pergamon Press, Oxford; 1966.

[29] Ritchie, J.D., Perdue, E.M. Proton-binding study of standard and reference fulvic acids, humic acids, and natural organic matter. Geochimica et Cosmochimica Acta

[30] Cabrera, A., Cox, L., Velarde, P., Cornejo, J. Terbuthylazine persistence in an organic amended soil. Journal of Environmental Science and Health, Part B 2008; 43(8)

[31] Moreno, J.L., García, C., Hernández, T. Toxic effect of cadmium and nickel on soil en‐ zymes and the influence of adding sewage sludge. European Journal of Soil Science

[32] Cabrera, A., Cox, L., Fernández-Hernández, A., García-Ortiz Civantos, C., Cornejo, J. Field appraisement of olive mills solid waste application in olive crops: Effect on her‐ bicide retention. Agriculture, Ecosystems and Environment 2009; 132(3-4) 260-266. [33] Bruna, F., Pavlovic, I., Celis, R., Barriga, C., Cornejo, J., Ulibarri, M.A. Organohydro‐ talcites as novel supports for the slow release of the herbicide terbuthylazine. Ap‐

[34] Maboeta, M.S., Reinecke, A.J., Reinecke, S.A. 1999. Effects of low levels of lead on growth and reproduction of the Asian earthworm *Perionyx excavates* (Oligochaeta).

[35] Mishra, P.C., Dash, M.C. Digestive enzymes of some earthworms. Experientia 1980;

[36] Habig, W.H., Pabst, M.J., Jakoby, W.B. Glutathione-s-transferases. Journal of Biologi‐

[37] Saint-Denis, M., Labrot, F., Narbonne, J.F., Ribera, D. Glutathione, glutathione-relat‐ ed enzymes, and catalase activities in the earthworm *Eisenia fetida andrei*. Archives of

[38] Statistical Graphics Corporation. Statgraphics 5.0. Statistical Graphics System, Educa‐ tional Institution Edition; Manugistics, Inc., Rockville, MD, USA p. 105; 1991.

Environmental Contamination and Toxicology 1998; 35(4) 602-614.

Ecotoxicology and Environmental Safety 1999; 44(3) 236-240.

vol. 6, Harpenden, UK; 1982.

ción; 1986.

34 Herbicides - Advances in Research

19(13) 1467-1476.

2003; 67(1) 85-96.

2003; 54(2) 377-386.

36 1156-1157.

plied Clay Science 2008; 42(1-2), 194-200.

cal Chemistry 1974; 249 7130-7139.

713-716.


**Chapter 3**

**Phytotoxicity of Phenolic Acids From Cereals**

Allelopathic interactions between individuals of different plant species or those of the some species are caused by plant-produced allelochemicals. Once released into the environment, passively or actively they can influence germination, growth and development of neighboring plant either negatively or positively [1, 2]. Most allelochemicals are characterized by multi‐ functional phytotoxicity and are often also important for general defense. Generally stressed donor plants increased allelopathic activity due to increased production of allelochemical. Factors such as placement of residues, nutrient status, environmental conditions and microbial

In recent years there has been an increasing focus on the prospects of exploiting allelopathy for controlling weeds but also insects and diseases. Allelopathy plays an important role in the agro ecosystem leading to a wide array of interactions between crop-crop, crop-weed and treecrop. Generally, these interactions are harmful to the receiver plants but provide a selective benefit to the donor. Several members of crops exhibit allelopathic interactions that play a

The allelochemicals are released largely by plant residues that are left in the fields after the harvest of a crop or through use of cover crops. Research on allelopathic interactions has been focused in agricultural crops as on option in the development of integrated weed management strategies, reducing environmental effects and the cost of crop protection [6-10]. Secondary metabolites with allelopathic properties are thought to protect plants against competing plants. For instance, allelopathic activity of decomposing wheat (*Titicum aestivun* L.), and oat (*Avena sativa* L.) straw on some crop species has been reported (11). Allelopathic potential of rye [12-14] and rice [6, 15, 16] has been extensively studied. In cereals such as, maize (*Zea may* L.), wheat (*Titicum aestivun* L.), rye (*Secale cereale* L.), barley (*Hordeum vulgare* L.), rice (*Oryza*

> © 2013 Bravo et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Bravo et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

significant role in the complex environment of the agro ecosystem.

Héctor R. Bravo, Sylvia V. Copaja and

Additional information is available at the end of the chapter

Madeleine Lamborot

http://dx.doi.org/10.5772/55942

activity also affect allelopathy [3-5].

**1. Introduction**
