**Pesticide Contamination in Groundwater and Streams Draining Vegetable Plantations in the Ofinso District, Ghana**

Benjamin O. Botwe1, William J. Ntow2 and Elvis Nyarko1 *1University of Ghana, Department of Oceanography & Fisheries 2University of California, Department of Plant Sciences 1Ghana 2USA* 

## **1. Introduction**

50 Soil Health and Land Use Management

USDA-NRCS. (2008). Overview and history of hydrologic units and the watershed

USGS. (2009). Federal guidelines, requirements, and procedures for the National Watershed

Washington, D.C. Available at: http://pubs.usgs.gov/tm/tm11a3/. Vereecken, H.; Kamai, T.; Harter, T.; Kasteel, R.; Hopmans, J. & Vanderborght, J. (2007).

http://www.ncgc.nrcs.usda.gov/products/datasets/watershed/history.html,

Conservation Service.

10.1029/2007GL031813.

accessed July 2008.

boundary dataset (WBD). Washington, D.C.: USDA Natural Resources

Boundary Dataset. U.S. Geological Survey, U.S. Department of the Interior, Reston, VA and Natural Resources Conservation Service, U.S. Department of Agriculture,

Explaining soil moisture variability as a function of mean soil moisture: a stochastic unsaturated flow perspective, *Geophysical Research Letters*, Vol. 34, L22402. Doi:

#### **1.1 Ghana's geographical location and climate**

Ghana, officially the Republic of Ghana and formerly the Gold Coast, is a West African country with a geographical location of 5°36′ N, 0°10′ E. It shares borders with Cote d'Ivoire to the west, Burkina Faso to the north and Togo to the east. To the south of the country is the Gulf of Guinea of the Atlantic Ocean. The climate is tropical equatorial ranging from the bimodal rainfall equatorial type in the south to the tropical unimodal monsoon type in the north. It is influenced by the hot, dry and dusty-laden air mass that moves from the northeast across the Sahara and by the tropical maritime air mass that moves from the south-west across the southern Atlantic ocean. The annual rainfall ranges from 1015 to 2300 mm with annual mean temperature and relative humidity of 30°C and 80% respectively (Ntow & Botwe, 2011). Ghana has a total land area of about 23,853, 900 ha and a population of about 24.2 million. The arable land covers an area of about 13,628,179 ha (approx. 57% of total land area) of which approximately 44% is under cultivation.

#### **1.2 Economic importance of agriculture in Ghana**

Agriculture is Ghana's most important economic sector, employing more than 60% of the labour force. Currently, agriculture contributes about 33% of Ghana's gross domestic product (GDP) and accounts for over 40% of export earnings. Ghana's agriculture is predominantly smallholder, traditional and rain-fed. The major agro-ecological zones in Ghana are Rain Forest, Deciduous Forest, Forest-Savannah Transition, Coastal Savannah and Northern (Interior) Savannah which comprises Guinea and Sudan Savannahs. The type of agricultural activity carried out in each zone is determined largely by rainfall. In the south, there is a major and a minor growing season due to the bimodal rainfall pattern in the Forest, Deciduous Forest, Transitional and Coastal Savannah zones whereas in the Northern Savannah, the unimodal rainfall pattern results in a single growing season. Within the agricultural sector, vegetable production plays an important socio-economic role, having developed from a mainly subsistence activity to a commercial activity. Vegetable production in Ghana typically occurs in intensely managed vegetable plantations characterized by an

Pesticide Contamination in Groundwater and

contamination evaluated.

Fig. 1. Map showing the study area

**1.5 Study area** 

Streams Draining Vegetable Plantations in the Ofinso District, Ghana 53

conducted in Ghana have focused on the organochlorine pesticides (Osafo & Frimpong, 1998; Ntow, 2001, 2005). However, pesticides from the organophosphate group, which are now commonly used in Ghana following the ban on persistent organochlorine pesticides, have not been determined in water quality studies. In this chapter, pesticide contamination in groundwater and streams draining vegetable plantations in the Ofinso District of Ghana are assessed and the ecotoxicological significance of the pesticides

The present study was conducted in the Ofinso District of the Ashanti Region of Ghana (Fig. 1). The Ofinso District is located in the extreme North-Western part of the Ashanti Region, with about half of its boundaries bordered by Brong Ahafo Region (in the north and west).

extensive network of drainage systems through which surplus water may flow out (Ntow *et al*., 2008). Vegetables cultivated in Ghana include tomato (*Lycopersicon esculentum*), eggplant (*Solanum melongena*), pepper (*Capsicum annum*) and onion (*Allium cepa*), although some regions are more efficient and specialised in the production of only one or two vegetable crops (Ntow, 2001).

#### **1.3 Pesticide use in vegetable cultivation in Ghana**

Vegetables generally attract a wide range of pests and diseases, and require intensive pest management (Dinham, 2003), which includes all aspects of the safe, efficient and economic use and handling of pesticides. In Ghana, pest and disease control practices in vegetable production involve the use of chemical pesticides. A total of 43 pesticides, comprising insecticides, fungicides and herbicides, have been found in use in vegetable farming in Ghana. Among these pesticides, the herbicides class of pesticides is the most used (44%), followed by insecticides (33%) and then fungicides (23%) (Ntow *et al*., 2006). Although it is recognized that better management of pesticides results in high crop productivity while greatly reducing adverse environmental impacts, most of the local farmers lack adequate training in the proper application of pesticides. Pest and disease control therefore involves relatively high inputs of highly toxic chemical pesticides which are most of the time misapplied (Ntow *et al*., 2006). The average pesticide application rate is estimated to be 0.08 litres active ingredient (a.i.) per hectare (Ntow *et al*., 2008). Misapplication and intensive use of pesticides in vegetable cultivation can result in pesticide contamination of the environment.

#### **1.4 Impacts of pesticide use in vegetable agro-ecosystems**

Water pollution by pesticides has long been recognized as a major environmental impact associated with agriculture due to the potential adverse effects on aquatic life and on humans if contamination extends to drinking waters (Skinner *et al*., 1997). Most vegetable farms in Ghana are sited a few meters from streams for easier access to water for irrigation purposes. The close proximity of streams to vegetable farms is of particular concern as there is high potential for pesticides to move offsite into surrounding streams via run-off through the extensive system of drainage canals that characterize these farms. Persistent pesticides, particularly the organochlorine group of pesticides, can be transferred to aquatic organisms at all trophic levels within the food chain due to their bioconcentration and bioaccumulation potential. Many organochlorine pesticides are known to mimic hormones and disrupt reproductive cycles of humans and wildlife (Colborn and Smolen, 1996) and therefore they can be detrimental to a wide variety of aquatic wildlife populations (Robinson, 1991). Even non-persistent pesticides, such as the pyrethroids, carbamates and organophosphate group of pesticides, can be highly toxic to aquatic life (Castillo *et al.*, 2006). Pesticides can also enter groundwater via seepage or soil percolation. Pesticides contamination of streams and groundwater also presents health threat to the rural communities as they depend on streams and groundwater for drinking and other domestic purposes.

Concerns over the adverse ecological and human health impacts of pesticides have led to the institution of very strict programs to control and monitor pesticide contamination in water sources in developed countries such as the United States and members states of the European Community (García de Llasera and Bernal-González, 2001). These programs have, however, not been implemented in most developing countries such as Ghana. Few studies conducted in Ghana have focused on the organochlorine pesticides (Osafo & Frimpong, 1998; Ntow, 2001, 2005). However, pesticides from the organophosphate group, which are now commonly used in Ghana following the ban on persistent organochlorine pesticides, have not been determined in water quality studies. In this chapter, pesticide contamination in groundwater and streams draining vegetable plantations in the Ofinso District of Ghana are assessed and the ecotoxicological significance of the pesticides contamination evaluated.

#### **1.5 Study area**

52 Soil Health and Land Use Management

extensive network of drainage systems through which surplus water may flow out (Ntow *et al*., 2008). Vegetables cultivated in Ghana include tomato (*Lycopersicon esculentum*), eggplant (*Solanum melongena*), pepper (*Capsicum annum*) and onion (*Allium cepa*), although some regions are more efficient and specialised in the production of only one or two vegetable

Vegetables generally attract a wide range of pests and diseases, and require intensive pest management (Dinham, 2003), which includes all aspects of the safe, efficient and economic use and handling of pesticides. In Ghana, pest and disease control practices in vegetable production involve the use of chemical pesticides. A total of 43 pesticides, comprising insecticides, fungicides and herbicides, have been found in use in vegetable farming in Ghana. Among these pesticides, the herbicides class of pesticides is the most used (44%), followed by insecticides (33%) and then fungicides (23%) (Ntow *et al*., 2006). Although it is recognized that better management of pesticides results in high crop productivity while greatly reducing adverse environmental impacts, most of the local farmers lack adequate training in the proper application of pesticides. Pest and disease control therefore involves relatively high inputs of highly toxic chemical pesticides which are most of the time misapplied (Ntow *et al*., 2006). The average pesticide application rate is estimated to be 0.08 litres active ingredient (a.i.) per hectare (Ntow *et al*., 2008). Misapplication and intensive use of pesticides in vegetable cultivation can result in pesticide contamination of the

Water pollution by pesticides has long been recognized as a major environmental impact associated with agriculture due to the potential adverse effects on aquatic life and on humans if contamination extends to drinking waters (Skinner *et al*., 1997). Most vegetable farms in Ghana are sited a few meters from streams for easier access to water for irrigation purposes. The close proximity of streams to vegetable farms is of particular concern as there is high potential for pesticides to move offsite into surrounding streams via run-off through the extensive system of drainage canals that characterize these farms. Persistent pesticides, particularly the organochlorine group of pesticides, can be transferred to aquatic organisms at all trophic levels within the food chain due to their bioconcentration and bioaccumulation potential. Many organochlorine pesticides are known to mimic hormones and disrupt reproductive cycles of humans and wildlife (Colborn and Smolen, 1996) and therefore they can be detrimental to a wide variety of aquatic wildlife populations (Robinson, 1991). Even non-persistent pesticides, such as the pyrethroids, carbamates and organophosphate group of pesticides, can be highly toxic to aquatic life (Castillo *et al.*, 2006). Pesticides can also enter groundwater via seepage or soil percolation. Pesticides contamination of streams and groundwater also presents health threat to the rural communities as they depend on streams

Concerns over the adverse ecological and human health impacts of pesticides have led to the institution of very strict programs to control and monitor pesticide contamination in water sources in developed countries such as the United States and members states of the European Community (García de Llasera and Bernal-González, 2001). These programs have, however, not been implemented in most developing countries such as Ghana. Few studies

crops (Ntow, 2001).

environment.

**1.3 Pesticide use in vegetable cultivation in Ghana** 

**1.4 Impacts of pesticide use in vegetable agro-ecosystems** 

and groundwater for drinking and other domestic purposes.

The present study was conducted in the Ofinso District of the Ashanti Region of Ghana (Fig. 1). The Ofinso District is located in the extreme North-Western part of the Ashanti Region, with about half of its boundaries bordered by Brong Ahafo Region (in the north and west).

Fig. 1. Map showing the study area

Pesticide Contamination in Groundwater and

events.

**2.2 Chemical analysis 2.2.1 Sample extraction** 

quantification of the pesticides.

acetone/cyclohexane (pesticide grade) mixture (1:9).

**2.2.2 Pesticide residue analysis** 

Streams Draining Vegetable Plantations in the Ofinso District, Ghana 55

pesticide residues were detected in primary samples. Sampling was conducted throughout rainy and dry seasons and was not timed to applications of different pesticides or to rain

The extraction and analyses of water samples were performed following the Association of Official Analytical Chemists 990.06 and 970.52 methods as described by Ntow *et al*. (2008). Briefly, water samples were extracted sequentially three times with 25 mL *n*-hexane each time. The extract was dried with anhydrous sodium sulphate and concentrated down to 10 mL by means of ultrasonic bath type concentrators (Turbo Vap II). Extract clean up was done, using a chromatographic column, packed with florisil, previously activated for 3 h in an oven at 130oC, and anhydrous sulphate (all rinsed with petroleum ether). The extract was transferred to the column. Three fractions were obtained after elution with 6, 15, 50% ethyl ether in petroleum ether. Maximal flux rate of elution was 5 mL/min. Each eluate was evaporated and the extracts (re-dissolved in 1.5 ml *n*-hexane and made up to 2 ml with more *n*-hexane) were injected into a gas chromatographic system for identification and

Extraction and analysis of sediment samples followed the method described by Ntow *et al.* (2008). Briefly sediment samples were well mixed to obtain a homogeneous sample and then transferred into a pan to air-dry at ambient temperature until a constant weight was obtained. The samples were later ground in a mortar into fine powder such that they could pass through 2 mm sieve. Five grams (dry weight) of the sediment samples were soxhlet extracted in methanol, and cleaned up in florisil in the same way as described above for water. Sampling protocol and analytical procedures were subjected to quality control

through field and laboratory blanks and spiking of samples with pesticide standards.

Laboratory glassware used in the sampling and analyses were cleaned as described by Ntow (2001). Pesticide grade solvents used for the analyses were *n*-hexane (>99%) and acetone (>99.9%) (Sigma, Munich, Germany); methanol (99.8%) and petroleum ether (BDH; VWR International, UK); dichloromethane and ethyl ether (Fluka; Munich, Germany). Deionised water was used from a Milli-Q water purification system (Millipore, Bedford, MA, USA) for blanks, sediment extraction, and spiked samples. Pesticide standards (> 98% purity) were obtained from Dr. Ehrenstorfer (Augsburg, Germany). Standard mixtures were prepared from individual pesticide stock solutions (50-100 mg in 100 ml acetone) and then diluted to working calibration standards at three concentration levels with

Measurement of pesticide compounds in water and sediment samples was performed on a GC-MS (Agilent 6890 Series GC System) coupled with an Agilent 5973N mass selective detector-electron impact ionization; and fused capillary column (HP-5MS) packed with 5% Phenyl Methyl Siloxane (30 m \* 0.25 mm I.D and film thickness 0.25 µm), which was operated in the selected ion-monitoring mode at the following conditions: injection port 250oC (splitless, pressure 22.62 psi; purge flow 50 mL/min; purge time 2.0 min; total flow 55.4 mL/min). Column oven: initial 70oC, held 2 min, programming rate 25oC/min (70 to 150oC); 10oC/min (150 to 200oC); 8oC/min (200 to 280oC) and held 10 min at 280oC. The

It is bordered to the east by Ejura-Sekyedumasi District, to the south by Afigya Sekyere, Ahafo Ano South and Atwima Districts. The district has 126 settlements and a population of about 35,190 with New Ofinso as its capital. The district has five towns namely Abofour, Nkenkasu, Afrancho, Akumadan and New Ofinso. The study area is within the Ofin, Pru and Afram river basins. In the present study, vegetable plantations were selected from Akumadan, Nkenkasu, and Afrancho. Agriculture is the main economic activity in these areas with over 70% of the active population being farmers. The district is well known for the cultivation of vegetable crops. Other major crops cultivated include cassava, maize, plantain and cocoa. More than 23 different active ingredients formulated as insecticides, herbicides and fungicides have been used in the cultivation of vegetables in the district.

The five most frequently used insecticides include two organophosphates (chlorpyrifos and dimethoate), two pyrethroids (lambda-cyhalothrin and cypermethrin), and one organochlorine (endosulfan). Farmers use these highly toxic pesticides under primitive field conditions with insufficient protective equipment and training. Pesticide applications occur frequently, all year round, and are relatively intensive (500-1000 ml/ha). Pesticides are also sprayed in combinations, with many farmers (60%) spraying their crops on calendar basis, at 7-day intervals (Ntow *et al.* 2006). Streams within the catchments of vegetable farmlands are vulnerable to pesticide contamination as a result of spray drift and surface runoff (Maule *et al*., 2007). The quality of these water resources is of critical interest as they serve as aquatic habitats and drinking water sources.

#### **2. Methods**

#### **2.1 Sampling of stream water, sediment and groundwater**

Twenty-one streams flowing in and/or around vegetable plantations, stream-bed sediments and 9 drinking water wells in the Ofinso District of the Ashanti Region of Ghana were sampled between February 2008 and January 2009 as part of a pesticide monitoring programme in vegetable agro-ecosystems in Ghana. Streams sampled included Akumadan, Nkenkasu and Afrancho (Fig. 1) which flow in and around vegetable farmlands. The other streams sampled in the district were ephemeral and these included Srani, Bosompong, Sukubrim, Siasu, Ankonom, and Naasu (not shown). For each stream, 1 L water samples were collected into 1-L glass amber coloured bottles with Teflon-lined caps from upstream, mid-stream and downstream. During the same period, stream-bed sediment samples of about 200 g were collected into wide-neck glass jars. Groundwater samples were extracted from drinking water wells located within farming communities at Akumadan, Nkenkasu, and Afrancho into 1-L glass amber coloured bottles with Teflon-lined caps. None of the wells sampled was in a farmed section of the study area. All the wells are shallow wells (< 15 m) and represent unconfined aquifers. The wells receive water from the soil and upper porous rock zones that characterise the Ofinso District. Prior to sampling, pumps were run for about 5 min to clear the casing of standing water and to bring in fresh water from the aquifer. During this period, field measurement parameters (e.g. temperature) were stabilised. The sampling bottles were rinsed with well water before taking the water samples. Three replicates were collected from each well. The samples were transported to the laboratory within 24 to 48 h on ice in clean ice chests and stored in the laboratory refrigerator at 4oC until analysed. The samples were extracted within 24 h of arrival at the laboratory. Field blanks were prepared with distilled water and were analysed only when pesticide residues were detected in primary samples. Sampling was conducted throughout rainy and dry seasons and was not timed to applications of different pesticides or to rain events.

#### **2.2 Chemical analysis 2.2.1 Sample extraction**

54 Soil Health and Land Use Management

It is bordered to the east by Ejura-Sekyedumasi District, to the south by Afigya Sekyere, Ahafo Ano South and Atwima Districts. The district has 126 settlements and a population of about 35,190 with New Ofinso as its capital. The district has five towns namely Abofour, Nkenkasu, Afrancho, Akumadan and New Ofinso. The study area is within the Ofin, Pru and Afram river basins. In the present study, vegetable plantations were selected from Akumadan, Nkenkasu, and Afrancho. Agriculture is the main economic activity in these areas with over 70% of the active population being farmers. The district is well known for the cultivation of vegetable crops. Other major crops cultivated include cassava, maize, plantain and cocoa. More than 23 different active ingredients formulated as insecticides, herbicides and fungicides have been used in the cultivation of vegetables in the district. The five most frequently used insecticides include two organophosphates (chlorpyrifos and dimethoate), two pyrethroids (lambda-cyhalothrin and cypermethrin), and one organochlorine (endosulfan). Farmers use these highly toxic pesticides under primitive field conditions with insufficient protective equipment and training. Pesticide applications occur frequently, all year round, and are relatively intensive (500-1000 ml/ha). Pesticides are also sprayed in combinations, with many farmers (60%) spraying their crops on calendar basis, at 7-day intervals (Ntow *et al.* 2006). Streams within the catchments of vegetable farmlands are vulnerable to pesticide contamination as a result of spray drift and surface runoff (Maule *et al*., 2007). The quality of these water resources is of critical interest as they serve as aquatic

Twenty-one streams flowing in and/or around vegetable plantations, stream-bed sediments and 9 drinking water wells in the Ofinso District of the Ashanti Region of Ghana were sampled between February 2008 and January 2009 as part of a pesticide monitoring programme in vegetable agro-ecosystems in Ghana. Streams sampled included Akumadan, Nkenkasu and Afrancho (Fig. 1) which flow in and around vegetable farmlands. The other streams sampled in the district were ephemeral and these included Srani, Bosompong, Sukubrim, Siasu, Ankonom, and Naasu (not shown). For each stream, 1 L water samples were collected into 1-L glass amber coloured bottles with Teflon-lined caps from upstream, mid-stream and downstream. During the same period, stream-bed sediment samples of about 200 g were collected into wide-neck glass jars. Groundwater samples were extracted from drinking water wells located within farming communities at Akumadan, Nkenkasu, and Afrancho into 1-L glass amber coloured bottles with Teflon-lined caps. None of the wells sampled was in a farmed section of the study area. All the wells are shallow wells (< 15 m) and represent unconfined aquifers. The wells receive water from the soil and upper porous rock zones that characterise the Ofinso District. Prior to sampling, pumps were run for about 5 min to clear the casing of standing water and to bring in fresh water from the aquifer. During this period, field measurement parameters (e.g. temperature) were stabilised. The sampling bottles were rinsed with well water before taking the water samples. Three replicates were collected from each well. The samples were transported to the laboratory within 24 to 48 h on ice in clean ice chests and stored in the laboratory refrigerator at 4oC until analysed. The samples were extracted within 24 h of arrival at the laboratory. Field blanks were prepared with distilled water and were analysed only when

habitats and drinking water sources.

**2.1 Sampling of stream water, sediment and groundwater** 

**2. Methods** 

The extraction and analyses of water samples were performed following the Association of Official Analytical Chemists 990.06 and 970.52 methods as described by Ntow *et al*. (2008). Briefly, water samples were extracted sequentially three times with 25 mL *n*-hexane each time. The extract was dried with anhydrous sodium sulphate and concentrated down to 10 mL by means of ultrasonic bath type concentrators (Turbo Vap II). Extract clean up was done, using a chromatographic column, packed with florisil, previously activated for 3 h in an oven at 130oC, and anhydrous sulphate (all rinsed with petroleum ether). The extract was transferred to the column. Three fractions were obtained after elution with 6, 15, 50% ethyl ether in petroleum ether. Maximal flux rate of elution was 5 mL/min. Each eluate was evaporated and the extracts (re-dissolved in 1.5 ml *n*-hexane and made up to 2 ml with more *n*-hexane) were injected into a gas chromatographic system for identification and quantification of the pesticides.

Extraction and analysis of sediment samples followed the method described by Ntow *et al.* (2008). Briefly sediment samples were well mixed to obtain a homogeneous sample and then transferred into a pan to air-dry at ambient temperature until a constant weight was obtained. The samples were later ground in a mortar into fine powder such that they could pass through 2 mm sieve. Five grams (dry weight) of the sediment samples were soxhlet extracted in methanol, and cleaned up in florisil in the same way as described above for water. Sampling protocol and analytical procedures were subjected to quality control through field and laboratory blanks and spiking of samples with pesticide standards.

Laboratory glassware used in the sampling and analyses were cleaned as described by Ntow (2001). Pesticide grade solvents used for the analyses were *n*-hexane (>99%) and acetone (>99.9%) (Sigma, Munich, Germany); methanol (99.8%) and petroleum ether (BDH; VWR International, UK); dichloromethane and ethyl ether (Fluka; Munich, Germany). Deionised water was used from a Milli-Q water purification system (Millipore, Bedford, MA, USA) for blanks, sediment extraction, and spiked samples. Pesticide standards (> 98% purity) were obtained from Dr. Ehrenstorfer (Augsburg, Germany). Standard mixtures were prepared from individual pesticide stock solutions (50-100 mg in 100 ml acetone) and then diluted to working calibration standards at three concentration levels with acetone/cyclohexane (pesticide grade) mixture (1:9).

## **2.2.2 Pesticide residue analysis**

Measurement of pesticide compounds in water and sediment samples was performed on a GC-MS (Agilent 6890 Series GC System) coupled with an Agilent 5973N mass selective detector-electron impact ionization; and fused capillary column (HP-5MS) packed with 5% Phenyl Methyl Siloxane (30 m \* 0.25 mm I.D and film thickness 0.25 µm), which was operated in the selected ion-monitoring mode at the following conditions: injection port 250oC (splitless, pressure 22.62 psi; purge flow 50 mL/min; purge time 2.0 min; total flow 55.4 mL/min). Column oven: initial 70oC, held 2 min, programming rate 25oC/min (70 to 150oC); 10oC/min (150 to 200oC); 8oC/min (200 to 280oC) and held 10 min at 280oC. The

Pesticide Contamination in Groundwater and

(Table 1).

α-Endosulfan β-endosulfan

Dieldrin Chlorpyrifos p,p'-DDE p,p'-DDT

pH

analyzed

Temperature (oC)

Turbidity (NTU) Moisture content (%) Total organic carbon (%)

Endosulfan sulphate

**3. Results and discussion** 

analyzed are presented in Table 2.

Total suspended solids (mg/l)

water were also suitable for aquatic life.

**3.2 Pesticide residue levels in groundwater** 

Streams Draining Vegetable Plantations in the Ofinso District, Ghana 57

toxicity values listed in Table 1, the acute and chronic risk ratios were calculated for the water samples. A ratio of 1 means the individual pesticide has reached its criteria concentration in the streams. Risk for acute toxicity is based on the highest pesticide concentration found compared to the LC50 (Table 1). Risk for chronic toxicity is calculated based on the average concentration of all positive observations and the water quality criteria

> crustaceans or fish (µgL-1) (EXTOXNET 1996)

> > 1.20 1.20 1.20 - 0.01 0.18 0.18

Water-quality criterion (µgL-1) (USEPA 1999)

> 0.056 0.056 0.056 0.056 0.041 0.001 0.001

5.6 - 6.8 - - - 18.0 - 26.3 2.1 – 13.6

Pesticides Main use Lowest LC50 for

Insecticide Insecticide Insecticide Insecticide Insecticide Insecticide Insecticide

Table 1. Reference toxicity values for pesticides analyzed

**3.1 Physicochemical characteristics of water and sediment samples analyzed** 

The physicohemical characteristics of groundwater, stream water and sediment samples

Physicochemical parameter Groundwater Stream water Sediment

5.8 - 6.4 21.4 - 22.7 0 0.74 - 2.93 - -

Table 2. Physicohemical characteristics of groundwater, stream water and sediment samples

The pH and turbidity of groundwater were within acceptable levels for human consumption. The pH, temperatures, levels of total suspended solids and turbidity of stream

Pesticide residues were not detected in all the groundwater samples analyzed (Table 3). The non-detection of pesticide residues in groundwater could be due to their high adsorption to soil particles which does not facilitate their infiltration into groundwater. This is an indication that groundwater consumption may not contribute to community exposure to these pesticides.

6.6 - 8.3 23.2 - 27.4 5.8 - 20.6 2.2-32.5 - -

carrier gas was nitrogen at 15 psi; detector make-up, 30 mL/min. The injection volume was 1 µL (Agilent 7683 Series injector). Selection of analysed pesticides was done on the basis of pesticide use information provided by Ntow *et al.* (2006). The pesticides analysed included α- and β-endosulfan, endosulfan sulphate, dieldrin, dichlorodiphenyltrichloroethane (p,p'- DDT), dichlorodiphenyldichloroethylene (p,p'-DDE) and chlorpyrifos. For quality control of gas chromatographic conditions, a checkout procedure was performed before sample analysis in which a standard mixture with α-endosulfan content of 400 ng/L was used. Calibration was carried out when the concentration of α-endosulfan in standard mixture deviated significantly from 400 ng/L. Also the linearity of detector response was checked with five standard solutions of concentration 200 - 1000 ng/L. The correlation coefficient, r, obtained was ≥ 0.94. Recovery of the different pesticides ranged between 79% and 104% and their detection limits varied between 0.001 and 0.01 μg/L. The residues are expressed as µg/L (ppb) for surface water and µg/kg dry weight (ppb) for sediment. Because most of the pesticides analysed by GC/MS had a method detection limit at or below 0.01 µg/L, the reporting limit was chosen as 0.01 µg/L for these compounds. This reporting limit was used in calculating incidences of occurrence. A pesticide that has been identified but not quantified is indicated as below the detection limit.

#### **2.2.3 Physicochemical analysis**

The pH and temperature of samples were determined in situ using a pH meter. The pH meter was first calibrated with standard pH buffers before immersing the probe into the water or sediment. Temperature was measured concurrently. Total suspended solids and turbidity in water were measured using a turbidity meter (2100P Turbidimeter, Hach Company, Loveland, CO, USA). Calibration of the turbidity meter was done by filtering some water samples through pre-weighed Whatman GF/F (0.45 μm pore-size) glass microfiber filters which were then dried at 60°C for 48 h and re-weighed to determine TSS. Water content (expressed as weight fraction of water) was determined by first weighing wet sediment samples, then ovendrying the sediment samples at 105°C until constant weight, and obtaining the weight difference. Total suspended solids and turbidity were measured concurrently (Ntow *et al.*, 2008). Total organic carbon was obtained from the percentage organic matter in the sediments as percentage loss-on-ignition after drying 1.0 g of the sediment samples (previously acidified for the removal of carbonates) at 550°C in a furnace (Mwamburi, 2003).

#### **2.3 Statistical analysis**

A paired Student's *t* test was performed to analyze significant differences between pesticide residue levels in stream water and stream-bed sediment. One-way analysis of variance (ANOVA) was performed to analyze significant differences in pesticide residue levels in water and sediment from different sites. Pearson correlation analysis was performed to determine the relationship between the levels of pesticide residues and sediment characteristics (total suspended solids and total organic carbon) at the 95% confidence level (*p* < 0.05)

#### **2.4 Ecotoxicological significance of measured pesticides in stream water**

The effects of pesticides on water quality are commonly assessed by comparing the observed concentrations of individual pesticide compounds in the aquatic system with criteria that have been established to protect the health of aquatic organisms (Castillo *et al.* 2000; Hoffman *et al.* 2000). By comparing the pesticide concentrations in this study with the toxicity values listed in Table 1, the acute and chronic risk ratios were calculated for the water samples. A ratio of 1 means the individual pesticide has reached its criteria concentration in the streams. Risk for acute toxicity is based on the highest pesticide concentration found compared to the LC50 (Table 1). Risk for chronic toxicity is calculated based on the average concentration of all positive observations and the water quality criteria (Table 1).


Table 1. Reference toxicity values for pesticides analyzed

## **3. Results and discussion**

56 Soil Health and Land Use Management

carrier gas was nitrogen at 15 psi; detector make-up, 30 mL/min. The injection volume was 1 µL (Agilent 7683 Series injector). Selection of analysed pesticides was done on the basis of pesticide use information provided by Ntow *et al.* (2006). The pesticides analysed included α- and β-endosulfan, endosulfan sulphate, dieldrin, dichlorodiphenyltrichloroethane (p,p'- DDT), dichlorodiphenyldichloroethylene (p,p'-DDE) and chlorpyrifos. For quality control of gas chromatographic conditions, a checkout procedure was performed before sample analysis in which a standard mixture with α-endosulfan content of 400 ng/L was used. Calibration was carried out when the concentration of α-endosulfan in standard mixture deviated significantly from 400 ng/L. Also the linearity of detector response was checked with five standard solutions of concentration 200 - 1000 ng/L. The correlation coefficient, r, obtained was ≥ 0.94. Recovery of the different pesticides ranged between 79% and 104% and their detection limits varied between 0.001 and 0.01 μg/L. The residues are expressed as µg/L (ppb) for surface water and µg/kg dry weight (ppb) for sediment. Because most of the pesticides analysed by GC/MS had a method detection limit at or below 0.01 µg/L, the reporting limit was chosen as 0.01 µg/L for these compounds. This reporting limit was used in calculating incidences of occurrence. A pesticide that has been identified but not

The pH and temperature of samples were determined in situ using a pH meter. The pH meter was first calibrated with standard pH buffers before immersing the probe into the water or sediment. Temperature was measured concurrently. Total suspended solids and turbidity in water were measured using a turbidity meter (2100P Turbidimeter, Hach Company, Loveland, CO, USA). Calibration of the turbidity meter was done by filtering some water samples through pre-weighed Whatman GF/F (0.45 μm pore-size) glass microfiber filters which were then dried at 60°C for 48 h and re-weighed to determine TSS. Water content (expressed as weight fraction of water) was determined by first weighing wet sediment samples, then ovendrying the sediment samples at 105°C until constant weight, and obtaining the weight difference. Total suspended solids and turbidity were measured concurrently (Ntow *et al.*, 2008). Total organic carbon was obtained from the percentage organic matter in the sediments as percentage loss-on-ignition after drying 1.0 g of the sediment samples (previously acidified

A paired Student's *t* test was performed to analyze significant differences between pesticide residue levels in stream water and stream-bed sediment. One-way analysis of variance (ANOVA) was performed to analyze significant differences in pesticide residue levels in water and sediment from different sites. Pearson correlation analysis was performed to determine the relationship between the levels of pesticide residues and sediment characteristics (total

The effects of pesticides on water quality are commonly assessed by comparing the observed concentrations of individual pesticide compounds in the aquatic system with criteria that have been established to protect the health of aquatic organisms (Castillo *et al.* 2000; Hoffman *et al.* 2000). By comparing the pesticide concentrations in this study with the

quantified is indicated as below the detection limit.

for the removal of carbonates) at 550°C in a furnace (Mwamburi, 2003).

suspended solids and total organic carbon) at the 95% confidence level (*p* < 0.05)

**2.4 Ecotoxicological significance of measured pesticides in stream water** 

**2.2.3 Physicochemical analysis** 

**2.3 Statistical analysis** 

## **3.1 Physicochemical characteristics of water and sediment samples analyzed**

The physicohemical characteristics of groundwater, stream water and sediment samples analyzed are presented in Table 2.


Table 2. Physicohemical characteristics of groundwater, stream water and sediment samples analyzed

The pH and turbidity of groundwater were within acceptable levels for human consumption. The pH, temperatures, levels of total suspended solids and turbidity of stream water were also suitable for aquatic life.

#### **3.2 Pesticide residue levels in groundwater**

Pesticide residues were not detected in all the groundwater samples analyzed (Table 3). The non-detection of pesticide residues in groundwater could be due to their high adsorption to soil particles which does not facilitate their infiltration into groundwater. This is an indication that groundwater consumption may not contribute to community exposure to these pesticides.

Pesticide Contamination in Groundwater and

Endosulfan (α, β and sulfate) Dieldrin DDE

Chlorpyrifos

Table 5).

Pesticide components

α- Endosulfan β-Endosulfan Endosulfansulphate Dieldrin p,p'-DDE p,p'-DDT Chlorpyrifos

Pesticide name Water solubility at

given temperature (mgL-1)

0.32 (22oC)


Table 4. Properties of pesticides detected in streams. Source: EXTOXNET (1996)

Mean ± SD (µgL-1)

0.062 ± 0.007 0.031 ± 0.011 0.031 ± 0.012

> - ND - -

water and stream-bed sediment. Source: Ntow (2001)

Table 5. Concentrations (Mean ± SD) and incidence ratios of pesticide residues in stream

Streams Draining Vegetable Plantations in the Ofinso District, Ghana 59

3.6

5.5 5.8 4.7

Stream water (n = 50) Sediment (n = 42)

Mean ± SD (µgKg-1 dw)

0.19 ± 0.02 0.13 ± 0.01 0.23 ± 0.01


I.R (%)

90.0 97.5 98.3

35.0 25.0 - 68.3

I.R (%)

> 64 60 78

> > - - - -

Endosulfan is banned or restricted in many countries because of its human health and environmental impacts. In the United States, for example, endosulfan is applied to grains, tea, fruits, vegetables, tobacco, and cotton (DeLorenzo *et al.* 2001). In Ghana, endosulfan has a restricted use that does not include vegetables (it has only been registered for use on cotton), yet it is used on vegetables. According to Ntow *et al.* (2006), endosulfan is one of the most commonly used pesticides in the study area. Different formulations of the active ingredient are sold in the study area under different trade names such as Thionex 35 EC/ULV, Thiodan 50 EC, Endosulfan, Endocoton, Caiman 350 EC, Phaser and Novasulfan 35 EC. Vegetable farmers in Ghana spray endosulfan on tomato, pepper, okra, egg-plant (garden eggs), cabbage and lettuce. Although there are numerous pests and diseases prominent on vegetables (for instance, there are 13 fungal pathogens on tomato alone), the use of endosulfan was not necessarily to control diseases. Application of endosulfan to control diseases was done on a trial-and-error basis because the local farmers were not able to identify the pests causing damage (Ntow *et al.*, 2006). The use of endosulfan on vegetables by Ghanaian farmers is of great concern due to the persistence and extreme toxicity of endosulfan to fish and aquatic invertebrates (Pérez-Ruzafa *et al*., 2000). The presence of endosulfan in stream water also has implications for public health as rural communities depend on stream water for drinking. The levels of endosulfan residues obtained in the present study are comparable to those obtained in a previous study by Ntow (2001) (see

Log*K*ow Soil adsorption

coefficient, *K*oc (mLg-1)

2,400


Soil halflife

50 d



Table 3. Concentrations (Mean ± SD) and incidence ratios of pesticide residues in groundwater, stream water and stream-bed sediment samples analyzed. [n = number of samples analyzed; SD = standard deviation; ND = below detection limit (0.01 µgL-1 or µgKg-1); I.R. = incidence ratio; dw = dry weight]

#### **3.3 Pesticide residue levels in stream water**

The mean concentrations and incidence of occurrence of pesticides detected in stream water are summarized in Table 3. Only 67 (35%) of the 192 stream water samples analyzed had pesticide residue detections. α-endosulfan, β-endosulfan and endosulfan sulphate were the only pesticide residues detected with mean concentrations of 0.027 ± 0.015, 0.021 ± 0.010, and 0.022 ± 0.010 µgL-1 (or ppb), respectively. The incidence of occurrence of these organochlorine pesticide residues were α-endosulfan (27.8%), β-endosulfan (13.9%) and endosulfan (21.5%). Technical endosulfan is a mixture of α- and β-endosulfan in a ratio of 7:3. Endosulfan sulfate is the principal metabolite of endosulfan and it is highly toxic. Endosulfan sulfate levels in stream water samples were nearly equal to those of the parent compounds (α- and β-endosulfan), suggesting current use of the pesticide. The occurrence of endosulfan residues in streams may be the result of direct overspray, spray drift, atmospheric transport of volatilized pesticides, agricultural runoff, pesticide misuse, and improper disposal of pesticide containers (Maule *et al*., 2007; Wan *et al*., 2005; Ntow *et al*., 2008). Inflows from shallow groundwater originating in the agricultural areas are however unlikely sources of pesticide contamination in the streams studied since pesticide residues were not detected in groundwater samples analyzed in the present study.

Pearson correlation analysis revealed an association (*r*2 > 0.6) between endosulfan residue concentration and total suspended solids in stream water for most sites. Thus, increase in the level of suspended solids (sediment) resulted in a corresponding increase in concentration of endosulfan. This partitioning behavior of endosulfan in the streams studied may be influenced by physicochemical properties. Endosulfan has low water solubility (0.32 mgL at 22oC) and high affinity for sediment as indicated by the high soil adsorption coefficient of 2,400 mLg-1 which can be attributed to its high octanol-water partitioning coefficient (log*K*ow = 3.6) (see Table 4). Therefore, with these properties, there is a high tendency for endosulfan to adsorb onto suspended sediments in the water column than to remain in solution as the study has shown.

I.R (%)



Table 3. Concentrations (Mean ± SD) and incidence ratios of pesticide residues in groundwater, stream water and stream-bed sediment samples analyzed. [n = number of samples analyzed; SD = standard deviation; ND = below detection limit (0.01 µgL-1 or

were not detected in groundwater samples analyzed in the present study.

Groundwater (n =81) Stream water (n = 192) Sediment (n = 180)

I.R (%)

27.8 13.9 21.5


Mean ± SD (µgKg-1 dw)

0.38 ± 0.24 0.18 ± 0.09 0.53 ± 0.24

0.16 ± 0.04 3.77 ± 1.90 ND 1.23 ± 0.40

I.R (%)

90.0 97.5 98.3

35.0 25.0 - 68.3

Mean ± SD (µgL-1)

0.027 ± 0.015 0.021 ± 0.010 0.022 ± 0.010

ND ND ND ND

The mean concentrations and incidence of occurrence of pesticides detected in stream water are summarized in Table 3. Only 67 (35%) of the 192 stream water samples analyzed had pesticide residue detections. α-endosulfan, β-endosulfan and endosulfan sulphate were the only pesticide residues detected with mean concentrations of 0.027 ± 0.015, 0.021 ± 0.010, and 0.022 ± 0.010 µgL-1 (or ppb), respectively. The incidence of occurrence of these organochlorine pesticide residues were α-endosulfan (27.8%), β-endosulfan (13.9%) and endosulfan (21.5%). Technical endosulfan is a mixture of α- and β-endosulfan in a ratio of 7:3. Endosulfan sulfate is the principal metabolite of endosulfan and it is highly toxic. Endosulfan sulfate levels in stream water samples were nearly equal to those of the parent compounds (α- and β-endosulfan), suggesting current use of the pesticide. The occurrence of endosulfan residues in streams may be the result of direct overspray, spray drift, atmospheric transport of volatilized pesticides, agricultural runoff, pesticide misuse, and improper disposal of pesticide containers (Maule *et al*., 2007; Wan *et al*., 2005; Ntow *et al*., 2008). Inflows from shallow groundwater originating in the agricultural areas are however unlikely sources of pesticide contamination in the streams studied since pesticide residues

Pearson correlation analysis revealed an association (*r*2 > 0.6) between endosulfan residue concentration and total suspended solids in stream water for most sites. Thus, increase in the level of suspended solids (sediment) resulted in a corresponding increase in concentration of endosulfan. This partitioning behavior of endosulfan in the streams studied may be influenced by physicochemical properties. Endosulfan has low water solubility (0.32 mgL at 22oC) and high affinity for sediment as indicated by the high soil adsorption coefficient of 2,400 mLg-1 which can be attributed to its high octanol-water partitioning coefficient (log*K*ow = 3.6) (see Table 4). Therefore, with these properties, there is a high tendency for endosulfan to adsorb onto suspended sediments in the water column than to

Pesticide components

α- Endosulfan β-Endosulfan Endosulfansulphate Dieldrin p,p'-DDE p,p'-DDT Chlorpyrifos

Mean ± SD (µgL-1)

ND ND ND

ND ND ND ND

µgKg-1); I.R. = incidence ratio; dw = dry weight]

**3.3 Pesticide residue levels in stream water** 

remain in solution as the study has shown.


Table 4. Properties of pesticides detected in streams. Source: EXTOXNET (1996)

Endosulfan is banned or restricted in many countries because of its human health and environmental impacts. In the United States, for example, endosulfan is applied to grains, tea, fruits, vegetables, tobacco, and cotton (DeLorenzo *et al.* 2001). In Ghana, endosulfan has a restricted use that does not include vegetables (it has only been registered for use on cotton), yet it is used on vegetables. According to Ntow *et al.* (2006), endosulfan is one of the most commonly used pesticides in the study area. Different formulations of the active ingredient are sold in the study area under different trade names such as Thionex 35 EC/ULV, Thiodan 50 EC, Endosulfan, Endocoton, Caiman 350 EC, Phaser and Novasulfan 35 EC. Vegetable farmers in Ghana spray endosulfan on tomato, pepper, okra, egg-plant (garden eggs), cabbage and lettuce. Although there are numerous pests and diseases prominent on vegetables (for instance, there are 13 fungal pathogens on tomato alone), the use of endosulfan was not necessarily to control diseases. Application of endosulfan to control diseases was done on a trial-and-error basis because the local farmers were not able to identify the pests causing damage (Ntow *et al.*, 2006). The use of endosulfan on vegetables by Ghanaian farmers is of great concern due to the persistence and extreme toxicity of endosulfan to fish and aquatic invertebrates (Pérez-Ruzafa *et al*., 2000). The presence of endosulfan in stream water also has implications for public health as rural communities depend on stream water for drinking. The levels of endosulfan residues obtained in the present study are comparable to those obtained in a previous study by Ntow (2001) (see Table 5).


Table 5. Concentrations (Mean ± SD) and incidence ratios of pesticide residues in stream water and stream-bed sediment. Source: Ntow (2001)

Pesticide Contamination in Groundwater and

to 78 d in the water-sediment system.

α-Endosulfan β-endosulfan Endosulfan

0

1

2

3

**Concentration (ppb)**

4

5

6

Streams Draining Vegetable Plantations in the Ofinso District, Ghana 61

(Reinert *et al*., 2002), which leads to the accumulation of pesticide residues in sediment over a period of time. The distribution of pesticide residues in water and sediment could be related to their physicochemical properties such as water solubility, soil adsorption coefficient and persistence as shown in Table 4. For example, endosulfan and DDE (organochlorine pesticides) have low water-solubility, high soil adsorption coefficients (*K*oc) and high persistence in soil, with half-lives between 50 days and 15 years (EXTOXNET, 1996). They are therefore expected to exhibit low degradation in sediment and so were frequently detected in sediments than water. These characteristics imply there could be a direct contribution to the streams from erosion of soil contaminated with these compounds (Munn & Gruber, 1997). The accumulation of chlorpyrifos in stream-bed sediment is in accordance with its high soil adsorption coefficient, *K*oc, of 6,070 mLg-1 and its half-life of 35

Fig. 2. Pesticide residue concentrations in stream water and underlying sediment

sulphate

There were also differences in the distribution patterns of endosulfan, dieldrin, DDE and chlorpyrifos in sediment which could be related to the differences in their physicochemical properties. DDE, with the highest *K*oc (100,000 mLg-1), recorded the highest levels in sediment, followed by chlorpyrifos (*K*oc = 6,070 mLg-1) while endosulfan with the lowest *K*oc (2,400 mLg-1) recorded the least concentration. The reverse trend was observed for stream water. The mean level of total endosulfan (α-endosulfan + β-endosulfan + endosulfan sulfate) in sediment from the present study (1.09 ± 0.57 µgKg-1 dw) was not significantly higher (*p* > 0.05) than that obtained from the previous study (0.54 ± 0.04 µgKg-1

**Pesticide residue**

Stream water Sediment

Dieldrin p,p'-DDT p,p'-DDE Chlorpyrifos

#### **3.4 Pesticide residue levels in stream-bed sediment**

The mean concentrations and incidence of occurrence of pesticides detected in stream-bed sediments are summarized in Table 3. Several pesticide residues were detected in the stream-bed sediment samples analyzed compared with stream water samples analyzed. αendosulfan, β-endosulfan, endosulfan sulfate occurred in at least 90% of all the sediment samples analyzed while dieldrin, *p,p*'-DDE and chlorpyrifos occurred in 25%, 35% and 68% of all the sediment samples analyzed, respectively. Chlorpyrifos is an organophosphate pesticide while all the other pesticide residues detected belong to the organochlorine group of pesticides.

DDT is well-known to persist in the environment, even in tropical environments (Kidd *et al.* 2001). Although *p,p*'-DDT was not detected in stream water and sediment, its metabolite *p,p*'-DDE was detected in sediment at an average concentration of 3.77 ± 1.90 µgKg-1 dry wt. DDE is more persistent in the environment than DDT. Thus, when the use of DDT in a country ceases, its levels are expected to decrease more rapidly while the levels of DDE increases, thereby producing an increasing DDE/DDT ratio. The DDE/DDT ratio is often used as an indicator of recent DDT inputs into the environment; a ratio < 1 indicates recent input (Ballschmiter & Wittlinger, 1991). The absence of DDT and presence of DDE in sediment could imply the disuse of the parent compound, DDT in Ghana. The relatively high levels of *p,p*'-DDE detected in the present study is a justification of the ban of DDT from agricultural use in Ghana. The non-detection of DDT could also confirm the efficacy of the ban on the agricultural use of DDT in Ghana.

Dieldrin and chlorpyrifos were also detected in sediment with mean concentrations of 0.16 ± 0.04 and 1.23 ± 0.40 µgKg-1 dw, respectively, although they were not detected in stream water samples. Apart from its usage, dieldrin can occur in the environment as a result of the degradation of a related pesticide, aldrin. Aldrin and dieldrin are persistent in the environment and they have been banned from agricultural use in Ghana (Ntow & Botwe, 2011). The occurrence of dieldrin in sediment could therefore be due to previous use of dieldrin and/or aldrin. Chlorpyrifos recorded the highest incidence of occurrence (68%) in sediment samples. Chlorpyrifos is a broad-spectrum organophosphorus pesticide. Chlorpyrifos, under the trade name Dursban 4E, is a registered insecticide in Ghana for the control of scale borers in cereals, vegetables and ornamentals, and for public health purposes. The occurrence of chlorpyrifos in sediment could be as a result of their current use in vegetable plantations. Residues of chlorpyrifos have also been measured in vegetables from the Ashanti Region (Amoah *et al*., 2006; Darko & Akoto, 2008).

Generally, the detected pesticides accumulated in sediment to several times their ambient water concentrations (Fig. 2). Thus, sediment is a better indicator of pesticide pollution than the overlying water. For example, endosulfan (α-endosulfan + β-endosulfan + endosulfan sulfate) accumulated to over 15 times its ambient water concentration. *p,p*'-DDE was also not detected in stream water although it occurred in relatively high concentrations in sediment (3.77 ± 1.90 µgKg-1). There was also a significant correlation (*r*2 > 0.6) between levels of pesticide residues and organic carbon content of sediment. This agrees well with the finding that sediment organic matter is the preferential site for the sorption of hydrophobic pollutants (Pignatello, 1998), which includes organochlorine pesticides.

The relatively higher levels of pesticides in sediment than the overlying water can be explained by the fact that pesticides are sequestered by sediments in aquatic systems

The mean concentrations and incidence of occurrence of pesticides detected in stream-bed sediments are summarized in Table 3. Several pesticide residues were detected in the stream-bed sediment samples analyzed compared with stream water samples analyzed. αendosulfan, β-endosulfan, endosulfan sulfate occurred in at least 90% of all the sediment samples analyzed while dieldrin, *p,p*'-DDE and chlorpyrifos occurred in 25%, 35% and 68% of all the sediment samples analyzed, respectively. Chlorpyrifos is an organophosphate pesticide while all the other pesticide residues detected belong to the

DDT is well-known to persist in the environment, even in tropical environments (Kidd *et al.* 2001). Although *p,p*'-DDT was not detected in stream water and sediment, its metabolite *p,p*'-DDE was detected in sediment at an average concentration of 3.77 ± 1.90 µgKg-1 dry wt. DDE is more persistent in the environment than DDT. Thus, when the use of DDT in a country ceases, its levels are expected to decrease more rapidly while the levels of DDE increases, thereby producing an increasing DDE/DDT ratio. The DDE/DDT ratio is often used as an indicator of recent DDT inputs into the environment; a ratio < 1 indicates recent input (Ballschmiter & Wittlinger, 1991). The absence of DDT and presence of DDE in sediment could imply the disuse of the parent compound, DDT in Ghana. The relatively high levels of *p,p*'-DDE detected in the present study is a justification of the ban of DDT from agricultural use in Ghana. The non-detection of DDT could also confirm the efficacy of

Dieldrin and chlorpyrifos were also detected in sediment with mean concentrations of 0.16 ± 0.04 and 1.23 ± 0.40 µgKg-1 dw, respectively, although they were not detected in stream water samples. Apart from its usage, dieldrin can occur in the environment as a result of the degradation of a related pesticide, aldrin. Aldrin and dieldrin are persistent in the environment and they have been banned from agricultural use in Ghana (Ntow & Botwe, 2011). The occurrence of dieldrin in sediment could therefore be due to previous use of dieldrin and/or aldrin. Chlorpyrifos recorded the highest incidence of occurrence (68%) in sediment samples. Chlorpyrifos is a broad-spectrum organophosphorus pesticide. Chlorpyrifos, under the trade name Dursban 4E, is a registered insecticide in Ghana for the control of scale borers in cereals, vegetables and ornamentals, and for public health purposes. The occurrence of chlorpyrifos in sediment could be as a result of their current use in vegetable plantations. Residues of chlorpyrifos have also been measured in vegetables

Generally, the detected pesticides accumulated in sediment to several times their ambient water concentrations (Fig. 2). Thus, sediment is a better indicator of pesticide pollution than the overlying water. For example, endosulfan (α-endosulfan + β-endosulfan + endosulfan sulfate) accumulated to over 15 times its ambient water concentration. *p,p*'-DDE was also not detected in stream water although it occurred in relatively high concentrations in sediment (3.77 ± 1.90 µgKg-1). There was also a significant correlation (*r*2 > 0.6) between levels of pesticide residues and organic carbon content of sediment. This agrees well with the finding that sediment organic matter is the preferential site for the sorption of hydrophobic pollutants (Pignatello, 1998), which includes organochlorine

The relatively higher levels of pesticides in sediment than the overlying water can be explained by the fact that pesticides are sequestered by sediments in aquatic systems

**3.4 Pesticide residue levels in stream-bed sediment** 

organochlorine group of pesticides.

the ban on the agricultural use of DDT in Ghana.

pesticides.

from the Ashanti Region (Amoah *et al*., 2006; Darko & Akoto, 2008).

(Reinert *et al*., 2002), which leads to the accumulation of pesticide residues in sediment over a period of time. The distribution of pesticide residues in water and sediment could be related to their physicochemical properties such as water solubility, soil adsorption coefficient and persistence as shown in Table 4. For example, endosulfan and DDE (organochlorine pesticides) have low water-solubility, high soil adsorption coefficients (*K*oc) and high persistence in soil, with half-lives between 50 days and 15 years (EXTOXNET, 1996). They are therefore expected to exhibit low degradation in sediment and so were frequently detected in sediments than water. These characteristics imply there could be a direct contribution to the streams from erosion of soil contaminated with these compounds (Munn & Gruber, 1997). The accumulation of chlorpyrifos in stream-bed sediment is in accordance with its high soil adsorption coefficient, *K*oc, of 6,070 mLg-1 and its half-life of 35 to 78 d in the water-sediment system.

Fig. 2. Pesticide residue concentrations in stream water and underlying sediment

There were also differences in the distribution patterns of endosulfan, dieldrin, DDE and chlorpyrifos in sediment which could be related to the differences in their physicochemical properties. DDE, with the highest *K*oc (100,000 mLg-1), recorded the highest levels in sediment, followed by chlorpyrifos (*K*oc = 6,070 mLg-1) while endosulfan with the lowest *K*oc (2,400 mLg-1) recorded the least concentration. The reverse trend was observed for stream water. The mean level of total endosulfan (α-endosulfan + β-endosulfan + endosulfan sulfate) in sediment from the present study (1.09 ± 0.57 µgKg-1 dw) was not significantly higher (*p* > 0.05) than that obtained from the previous study (0.54 ± 0.04 µgKg-1

Pesticide Contamination in Groundwater and

significant.

aquatic systems.

**4. Conclusion** 

0

2

4

6

**Chronic risk ratio**

8

10

12

Streams Draining Vegetable Plantations in the Ofinso District, Ghana 63

detected in water at the quantification limit of 0.01 µgL-1 in this study, the toxicity factor was considered relevant to estimate since the quantification limits for these pesticides were generally above their respective water quality criteria. For example, the water quality criterion for DDT and DDE is 0.001 µgL-1 (Table 5) and the quantification limit was 0.01 µgL-1. This means that when DDT and DDE are detected, they have already exceeded their water quality criteria many times (see Figs. 3 & 4). The quantification limit was therefore used to calculate the toxicity factors. Thus, any occurrence of DDT and/or DDE in the streams is

Endosulfan, dieldrin, DDE and chlorpyrifos are among the pesticides that are very toxic to fish and many aquatic invertebrate species. There were no records of fish or amphibian kills in the streams at the time of the study. However, simultaneous exposure to multiple contaminants is known to produce an additive, and sometimes even synergistic and complex effects in organisms which can affect the abundance and diversity of non-target species and alter trophic interactions (Rovedatti *et al.*, 2001). Sediment is an important reservoir of contaminants, acting as both an ultimate sink and potential source via a series of biogeochemical processes (Guo *et al*., 2009). Pesticide contamination of sediments may thus lead to exposure of sediment-dwelling organisms to repeated pulses or fluctuating concentrations of pesticides (Reinert *et al*., 2002). There is therefore the need to assess the impact of water and sediment contamination on species abundance and diversity in these

Fig. 4. Chronic risk ratios for detected pesticides in stream water

sulphate

α-Endosulfan β-endosulfan Endosulfan

The results of this study have provided an insight into the levels of pesticide residue contamination in streams flowing in and around vegetable plantations in the Ofinso District of Ghana. Among the pesticides detected, endosulfan was the compound with the highest

**Pesticide residue**

Dieldrin p,p'-DDT p,p'-DDE Chlorpyrifos

dw) by Ntow (2001). However, the mean sediment DDE level obtained from the present study (3.77 ± 1.90 µgKg -1 dw) was significantly higher (p < 0.05) than that obtained from the previous study by Ntow (2001), possibly due to the accumulation of the residue in the environment over time.

#### **3.5 Ecotoxicological significance of measured pesticides in stream water**

To evaluate the ecotoxicological significance of pesticides contamination in streams, acute (ARR) and chronic (CRR) risk ratios were calculated for the water samples by comparing the pesticide concentrations in the samples with their toxicity values (Table 1).

The calculated risk ratios for acute toxicity are shown in Fig. 3. It was found that none of the detected pesticides had an acute risk ratio greater than 1. Using the quantification limit of 0.01 µgL-1, chlorpyrifos had a value of 1 in the streams. This means that when chlorpyrifos is detected in water, its concentration is already equal to its acute risk criteria. Thus, any occurrence of chlorpyrifos in water could pose a risk of acute toxicity to fish and crustaceans, and especially, species such as cladocerans, which have been observed to be highly sensitive to chlorpyrifos (Brock *et al*., 1992; van Wijngaarden *et al*., 2005). According to the fringing communities, fish is scarce in the streams within the catchments although fingerlings and other aquatic organisms such as frogs and crabs are present. Considering that the maximum concentrations found in this study are not the highest possible concentrations that can occur, compounds with a factor > 0.1 could pose a moderate risk of acute toxicity. Also, for many compounds, there is not a large data set of toxicity values for aquatic organisms of different trophic levels. Furthermore, the great majority of compounds have not been tested with tropical organisms.

Fig. 3. Acute risk ratios for detected pesticides in stream water

The calculated risk ratios for chronic toxicity are shown in Fig. 4. It was found that DDT and DDE exceeded their chronic risk criteria in the streams. Although DDT and DDE were not

dw) by Ntow (2001). However, the mean sediment DDE level obtained from the present study (3.77 ± 1.90 µgKg -1 dw) was significantly higher (p < 0.05) than that obtained from the previous study by Ntow (2001), possibly due to the accumulation of the residue in the

To evaluate the ecotoxicological significance of pesticides contamination in streams, acute (ARR) and chronic (CRR) risk ratios were calculated for the water samples by comparing the

The calculated risk ratios for acute toxicity are shown in Fig. 3. It was found that none of the detected pesticides had an acute risk ratio greater than 1. Using the quantification limit of 0.01 µgL-1, chlorpyrifos had a value of 1 in the streams. This means that when chlorpyrifos is detected in water, its concentration is already equal to its acute risk criteria. Thus, any occurrence of chlorpyrifos in water could pose a risk of acute toxicity to fish and crustaceans, and especially, species such as cladocerans, which have been observed to be highly sensitive to chlorpyrifos (Brock *et al*., 1992; van Wijngaarden *et al*., 2005). According to the fringing communities, fish is scarce in the streams within the catchments although fingerlings and other aquatic organisms such as frogs and crabs are present. Considering that the maximum concentrations found in this study are not the highest possible concentrations that can occur, compounds with a factor > 0.1 could pose a moderate risk of acute toxicity. Also, for many compounds, there is not a large data set of toxicity values for aquatic organisms of different trophic levels. Furthermore, the great majority of compounds

**3.5 Ecotoxicological significance of measured pesticides in stream water** 

pesticide concentrations in the samples with their toxicity values (Table 1).

environment over time.

have not been tested with tropical organisms.

0

0.2

0.4

0.6

**Acute risk ratio**

0.8

1

1.2

Fig. 3. Acute risk ratios for detected pesticides in stream water

sulphate

α-Endosulfan β-endosulfan Endosulfan

The calculated risk ratios for chronic toxicity are shown in Fig. 4. It was found that DDT and DDE exceeded their chronic risk criteria in the streams. Although DDT and DDE were not

**Pesticide residue**

Dieldrin p,p'-DDT p,p'-DDE Chlorpyrifos

detected in water at the quantification limit of 0.01 µgL-1 in this study, the toxicity factor was considered relevant to estimate since the quantification limits for these pesticides were generally above their respective water quality criteria. For example, the water quality criterion for DDT and DDE is 0.001 µgL-1 (Table 5) and the quantification limit was 0.01 µgL-1. This means that when DDT and DDE are detected, they have already exceeded their water quality criteria many times (see Figs. 3 & 4). The quantification limit was therefore used to calculate the toxicity factors. Thus, any occurrence of DDT and/or DDE in the streams is significant.

Endosulfan, dieldrin, DDE and chlorpyrifos are among the pesticides that are very toxic to fish and many aquatic invertebrate species. There were no records of fish or amphibian kills in the streams at the time of the study. However, simultaneous exposure to multiple contaminants is known to produce an additive, and sometimes even synergistic and complex effects in organisms which can affect the abundance and diversity of non-target species and alter trophic interactions (Rovedatti *et al.*, 2001). Sediment is an important reservoir of contaminants, acting as both an ultimate sink and potential source via a series of biogeochemical processes (Guo *et al*., 2009). Pesticide contamination of sediments may thus lead to exposure of sediment-dwelling organisms to repeated pulses or fluctuating concentrations of pesticides (Reinert *et al*., 2002). There is therefore the need to assess the impact of water and sediment contamination on species abundance and diversity in these aquatic systems.

Fig. 4. Chronic risk ratios for detected pesticides in stream water

## **4. Conclusion**

The results of this study have provided an insight into the levels of pesticide residue contamination in streams flowing in and around vegetable plantations in the Ofinso District of Ghana. Among the pesticides detected, endosulfan was the compound with the highest

Pesticide Contamination in Groundwater and

1753–1763

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incidence of occurrence in both water and sediment, which is also the most frequently used pesticide in the study area. Sediment samples exhibited greater number and higher concentrations of pesticides residues than stream water samples. Although acute and chronic risk ratios indicated that the concentrations of the detected pesticide residues in streams did not surpass aquatic quality criteria, the presence of endosulfan in stream water has implications for public health. The use of endosulfan in agriculture should continue to be carefully monitored given its persistence, bioaccumulation, and continued release into streams. An extension of both the study areas and range of pesticides residues analyzed should be considered in future work.

## **5. Acknowledgement**

The authors wish to express their gratitude to the Royal Netherlands Academy of Arts and Sciences (KNAW) for financial support. The Kinneret Limnological Laboratory, Migdal, Israel, is acknowledged for technical assistance in the use of GC/MS.

#### **6. References**


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The authors wish to express their gratitude to the Royal Netherlands Academy of Arts and Sciences (KNAW) for financial support. The Kinneret Limnological Laboratory, Migdal,

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**5. Acknowledgement** 

**6. References** 


**5** 

 *Argentina* 

**Fire Impact on Several Chemical and** 

*Universidad Nacional de Córdoba, Córdoba* 

*Departamento de Recursos Naturales, Facultad de Ciencias Agropecuarias,* 

**Physicochemical Parameters in a Forest Soil** 

Andrea Rubenacker, Paola Campitelli, Manuel Velasco and Silvia Ceppi

Cordoba is a Mediterranean State, with semiarid climate, dry autumn and winter, in which the wild fire can take place, especially at the end of dry season. Forest fires happen frequently in the mountain zones of the province of Córdoba, Argentina, which are located at west and south-west region. The vegetation, in the south-west zone, are principally *Pinus halepensis Mill.; Pinus elliottii* implanted and the native vegetation cover is *Stipa caudata, Piptochaetium hackelii, P. napostaense y* Briza *subaristata*, between others. Taxonomically the

In the west area the native vegetation is principally *Acacia caven, Festuca hieronymi, Stipa, Poa* 

Forest wild fires constitute a serious environmental problem, not only due to the destruction of vegetation but also because the degradation that may be induced in a soil as a consequence of the change produced in its properties. Wild fire can strongly modify the abiotic and biotic characteristics of soil, altering its structure, chemical and physicochemical properties, carbon content and macronutrient levels. The degree of the alteration produced depends on the frequency and intensity of fire, all these modifications being particularly

Organic matter is a key factor for forest soil. It has a direct and /or an indirect influence on all physical and chemical characteristics of the soil. While low severity fires, such as those prescribed for forest management, have been reported to have transient but positive effect on soil fertility, severe wildfire result in significant losses of soil organic matter, and nutrient, and deterioration of the overall physical-chemical properties of soil that determine

Fire may directly consume part or all of the standing plant material and litter as well as the organic matter in the upper layer of the soil. One of the most important soil change, during the burning is the alteration in the organic matter content therefore, the nutrient contained in the organic matter are either more available or can be volatilized and lost from the site. The soluble nutrient would be loss for erosion or leaching if they are not immediately

Humic substances are one of the most important fractions of the organic matter and are considered the most abundant organic component in nature and largely contribute to soil structuring and stability, to its permeability for water and gases, to its water holding

its fertility, such as porosity, structure among others (Certini, 2005).

**1. Introduction** 

soil corresponds to an Ustorthent.

important in the surface horizons.

absorbed by plants or retained by soil.

*stukerti,* between others. The soil is an Argiustoll.


## **Fire Impact on Several Chemical and Physicochemical Parameters in a Forest Soil**

Andrea Rubenacker, Paola Campitelli, Manuel Velasco and Silvia Ceppi *Departamento de Recursos Naturales, Facultad de Ciencias Agropecuarias, Universidad Nacional de Córdoba, Córdoba Argentina* 

#### **1. Introduction**

66 Soil Health and Land Use Management

Robinson, A. Y. (1991). Sustainable Agriculture: The Wildlife Connection. *American Journal of* 

Rovedatti, M. G.; Castañé, P. M.; Topalián, M. L. & Salibián, A. (2001). Monitoring of

United States Environmental Protection Agency (USEPA). (1999). National recommended

van Wijngaarden, R. P. A.; Brock, T. C. M. & Douglas, M. T. (2005). Effects of chlorpyrifos in

ecotoxicological thresholds. *Pest Management Science*, Vol. 61, pp. 923–935. Wan, M. T.; Kuo, J.; Buday, C.; Schroeder, G.; Van Aggelen, G. & Pasternak, J. (2005).

water quality criteria. USEPA 822-Z-99-001. Washington, DC.

River (Buenos Aires, Argentina). *Water Research*, Vol. 35, pp. 3457-3461. Skinner, J. A.; Lewis, K. A.; Bardon, K. S.; Tucker, P.; Catt, J. A. & Chambers, B. J. (1997). An

organochlorine and organophosphorus pesticides in the water of the Reconquista

overview of the environmental impact of agriculture in the U.K. *Journal of* 

freshwater model ecosystems: the influence of experimental conditions on

Toxicity of α-, β-, (α + β)-endosulfan and their formulated and degradation products to *Daphnia magna*, *Hyalella azteca*, *Oncorhynchus mykiss*, *Oncorhynchus kisutch*, and biological implications in streams. *Environmental Toxicology &* 

*Alternative Agriculture*, Vol. 6, No. 4, pp. 161-167.

*Environmental Management*, Vol. 50, pp. 111–128

*Chemistry*, Vol. 24, pp. 1146–1154.

Cordoba is a Mediterranean State, with semiarid climate, dry autumn and winter, in which the wild fire can take place, especially at the end of dry season. Forest fires happen frequently in the mountain zones of the province of Córdoba, Argentina, which are located at west and south-west region. The vegetation, in the south-west zone, are principally *Pinus halepensis Mill.; Pinus elliottii* implanted and the native vegetation cover is *Stipa caudata, Piptochaetium hackelii, P. napostaense y* Briza *subaristata*, between others. Taxonomically the soil corresponds to an Ustorthent.

In the west area the native vegetation is principally *Acacia caven, Festuca hieronymi, Stipa, Poa stukerti,* between others. The soil is an Argiustoll.

Forest wild fires constitute a serious environmental problem, not only due to the destruction of vegetation but also because the degradation that may be induced in a soil as a consequence of the change produced in its properties. Wild fire can strongly modify the abiotic and biotic characteristics of soil, altering its structure, chemical and physicochemical properties, carbon content and macronutrient levels. The degree of the alteration produced depends on the frequency and intensity of fire, all these modifications being particularly important in the surface horizons.

Organic matter is a key factor for forest soil. It has a direct and /or an indirect influence on all physical and chemical characteristics of the soil. While low severity fires, such as those prescribed for forest management, have been reported to have transient but positive effect on soil fertility, severe wildfire result in significant losses of soil organic matter, and nutrient, and deterioration of the overall physical-chemical properties of soil that determine its fertility, such as porosity, structure among others (Certini, 2005).

Fire may directly consume part or all of the standing plant material and litter as well as the organic matter in the upper layer of the soil. One of the most important soil change, during the burning is the alteration in the organic matter content therefore, the nutrient contained in the organic matter are either more available or can be volatilized and lost from the site. The soluble nutrient would be loss for erosion or leaching if they are not immediately absorbed by plants or retained by soil.

Humic substances are one of the most important fractions of the organic matter and are considered the most abundant organic component in nature and largely contribute to soil structuring and stability, to its permeability for water and gases, to its water holding

Fire Impact on Several Chemical and Physicochemical Parameters in a Forest Soil 69

sampling in the unburned and burned soil, respectively. The soil was taxonomically characterized as Ustorthent. The samples were air-dried, crushed and passed through a 2

The humic acids (HA) analyzed were extracted from the burned (HA-BS) and unburned soil

The samples of burned and unburned soil were analyzed for pH at a rate 1:2.5 (w:v), electric conductivity (EC), total nitrogen content (TN) by the Kjeldahl method, phosphorus available (P) by Bray & Kurtz method (1945), total organic carbon (TOC) by combustion at 540 0C for 4 h (Abad, et al., 2002) and oxidable carbon (Cox) by the methodology proposed by de Richter & Von Wistinghausen (1981). Organic light fraction (OLF) were determined according the method proposed by Janzen et al., 1992, the C and N content of the OLF by dry combustion

The carbon content of humic substances (CHS), humic acids (CHA) and fulvic acids (CFA) were determined according to the technique proposed by Syms & Haby 1971. The carbon content of each fraction (CHS, CHA and CFA) were calculated as percentage of the TOC, therefore, the % CHA correspond to the Humification Index (HI) (Roletto et al., 1985;

The apolar or free lipidic fraction (FLF) were extracted with petroleum ether (40-60 0C) in 250 ml Soxhtel loaded with 50 g of soil; the extraction phase was renewed every 12h. The total extract was dehydrated with anhydrous Na2SO4 evaporated under reduced pressure to approximately 50 ml, dried under N2 stream at room temperature (20-25 0C), and finally

The spectroscopy characteristics of the alkaline extract of both soil samples, the absorbance at different wavelength (280, 470 y 664 nm), were determined by the methodology proposed by Sapeck & Sapeck (1999). The ratio E2/E6, E4/E6 and E4/E6 were calculated from the corresponding absorbance value of the alkaline extract. The measures were determined

HA from burned and unburned soil were extracted with NaOH 0.1 mol L-1, purified with HCl:HF (1:3) and dried at low temperature until constant weight, according to the procedure recommended by Chen et al. (1978). All solutions were prepared with tridistilled

HA ash content was measured by heating it at 550 0C for 24 h. The elemental composition for C, H, N, S was determined by an analyzer instrument Carlo Erba 1108, using isothiourea as standard. Oxygen was calculated by difference: O%= 100 - (%C+%H+%N+%S) (ash and

The absorbance of the extracted HA were measured on a solution containing 3.0 mg of each HA in 10 mL of 0.05 mol L-1 NaHCO3 at different wavelength (280, 470 y 664 nm)

weighted, following the methodology proposed by Zancada et al., 2004.

mm sieves before all the analytical analysis

using a Perkin Elmer CN Elemental Analizer.

using Spectronic 20 Genesys Spectrophotometer.

water and all the reagents were ACS reagent grade.

**2.2.1 Humic Acids isolation** 

**2.2.2 Humic Acids analyses** 

**2.2.3 Spectroscopic characteristics** 

moisture-free basis).

(HA-UBS)

**2.2 Methods** 

Ciavatta et al., 1988).

capacity, to the nutrient availability, to the pH buffering and to the interaction with metal ions (Schnitzer 2000; Hayes & Malcom, 2001, Campitelli & Ceppi 2008)

Depending on the fire severity the organic matter may change not only their level, but also, their different fractions, i.e. the humic substances (humic and fulvic acids) content and their principal characteristics (Vergnoux et al., 2011a, 2011b; Duguy & Rovira 2010).

The fire could induce transformation in the solubility of humic substances in different media, alkali or acid, and of their fraction (humic and fulvic acids). Thus, it could means that the humic and fulvic acids suffer structural modification, provably of the peripheral chains and the oxygenated moieties.

The study of the burned soil is necessary to analyze the soil degradation, not only to estimate the modification of the nutrient content but either the physicochemical characteristics of the organic matter, specially analyzing the humic substances and their fractions.

Humic acids have an important role in soil structure and nutrient capacity due to their surface charge development which may change due to the fire event. Some researchers have mainly shown changes in the aromaticity and in the oxygen-containing functional groups (Almedros et al., 2003; Gonzalez-Vila & Almendros 2009; Kniker et al., 2005; Vergnoux et al., 2007).

Most of the researches are focusing in the nutrient content, carbon content and their fraction, the fate of nutrient after fire, the effect of erosion, in general, the effect of fire disturbance of the soil properties. Moreover, a single parameter is insufficient to give an accurate evaluation of soil alteration. That is why several parameters need to be taken into consideration (Vergnoux et al., 2009).

Because the abundance and importance in soil of the organic matter and their fraction, mainly the humic acids, is necessary to focus the study not only onto the principal nutrient content, but also, on the characteristics of the humic acids and compare it with those extracted from the same unburned soil.

The objective of this research was to study, through different analytical techniques, i) several soil parameters related to chemical soil fertility and ii) chemical and physicochemical properties of organic carbon and their different fractions, focusing mainly, on the humic acids extracted from the forest soil after the fire event in order to compare it with the humic acids from the unburned soil.

## **2. Materials**

#### **2.1 Study site and sampling**

The area selected was the south-west area of the province of Cordoba, named as San Agustín (Departamento de Calamuchita).

The annual average rainfall is about 600-800 mm. The mean temperature in the area fluctuates from 9 0C in winter to 20 0C in summer.

The tree stratum are principally *Pinus halepensis Mill.; Pinus elliottii* implanted and native vegetation cover is *Stipa caudata, Piptochaetium hackelii, P. napostaense y* Briza *subaristata*, between others. Three composite samples (10 subsamples) were taken from the upper layer (0-10 cm) of soil, some days after the wildfire occurrence and before any rainfall event. The samples were taken from the burned (BS) and adjacent unburned soil (UBS), at the same sampling moment. Litter and the ash were not removed from the soil surface before sampling in the unburned and burned soil, respectively. The soil was taxonomically characterized as Ustorthent. The samples were air-dried, crushed and passed through a 2 mm sieves before all the analytical analysis

The humic acids (HA) analyzed were extracted from the burned (HA-BS) and unburned soil (HA-UBS)

## **2.2 Methods**

68 Soil Health and Land Use Management

capacity, to the nutrient availability, to the pH buffering and to the interaction with metal

Depending on the fire severity the organic matter may change not only their level, but also, their different fractions, i.e. the humic substances (humic and fulvic acids) content and their

The fire could induce transformation in the solubility of humic substances in different media, alkali or acid, and of their fraction (humic and fulvic acids). Thus, it could means that the humic and fulvic acids suffer structural modification, provably of the peripheral chains

The study of the burned soil is necessary to analyze the soil degradation, not only to estimate the modification of the nutrient content but either the physicochemical characteristics of the organic matter, specially analyzing the humic substances and their

Humic acids have an important role in soil structure and nutrient capacity due to their surface charge development which may change due to the fire event. Some researchers have mainly shown changes in the aromaticity and in the oxygen-containing functional groups (Almedros et al., 2003; Gonzalez-Vila & Almendros 2009; Kniker et al., 2005; Vergnoux et al.,

Most of the researches are focusing in the nutrient content, carbon content and their fraction, the fate of nutrient after fire, the effect of erosion, in general, the effect of fire disturbance of the soil properties. Moreover, a single parameter is insufficient to give an accurate evaluation of soil alteration. That is why several parameters need to be taken into

Because the abundance and importance in soil of the organic matter and their fraction, mainly the humic acids, is necessary to focus the study not only onto the principal nutrient content, but also, on the characteristics of the humic acids and compare it with those

The objective of this research was to study, through different analytical techniques, i) several soil parameters related to chemical soil fertility and ii) chemical and physicochemical properties of organic carbon and their different fractions, focusing mainly, on the humic acids extracted from the forest soil after the fire event in order to compare it with the humic

The area selected was the south-west area of the province of Cordoba, named as San

The annual average rainfall is about 600-800 mm. The mean temperature in the area

The tree stratum are principally *Pinus halepensis Mill.; Pinus elliottii* implanted and native vegetation cover is *Stipa caudata, Piptochaetium hackelii, P. napostaense y* Briza *subaristata*, between others. Three composite samples (10 subsamples) were taken from the upper layer (0-10 cm) of soil, some days after the wildfire occurrence and before any rainfall event. The samples were taken from the burned (BS) and adjacent unburned soil (UBS), at the same sampling moment. Litter and the ash were not removed from the soil surface before

ions (Schnitzer 2000; Hayes & Malcom, 2001, Campitelli & Ceppi 2008)

and the oxygenated moieties.

consideration (Vergnoux et al., 2009).

extracted from the same unburned soil.

acids from the unburned soil.

**2.1 Study site and sampling** 

Agustín (Departamento de Calamuchita).

fluctuates from 9 0C in winter to 20 0C in summer.

**2. Materials** 

fractions.

2007).

principal characteristics (Vergnoux et al., 2011a, 2011b; Duguy & Rovira 2010).

The samples of burned and unburned soil were analyzed for pH at a rate 1:2.5 (w:v), electric conductivity (EC), total nitrogen content (TN) by the Kjeldahl method, phosphorus available (P) by Bray & Kurtz method (1945), total organic carbon (TOC) by combustion at 540 0C for 4 h (Abad, et al., 2002) and oxidable carbon (Cox) by the methodology proposed by de Richter & Von Wistinghausen (1981). Organic light fraction (OLF) were determined according the method proposed by Janzen et al., 1992, the C and N content of the OLF by dry combustion using a Perkin Elmer CN Elemental Analizer.

The carbon content of humic substances (CHS), humic acids (CHA) and fulvic acids (CFA) were determined according to the technique proposed by Syms & Haby 1971. The carbon content of each fraction (CHS, CHA and CFA) were calculated as percentage of the TOC, therefore, the % CHA correspond to the Humification Index (HI) (Roletto et al., 1985; Ciavatta et al., 1988).

The apolar or free lipidic fraction (FLF) were extracted with petroleum ether (40-60 0C) in 250 ml Soxhtel loaded with 50 g of soil; the extraction phase was renewed every 12h. The total extract was dehydrated with anhydrous Na2SO4 evaporated under reduced pressure to approximately 50 ml, dried under N2 stream at room temperature (20-25 0C), and finally weighted, following the methodology proposed by Zancada et al., 2004.

The spectroscopy characteristics of the alkaline extract of both soil samples, the absorbance at different wavelength (280, 470 y 664 nm), were determined by the methodology proposed by Sapeck & Sapeck (1999). The ratio E2/E6, E4/E6 and E4/E6 were calculated from the corresponding absorbance value of the alkaline extract. The measures were determined using Spectronic 20 Genesys Spectrophotometer.

#### **2.2.1 Humic Acids isolation**

HA from burned and unburned soil were extracted with NaOH 0.1 mol L-1, purified with HCl:HF (1:3) and dried at low temperature until constant weight, according to the procedure recommended by Chen et al. (1978). All solutions were prepared with tridistilled water and all the reagents were ACS reagent grade.

#### **2.2.2 Humic Acids analyses**

HA ash content was measured by heating it at 550 0C for 24 h. The elemental composition for C, H, N, S was determined by an analyzer instrument Carlo Erba 1108, using isothiourea as standard. Oxygen was calculated by difference: O%= 100 - (%C+%H+%N+%S) (ash and moisture-free basis).

#### **2.2.3 Spectroscopic characteristics**

The absorbance of the extracted HA were measured on a solution containing 3.0 mg of each HA in 10 mL of 0.05 mol L-1 NaHCO3 at different wavelength (280, 470 y 664 nm)

Fire Impact on Several Chemical and Physicochemical Parameters in a Forest Soil 71

The results of the principal chemical parameters are shown in table 1. The concentration of the cations, such as Na+ and K+ were not altered by fire, Ca2+ content slightly increase after fire, probably due to their release from the litter layer and Mg2+ decrease. The increase observed in the availability of Ca2+, may be remarkably in a fire event, but ephemerally

Sample **pH EC TN P TOC Cox TOC/TN Na K Ca Mg CIC**  Unburned soil (UBS) 6.20a 0.60a 6.6a 23.0a 105a 24.2a 15.9a 0.22a 1.15a 10.25a 2.25b 23.5a Burned soil (BS) 6.53a 1.19b 7.1a 52.4b 128b 24.9a 18.0b 0.22a 1.03a 11.1a 1.5a 23.6a EC: dSm-1; P: mg kg-1; TOC, Cox: g kg-1; CIC, Na, K, Ca Mg: cmol kg-1 Different letters (a–b) in the same

The effect of burning onto soil Total Nitrogen (TN) content present a paradox, which have been debated for years (Neary et al., 1999; Knicker & Skjemstad,, 2000). Fisher & Binkley (2000) found that the immediate response of soil N to heating is a decrement because some loss through volatilization; Certini (2005), suggested that organic N could be volatilizes and in part mineralized to ammonium. Santin et al. (2008) found that the TN after fire increase and González-Vila et al. (2009) suggest that wildfire promote the accumulation of

onto negative charge of mineral and/or organic surface, but with time transformed to NO3-

Nitrate, without any plant uptake, will be lost from the ecosystem either by denitrification

The TN content increased slightly in the burned soil, but this change is not statistically significant; this behavior may be due to the nitrogen supplied by the burned litter and/or

The forest fires have not necessarily the same impact on soil P as on N, because losses of P through volatilization or leaching are small. The combustion of vegetation and litter causes modification on biogeochemical cycle of P. Burning convert the organic pool of soil P to orthophosphate, which is the form of P available to biota. Furthermore, the peak of P bioavailability is around pH 6.5. These could be the reason for which an enrichment of P is observed in the studied burned soil, but this enrichment will decline soon, because it precipitates as slightly available mineral forms (Certini, 2005; Cade-Menun et al., 2000). In agreement with this suggestion, the increase in the available P content in this burned soil

Cation Exchange Capacity (CIC), on average, decrease after a fire event due to the loss of organic matter (Certini 2005; Badía & Martí, 2003), in this soil CIC was not changed,

In general the soil pH increase by soil heating as a result of organic acids denaturalization, this increase take place when the temperatures are higher than 450 or 5000C, in coincidence with the complete combustion of fuel and the bases release (Arocena & Opio 2003; Knicker,

+ or NO3- , the NH4

+ could be adsorbed

.

column indicate significant differences (p<0.05) according to Tukey test.

recalcitrant organic-N forms. The N, would be as NH4

or leaching (Certini, 2005; Knicker, 2007).

could be due to the soil pH value (table 1).

probably because the Cox content is the same before and after fire.

the ash contained in the sample.

Table 1. Principal chemical characteristic of burned and unburned soil

**3. Results and discussion** 

**3.1 Soil characterization 3.1.1 Main properties** 

(Certini, 2005).

according the methodology proposed by Kononova, 1982; Zbytniewski & Buszewski, 2005; Sellami et al., 2008. From the absorbance value then were calculated the E2/E4, E2/E6 and E4/E6 ratio

#### **2.2.4 Potentiometric titration**

Potentiometric titrations were carried out according to the technique proposed by Campitelli et al. (2003), which is briefly: HA solution of each samples were prepared by dissolving HA (≈ 50 mg) with minimum volume of NaOH solution (0.1 mol L-1) and adding water up to the final volume (50 ml). An aliquot containing the desired amount of HA (≈7–8 mg) was transferred to the titration flask containing 10 mL of tridistilled water. The titrant (HCl =0.05 mol L-1) was added from an automatic burette (Schott Geräte T80/20) at a titrant rate of 0.1 ml/40 s. This rate was chosen taking into consideration that the variation of pH values should range between 0.02 and 0.04 pH units. The pH values were measured with an Orion Research 901 pH meter equipped with a glass-combined electrode (Orion 9103 BN). All titrations were performed in KCl 0.01 mol L-1 as background electrolyte. The same titration was followed in absence of HA (reference or blank titration) for each titration curve, in order to subtracts it from the raw data titration, and thus obtain the charge developed by the HA sample. Each HA solution, with the corresponding blank solution, was titrated by triplicate and the reported data representing the average values. All the reagents were ACS reagent grade.

#### **2.2.5 Capillary zone electrophoresis**

Capillary zone electrophoresis (CZE) experiments were performed on an Agilent Technology Capillary electrophoresis system equipped with a diode array. Operation of the instrument, data collection and analysis were controlled by Agilent ChemStation software. The polarity was negative, voltage of -30kV, temperature 25 0C, total run time 30 min (for time migration higher than 30 min no significant peak were observed). The samples were injected hydrodynamically using pressure of 5000 Pa for 20 s. The absorbance was monitored at four different wavelengths (210 nm, 230 nm, 260 nm and 450nm) and 260 nm was selected to report.

Each HA electropherogram was carried out by triplicate and the reported data representing the average values. The dimensions of the fused-silica capillary were 75 µm internal diameter; 73.0 cm total length and 64.5 cm effective length. All the solutions and background electrolyte (BGE) were prepared from analytical (p.a. or HPLC) chemicals and ultra pure water. BGE was buffer borate 20 mmol L-1 at pH=9.3, the concentration of the HA solutions were 1000 ppm. At the beginning of daily work, the capillary was washed for 5 min with 0.1 mol L-1 NaOH solutions, followed by 5 min washing with ultra pure water and 20 min with BGE at 25oC and 104 Pa. At the end of the daily work, the capillary was rinsed with BGE for 5 min and water for 10 min, at the same temperature and pressure condition.

The capillary was treated before each sampling injection, as following, pre-condition: 2 min with NaOH 0.1 mol L-1 at 104 Pa, followed by washing with BGE for 3 min at 104 Pa, and finally waiting for 1 min. Post-run conditions were: 1 min with NaOH 0.1 mol L-1 at 104 Pa, followed by 5 min with water at the same pressure.

## **3. Results and discussion**

## **3.1 Soil characterization**

#### **3.1.1 Main properties**

70 Soil Health and Land Use Management

according the methodology proposed by Kononova, 1982; Zbytniewski & Buszewski, 2005; Sellami et al., 2008. From the absorbance value then were calculated the E2/E4, E2/E6 and

Potentiometric titrations were carried out according to the technique proposed by Campitelli et al. (2003), which is briefly: HA solution of each samples were prepared by dissolving HA (≈ 50 mg) with minimum volume of NaOH solution (0.1 mol L-1) and adding water up to the final volume (50 ml). An aliquot containing the desired amount of HA (≈7–8 mg) was transferred to the titration flask containing 10 mL of tridistilled water. The titrant (HCl =0.05 mol L-1) was added from an automatic burette (Schott Geräte T80/20) at a titrant rate of 0.1 ml/40 s. This rate was chosen taking into consideration that the variation of pH values should range between 0.02 and 0.04 pH units. The pH values were measured with an Orion Research 901 pH meter equipped with a glass-combined electrode (Orion 9103 BN). All titrations were performed in KCl 0.01 mol L-1 as background electrolyte. The same titration was followed in absence of HA (reference or blank titration) for each titration curve, in order to subtracts it from the raw data titration, and thus obtain the charge developed by the HA sample. Each HA solution, with the corresponding blank solution, was titrated by triplicate and the reported data representing the average values. All the reagents were ACS

Capillary zone electrophoresis (CZE) experiments were performed on an Agilent Technology Capillary electrophoresis system equipped with a diode array. Operation of the instrument, data collection and analysis were controlled by Agilent ChemStation software. The polarity was negative, voltage of -30kV, temperature 25 0C, total run time 30 min (for time migration higher than 30 min no significant peak were observed). The samples were injected hydrodynamically using pressure of 5000 Pa for 20 s. The absorbance was monitored at four different wavelengths (210 nm, 230 nm, 260 nm and 450nm) and 260 nm

Each HA electropherogram was carried out by triplicate and the reported data representing the average values. The dimensions of the fused-silica capillary were 75 µm internal diameter; 73.0 cm total length and 64.5 cm effective length. All the solutions and background electrolyte (BGE) were prepared from analytical (p.a. or HPLC) chemicals and ultra pure water. BGE was buffer borate 20 mmol L-1 at pH=9.3, the concentration of the HA solutions were 1000 ppm. At the beginning of daily work, the capillary was washed for 5 min with 0.1 mol L-1 NaOH solutions, followed by 5 min washing with ultra pure water and 20 min with BGE at 25oC and 104 Pa. At the end of the daily work, the capillary was rinsed with BGE for 5 min and water for 10 min, at the same temperature and pressure

The capillary was treated before each sampling injection, as following, pre-condition: 2 min with NaOH 0.1 mol L-1 at 104 Pa, followed by washing with BGE for 3 min at 104 Pa, and finally waiting for 1 min. Post-run conditions were: 1 min with NaOH 0.1 mol L-1 at 104 Pa,

E4/E6 ratio

reagent grade.

was selected to report.

condition.

**2.2.5 Capillary zone electrophoresis** 

followed by 5 min with water at the same pressure.

**2.2.4 Potentiometric titration** 

The results of the principal chemical parameters are shown in table 1. The concentration of the cations, such as Na+ and K+ were not altered by fire, Ca2+ content slightly increase after fire, probably due to their release from the litter layer and Mg2+ decrease. The increase observed in the availability of Ca2+, may be remarkably in a fire event, but ephemerally (Certini, 2005).


EC: dSm-1; P: mg kg-1; TOC, Cox: g kg-1; CIC, Na, K, Ca Mg: cmol kg-1 Different letters (a–b) in the same column indicate significant differences (p<0.05) according to Tukey test.

Table 1. Principal chemical characteristic of burned and unburned soil

The effect of burning onto soil Total Nitrogen (TN) content present a paradox, which have been debated for years (Neary et al., 1999; Knicker & Skjemstad,, 2000). Fisher & Binkley (2000) found that the immediate response of soil N to heating is a decrement because some loss through volatilization; Certini (2005), suggested that organic N could be volatilizes and in part mineralized to ammonium. Santin et al. (2008) found that the TN after fire increase and González-Vila et al. (2009) suggest that wildfire promote the accumulation of recalcitrant organic-N forms. The N, would be as NH4 + or NO3- , the NH4 + could be adsorbed onto negative charge of mineral and/or organic surface, but with time transformed to NO3- . Nitrate, without any plant uptake, will be lost from the ecosystem either by denitrification or leaching (Certini, 2005; Knicker, 2007).

The TN content increased slightly in the burned soil, but this change is not statistically significant; this behavior may be due to the nitrogen supplied by the burned litter and/or the ash contained in the sample.

The forest fires have not necessarily the same impact on soil P as on N, because losses of P through volatilization or leaching are small. The combustion of vegetation and litter causes modification on biogeochemical cycle of P. Burning convert the organic pool of soil P to orthophosphate, which is the form of P available to biota. Furthermore, the peak of P bioavailability is around pH 6.5. These could be the reason for which an enrichment of P is observed in the studied burned soil, but this enrichment will decline soon, because it precipitates as slightly available mineral forms (Certini, 2005; Cade-Menun et al., 2000). In agreement with this suggestion, the increase in the available P content in this burned soil could be due to the soil pH value (table 1).

Cation Exchange Capacity (CIC), on average, decrease after a fire event due to the loss of organic matter (Certini 2005; Badía & Martí, 2003), in this soil CIC was not changed, probably because the Cox content is the same before and after fire.

In general the soil pH increase by soil heating as a result of organic acids denaturalization, this increase take place when the temperatures are higher than 450 or 5000C, in coincidence with the complete combustion of fuel and the bases release (Arocena & Opio 2003; Knicker,

Fire Impact on Several Chemical and Physicochemical Parameters in a Forest Soil 73

incompletely burnt plants necromass, or a post-fire enhancement of the litter from decaying fire-affected vegetation production (Knicker et al., 2005a; 2007; Gonzalez-Perez et al., 2004;

The light fraction (LF) content (Table 2), which represents all residues, with a density value lower than 1.7 g ml-1, on the top soil before and after the wildfire event, could be the reason for the high value of the TOC observed. This fraction (LF) increases after fire in the same way as the TOC, around 28%; which represent one possible source of organic material (incomplete burnt plants) that would be incorporated to the native pool of soil organic matter and thus a way to a progressive stabilization of the different organic compounds produced by the fire effect, such as, aliphatic compounds, polysaccharides, peptides of plant and microbial origin and other organic compound generated by fire. The carbon content slightly increases and nitrogen content decreases significantly (≈ 27%) after fire in the LF. The C/N ratio indicate that this fraction is formed by an unstable organic fraction, composed by debris with incomplete combustion, thus, it could produce a nitrogen immobilization during the stabilization and the incorporation to the native soil organic

The carbon content of each fraction (CHS, CHA and CFA) were calculated as a percentage of the TOC, therefore, the % CHA correspond to the Humification Index (HI) (Roletto et al.,

Vergnoux et al.(2011a, 2011b), found that the different fraction of the humic substances

Gonzalez-Perez et al.(2004); Kincker et al.(2005). Other studies suggest that during the wildfire a humic-like fraction can be produced from burned plant biomass and thus it would be extractable in alkaline solution. In general, medium heating, i.e. temperatures not higher than 2500C, leads to increase complexity of the organic matter: newly formed compounds, oxidation and thermal fixation of alkyl moieties, etc. (Almendros et al., 1992;

The organic carbon content of each fraction (CHS, CHA, and CFA) of the burned and

Unburned soil (UBS) 1.92a 0.68a 1.23b 1.80b 56.7a 1.54b 17.35a 11.2a 0.24a Burned soil (BS) 1.84a 0.79b 1.06a 1.34a 66.9b 1.12a 18.37a 16.4b 0.39b CHS, CFA, CHA, LF and FLF: expressed as % in function of 100 g of TOC Different letters (a–b) in the

Table 2. Carbon content in each humic substances fraction (CHS, CFA, and CHA), carbon light fraction content (LF), nitrogen and carbon content in the carbon light fraction, and the

The variation in the CHS content after the wildfire is not statistically significant; this could be due to the original humic materials transformations into an alkali-insoluble macromolecule material (Gonzalez-Perez et al., 2004; Fernandez et al., 2004), which is in agreement with the amount of hydrophobic fraction (FLF) found in both soil samples (table 2).

same column indicate significant differences (p< 0.05) according to Tukey test.

free lipidic fraction (FLF) content in burned and unburned soil

**Sample CHS CFA CHA(HI) CHA/CFA LF N% C% C/N FLF** 

decrease after fire, in agreement with Almendros et al.(1990); Fernandez et al.(1997);

Santin et al., 2008)

matter.

**3.2 Organic matter fractions analysis** 

1985; Ciavatta et al., 1988).

Gonzalez-Perez et al., 2004).

unburned soil is shown in table 2.

2007; Certini 2005). For the soil analyzed, the increasing observed in soil pH after the fire event was slight (around 5%), this is in agreement with the cation (Na, K, Ca, Mg) content which were not largely modified by the heating soil, suggesting that the temperature did not raise up to 4500C or greater.

The electric conductivity (EC) increase in the burned soil, it could be assigned to the release of inorganic ions from the combusted organic matter present as litter or ash; this increase could be temporary (Kutiel & Imbar 1993; Hernandez, et al.,1997; Certini, 2005).

## **3.1.2 Organic matter**

The most intuitive expected change in the soils during a fire event is the loss of organic matter. This change depends on the fire severity, vegetation type, soil texture and even slope. The impact on the organic matter consist of slight distillation (volatilization of minor constituents), charring or complete oxidation. Substantial consumption of organic matter begins in the 200-250 0C range to complete at around 450-500 0C (Fernandez et al., 1997; Giovannini et al., 1988; Certini, 2005; Knicker, 2007).

The influence of fire on the organic matter content have been reported a wide range of effects, showing even contrasting results (Gonzalez-Perez et al., 2004; Czimczik et al., 2005, Dai et al., 2005; Knicker et al., 2005; Alexis et al., 2007)

The oxidable organic carbon (Cox) content was not altered by fire, but, total organic carbon (TOC) increase around 21%, this behavior could be attributed to the accumulation of recalcitrant hydrophobic fraction of organic matter (Gonzalez-Perez et al., 2004; Santin et al., 2008).

The organic fraction extracted with petroleum ether, the soil free lipids, represents a diverse group of hydrophobic substances ranging from simple compounds such as fatty acids, to more complex substances as sterols, terpenes, polynuclear hydrocarbons, chlorophylls, fats, waxes and resins. The hydrophobic fraction extracted (FLF) from the sample after fire was greater (≈ 38%) than that quantified for the sample of the control soil (table 2), in agreement with those found by Almendros et al. (1988), for a soil under *Pinus pinea*. Although, such compounds occur in fire unaffected soil, their abundance is increased by fire due to greater stability of lipids and lignin derivatives but also due to the neoformation of aromatic polymers (Almendros et al., 2003; Fernandez et al., 2004; Knicker et al., 2005a).

The high TOC content before and after fire event could be due to the sampling methodology, taking the soil sample with all the litter and grass in soil before fire and litter from decaying fire affected vegetation. The increase in the TOC content suggests that this fire event contributes to an enhancement of the organic matter, through the incomplete combust vegetation and thus contributes to a soil TOC increase. With the time residence in the soil this unstable organic matter could be incorporated to the stable pool of organic matter, this behavior is related to the process of accumulation of organic compounds in soil controlled by their chemical affinity with the native organic matter. The randomness of the process and the heterogeneity of the organic molecules, probable produced by fire, lead to the accumulation of organic matter in which hydrophilic association may be contiguous with hydrophobic domains or contained in one other, and thus the native organic matter pool could behave as sink of the decaying fire affected vegetation. (Spaccini et al., 2000; Santin et al., 2008; Gonzales-Perez et al., 2004; Knicker et al., 2005).

The increase in the TOC/TN ratio (Table 1) after the fire event is due, principally, to the TOC increase more than to the TN change after fire. This could confirm the accumulation of incompletely burnt plants necromass, or a post-fire enhancement of the litter from decaying fire-affected vegetation production (Knicker et al., 2005a; 2007; Gonzalez-Perez et al., 2004; Santin et al., 2008)

The light fraction (LF) content (Table 2), which represents all residues, with a density value lower than 1.7 g ml-1, on the top soil before and after the wildfire event, could be the reason for the high value of the TOC observed. This fraction (LF) increases after fire in the same way as the TOC, around 28%; which represent one possible source of organic material (incomplete burnt plants) that would be incorporated to the native pool of soil organic matter and thus a way to a progressive stabilization of the different organic compounds produced by the fire effect, such as, aliphatic compounds, polysaccharides, peptides of plant and microbial origin and other organic compound generated by fire. The carbon content slightly increases and nitrogen content decreases significantly (≈ 27%) after fire in the LF. The C/N ratio indicate that this fraction is formed by an unstable organic fraction, composed by debris with incomplete combustion, thus, it could produce a nitrogen immobilization during the stabilization and the incorporation to the native soil organic matter.

#### **3.2 Organic matter fractions analysis**

72 Soil Health and Land Use Management

2007; Certini 2005). For the soil analyzed, the increasing observed in soil pH after the fire event was slight (around 5%), this is in agreement with the cation (Na, K, Ca, Mg) content which were not largely modified by the heating soil, suggesting that the temperature did

The electric conductivity (EC) increase in the burned soil, it could be assigned to the release of inorganic ions from the combusted organic matter present as litter or ash; this increase

The most intuitive expected change in the soils during a fire event is the loss of organic matter. This change depends on the fire severity, vegetation type, soil texture and even slope. The impact on the organic matter consist of slight distillation (volatilization of minor constituents), charring or complete oxidation. Substantial consumption of organic matter begins in the 200-250 0C range to complete at around 450-500 0C (Fernandez et al., 1997;

The influence of fire on the organic matter content have been reported a wide range of effects, showing even contrasting results (Gonzalez-Perez et al., 2004; Czimczik et al., 2005,

The oxidable organic carbon (Cox) content was not altered by fire, but, total organic carbon (TOC) increase around 21%, this behavior could be attributed to the accumulation of recalcitrant hydrophobic fraction of organic matter (Gonzalez-Perez et al., 2004; Santin et al.,

The organic fraction extracted with petroleum ether, the soil free lipids, represents a diverse group of hydrophobic substances ranging from simple compounds such as fatty acids, to more complex substances as sterols, terpenes, polynuclear hydrocarbons, chlorophylls, fats, waxes and resins. The hydrophobic fraction extracted (FLF) from the sample after fire was greater (≈ 38%) than that quantified for the sample of the control soil (table 2), in agreement with those found by Almendros et al. (1988), for a soil under *Pinus pinea*. Although, such compounds occur in fire unaffected soil, their abundance is increased by fire due to greater stability of lipids and lignin derivatives but also due to the neoformation of aromatic

The high TOC content before and after fire event could be due to the sampling methodology, taking the soil sample with all the litter and grass in soil before fire and litter from decaying fire affected vegetation. The increase in the TOC content suggests that this fire event contributes to an enhancement of the organic matter, through the incomplete combust vegetation and thus contributes to a soil TOC increase. With the time residence in the soil this unstable organic matter could be incorporated to the stable pool of organic matter, this behavior is related to the process of accumulation of organic compounds in soil controlled by their chemical affinity with the native organic matter. The randomness of the process and the heterogeneity of the organic molecules, probable produced by fire, lead to the accumulation of organic matter in which hydrophilic association may be contiguous with hydrophobic domains or contained in one other, and thus the native organic matter pool could behave as sink of the decaying fire affected vegetation. (Spaccini et al., 2000;

The increase in the TOC/TN ratio (Table 1) after the fire event is due, principally, to the TOC increase more than to the TN change after fire. This could confirm the accumulation of

polymers (Almendros et al., 2003; Fernandez et al., 2004; Knicker et al., 2005a).

Santin et al., 2008; Gonzales-Perez et al., 2004; Knicker et al., 2005).

could be temporary (Kutiel & Imbar 1993; Hernandez, et al.,1997; Certini, 2005).

not raise up to 4500C or greater.

Giovannini et al., 1988; Certini, 2005; Knicker, 2007).

Dai et al., 2005; Knicker et al., 2005; Alexis et al., 2007)

**3.1.2 Organic matter** 

2008).

The carbon content of each fraction (CHS, CHA and CFA) were calculated as a percentage of the TOC, therefore, the % CHA correspond to the Humification Index (HI) (Roletto et al., 1985; Ciavatta et al., 1988).

Vergnoux et al.(2011a, 2011b), found that the different fraction of the humic substances decrease after fire, in agreement with Almendros et al.(1990); Fernandez et al.(1997);

Gonzalez-Perez et al.(2004); Kincker et al.(2005). Other studies suggest that during the wildfire a humic-like fraction can be produced from burned plant biomass and thus it would be extractable in alkaline solution. In general, medium heating, i.e. temperatures not higher than 2500C, leads to increase complexity of the organic matter: newly formed compounds, oxidation and thermal fixation of alkyl moieties, etc. (Almendros et al., 1992; Gonzalez-Perez et al., 2004).

The organic carbon content of each fraction (CHS, CHA, and CFA) of the burned and unburned soil is shown in table 2.


CHS, CFA, CHA, LF and FLF: expressed as % in function of 100 g of TOC Different letters (a–b) in the same column indicate significant differences (p< 0.05) according to Tukey test.

Table 2. Carbon content in each humic substances fraction (CHS, CFA, and CHA), carbon light fraction content (LF), nitrogen and carbon content in the carbon light fraction, and the free lipidic fraction (FLF) content in burned and unburned soil

The variation in the CHS content after the wildfire is not statistically significant; this could be due to the original humic materials transformations into an alkali-insoluble macromolecule material (Gonzalez-Perez et al., 2004; Fernandez et al., 2004), which is in agreement with the amount of hydrophobic fraction (FLF) found in both soil samples (table 2).

Fire Impact on Several Chemical and Physicochemical Parameters in a Forest Soil 75

The values of the quotient E2/E6 and E4/E6 are around 20-30% greater for both fraction (CHS and CFA) in the burned soil than in the unburned (Table 3). This variation suggest that the temperatures reached during the fire event, probably around 250-3000C, produced some degree of disaggregation effect and also, the increasing in the quotient value could be due to the newly organic compounds produced by the litter and vegetal residues burned

**Sample E2/E4 E2/E6 E4/E6 E2/E4 E2/E6 E4/E6** 

 CHS CHS CHS CFA CFA CFA Unburned soil (UBS) 7.7a 0.67a 5.2a 28.65a 0.63a 18.1a Burned soil (BS) 7.5a 0.84b 6.33b 29.4a 0.81b 23.9b Different letters (a–b) in the same column indicate significant differences (p<0.05) according to Tukey

The greater content obtained for the CFA (table 2) is in agreement with the disaggregation

Elemental composition (ash and moisture-free basis) O/C, H/C (atomic ratios) and E2/E4, E2/E6 and E4/E6 ratio of the HA extracted from unburned and burned soil are shown in

The increase in the carbon content after fire could be produced by the incorporation of the incompletely burned necromass to the original supramolecular structure. The decrease in the oxygen content after fire suggests that the environment could have reducing

The atomic ratio of O/C and H/C are often used to monitor structural changes of humic

**Sample C H N O S O/C H/C E280 E460 E660 E4/E6**  HA-UBS 49.67a 5.46a 4.97a 39.46b < 0.4a 0.59b 1.32b 1.78a 0.34a 0.09a 3.77a HA-BS 53.89b 5.28a 4.93a 35.45a < 0.4a 0.49a 1.18a 2.15b 0.47b 0.12b 3.92a Different letters (a–b) in the same column indicate significant differences (p<0.05) according to Tukey

Table 4. Elemental composition (ash and moisture-free basis) O/C, H/C (atomic ratios) and

The decrease in the atomic H/C ratio observed for HA-BS, suggest a diminution in the peripheral aliphatic chains with low thermal stability and thus, an increase in the aromaticity because this domains was found resistant to the effects of fire. The decrease in the O/C ratio indicates a substantial loss of oxygen-containing functional groups. The mains change observed in HA heated in laboratory or in natural fire are the dehydration and

Table 3. Alkaline extracts Absorbance ratio of burned and unburned soil samples

observed through the E2/E6 and E4/E6 values after the fire event.

substances (Gonzalez-Perez et al., 2004; Adani et al., 2006).

E2/E4, E2/E6 and E4/E6 ratio of the HA studied

**3.4 Humic Acids characterization 3.4.1 Elemental composition** 

during the wildfire.

test.

Table 4.

properties.

test.

The CFA increases around 15% after fire and CHA decrease around 12% in the soil exposed to high temperatures. The increase of the CFA content indicate the newly formed compounds, with more aliphatic chains, in general, with less molecular size, produced by the breakup of the more aggregated structures of the humic acids and thus, the carbon humic fraction decrease. The Humification Index (HI) (Table 2) is reduced about 12% indicating, also, the alteration in the humic substances by wildfire.

The ratio CHA/CFA (Table 2), also known as "degree of polymerization or polymerization index", decrease around 25% in the burned soil, reflecting the breakdown of the complex and more aggregated structures of unheated soil humic fraction, indicating that the wildfire lead to an important change in the structure and the properties of the humic substances fraction (Debano et al., 2000; Shakeesby &Doerr 2006).

## **3.3 Spectroscopic properties of soil alkaline extracts**

The scattering of monochromatic light in a diluted solution of macromolecules or colloidal particles is closely related to weight, size, aggregation and interaction of particles in solution. The UV-Visible absorption of humic substances was used to evaluate the condensation degree of the aromatic compounds (Chen el al., 1977; Stevenson, 1982; Polak et al., 2009).

Sutton & Sposito (2005), suggest that the apparent size of humic materials do not change due to tight coiling (or uncoiling), but instead change due to disaggregation (or aggregation) of clusters of small molecules.

The absorption at 280 nm was also introduced to represent total aromaticity, because the ππ\* electron transition occurs in this UV region, for phenolic arenes, benzoic acids, aniline derivatives polyenes and polycyclic aromatic hydrocarbon with two or more rings (Uyguner & Bekbolet, 2005).

The absorption at 470 nm is related with the fragment produced for the depolimerization or disaggregation of the supramolecular structure or material with a low humification degree (Sellami et al., 2008; Zbytniewski & Buszewski, 2004).

The absorbance at 664 nm is characteristics of high oxygen content, aromatic compound, strongly humified material with a high degree of condensed groups (Sellami et al., 2008).

Lipski et al.(1999) defined E2/E4 ratio (the ratio of absorbance at 280 and 400 nm) to characterize the degradation of phenolic/quinoid core of humic acids to simpler carboxylic aromatic compounds. This ratio may represent an alternative parameter for the elucidation of the photocatalytic degradation efficiency.

The value of the quotient E4/E6 (the ratio of absorbance at 400 and 665nm) and E2/E6 (the ratio of absorbance 280 and 665 nm) coefficient are related with aromatic condensation; suggest the aggregation level, phenolic and benzene-carboxylic group content, among other characteristics. A low ratio reflects a high degree of aromaticity, aggregation and high humification level; large values are associated with the presence of smaller size organic molecules, more aliphatic structures, high content of functional groups, high disaggregation level (Chen et al., 1977; Pertusati & Prado, 2007, Zbytniewski & Buszewski, 2004).

The value of the coefficient E2/E4 for CHS and CFA (Table 3) obtained in the alkaline extracts for the burned and unburned soil, don't have a great variation, suggesting that the degradation of core structure of humic substances, depolymerization or the disaggregation of the supramolecular structure was not significant, probably several aggregate disruption was produced by heating the soil (Uyguner & Bekbolet, 2005; Sutton & Sposito, 2005).

The values of the quotient E2/E6 and E4/E6 are around 20-30% greater for both fraction (CHS and CFA) in the burned soil than in the unburned (Table 3). This variation suggest that the temperatures reached during the fire event, probably around 250-3000C, produced some degree of disaggregation effect and also, the increasing in the quotient value could be due to the newly organic compounds produced by the litter and vegetal residues burned during the wildfire.


Different letters (a–b) in the same column indicate significant differences (p<0.05) according to Tukey test.

Table 3. Alkaline extracts Absorbance ratio of burned and unburned soil samples

The greater content obtained for the CFA (table 2) is in agreement with the disaggregation observed through the E2/E6 and E4/E6 values after the fire event.

## **3.4 Humic Acids characterization**

#### **3.4.1 Elemental composition**

74 Soil Health and Land Use Management

The CFA increases around 15% after fire and CHA decrease around 12% in the soil exposed to high temperatures. The increase of the CFA content indicate the newly formed compounds, with more aliphatic chains, in general, with less molecular size, produced by the breakup of the more aggregated structures of the humic acids and thus, the carbon humic fraction decrease. The Humification Index (HI) (Table 2) is reduced about 12%

The ratio CHA/CFA (Table 2), also known as "degree of polymerization or polymerization index", decrease around 25% in the burned soil, reflecting the breakdown of the complex and more aggregated structures of unheated soil humic fraction, indicating that the wildfire lead to an important change in the structure and the properties of the humic substances

The scattering of monochromatic light in a diluted solution of macromolecules or colloidal particles is closely related to weight, size, aggregation and interaction of particles in solution. The UV-Visible absorption of humic substances was used to evaluate the condensation degree of the aromatic compounds (Chen el al., 1977; Stevenson, 1982; Polak et

Sutton & Sposito (2005), suggest that the apparent size of humic materials do not change due to tight coiling (or uncoiling), but instead change due to disaggregation (or aggregation)

The absorption at 280 nm was also introduced to represent total aromaticity, because the ππ\* electron transition occurs in this UV region, for phenolic arenes, benzoic acids, aniline derivatives polyenes and polycyclic aromatic hydrocarbon with two or more rings

The absorption at 470 nm is related with the fragment produced for the depolimerization or disaggregation of the supramolecular structure or material with a low humification degree

The absorbance at 664 nm is characteristics of high oxygen content, aromatic compound, strongly humified material with a high degree of condensed groups (Sellami et al., 2008). Lipski et al.(1999) defined E2/E4 ratio (the ratio of absorbance at 280 and 400 nm) to characterize the degradation of phenolic/quinoid core of humic acids to simpler carboxylic aromatic compounds. This ratio may represent an alternative parameter for the elucidation

The value of the quotient E4/E6 (the ratio of absorbance at 400 and 665nm) and E2/E6 (the ratio of absorbance 280 and 665 nm) coefficient are related with aromatic condensation; suggest the aggregation level, phenolic and benzene-carboxylic group content, among other characteristics. A low ratio reflects a high degree of aromaticity, aggregation and high humification level; large values are associated with the presence of smaller size organic molecules, more aliphatic structures, high content of functional groups, high disaggregation

The value of the coefficient E2/E4 for CHS and CFA (Table 3) obtained in the alkaline extracts for the burned and unburned soil, don't have a great variation, suggesting that the degradation of core structure of humic substances, depolymerization or the disaggregation of the supramolecular structure was not significant, probably several aggregate disruption was produced by heating the soil (Uyguner & Bekbolet, 2005; Sutton & Sposito, 2005).

level (Chen et al., 1977; Pertusati & Prado, 2007, Zbytniewski & Buszewski, 2004).

indicating, also, the alteration in the humic substances by wildfire.

fraction (Debano et al., 2000; Shakeesby &Doerr 2006).

**3.3 Spectroscopic properties of soil alkaline extracts** 

(Sellami et al., 2008; Zbytniewski & Buszewski, 2004).

of the photocatalytic degradation efficiency.

al., 2009).

of clusters of small molecules.

(Uyguner & Bekbolet, 2005).

Elemental composition (ash and moisture-free basis) O/C, H/C (atomic ratios) and E2/E4, E2/E6 and E4/E6 ratio of the HA extracted from unburned and burned soil are shown in Table 4.

The increase in the carbon content after fire could be produced by the incorporation of the incompletely burned necromass to the original supramolecular structure. The decrease in the oxygen content after fire suggests that the environment could have reducing properties.

The atomic ratio of O/C and H/C are often used to monitor structural changes of humic substances (Gonzalez-Perez et al., 2004; Adani et al., 2006).


Different letters (a–b) in the same column indicate significant differences (p<0.05) according to Tukey test.

Table 4. Elemental composition (ash and moisture-free basis) O/C, H/C (atomic ratios) and E2/E4, E2/E6 and E4/E6 ratio of the HA studied

The decrease in the atomic H/C ratio observed for HA-BS, suggest a diminution in the peripheral aliphatic chains with low thermal stability and thus, an increase in the aromaticity because this domains was found resistant to the effects of fire. The decrease in the O/C ratio indicates a substantial loss of oxygen-containing functional groups. The mains change observed in HA heated in laboratory or in natural fire are the dehydration and

Fire Impact on Several Chemical and Physicochemical Parameters in a Forest Soil 77

Fig. 1. (a): Charge-pH curves of humic acids extracted from burned (HA-BS) and unburned

The charge development of HA isolated from the burned soil (HA-BS) is greater than for the humic acids extracted from unburned soil (HA-UBS) in the region of pH 6 to 11 and lower at the more acidic region (3 to 6). Total acidity is about 60% greater in the HA extracted from burned soil than those of the unburned soil. In the acidic pH region (3 to 6) the lower charge development for HA isolated from burned soil could be due to the loss of strong acidic sites

soil (HA-UBS). The charge developments were calculated on the basis of the sixth polynomial equation (with R2 values exceeding 0.999 in all cases). (Charge development were calculated taking into account the ash content); (b): Apparent proton-affinity distribution of humic acids extracted from from burned (HA-BS) and unburned soil (HA-UBS) obtained from the first derivatives through charge-pH curves [d(−Q)/d (pH)] smoothing with sixth degree polynomial equation through the experimental data in the

produced by the disruption of the supramolecular structure.

range of 3–10

decarboxyilation which explain the progressive alteration in the colloidal properties of soil affected by fire (Gonzalez-Perez et al., 2004).

#### **3.4.2 Spectroscopic properties**

UV-Visible spectra were recorded for both HA analyzed, the specific absorbance decreases steadily with increasing wavelength. The spectra are close to those presented in other studies related to the chemical nature of humic acids (Senesi et al., 1989; Fuentes et al., 2006). The absorption properties are conventional and versatile for the characterization and were used to evaluate the condensation degree of the humic aromatic nuclei. Various absorption wavelengths at 270, 280, 300, 400, 465 nm, among other, and their ratios have been cited for the spectral differentiation of humic substances (Sellami et al., 2008; Uyguner et al., 2005).

By analyzing the absorption spectrum of UV-Visible, three important regions were observed at 280, 460 and 660 nm. The absorbance at 280 nm (E280) is related to lignin, aniline derivatives, polyenes and polycyclic aromatic hydrocarbon with two or more rings (Uyguner & Bekbolet, 2005). The absorbance at 460 nm (E460) is the result of organic macromolecules with a low polymerization degree, and the absorbance at 660 nm (E660) is characteristic of high oxygen content, aromatic compound, high size and molecular weight (Sellami et al., 2008; Uyguner et al., 2005).

The absorbance of the HA extracted from the burned soil is greater than the absorbance of the HA isolated from the unburned soil, similar to those obtained for Vergnoux et al.(2011a). This behavior indicate that the HA isolated from the soil exposed to high temperatures have greater content of different fraction of organic compounds. The increase of the absorption at 280 nm (Table 4) indicate the presence of fraction like lignin derivatives and compounds with aliphatic chains; the absorption at 460 (Table 4) suggest the increment of compounds with a low polymerization degree or less condensed structural domains and the increment of the absorption at 660 nm (Table 4) suggest the increase of aromatic compounds with great microbial and /or chemical resistance, structures that have refractory character (Vergnoux et al. 2011a; Sellami et al., 2008; Santin et al 2008; Gonzalez-Perez et al., 2004).

The growth observed in the content of all these fractions could be due through the incorporation of the compounds produced by an incomplete combustion of the vegetation, and therefore, a considerable amount of newly formed C forms were adding together to the thermal modified C forms previously existing in the ecosystem (Cofer et al., 1997; Gonzalez-Perez et al., 2004). Through the E4/E6 value for both HA, burned and unburned HA, (3.92 and 3.77 respectively), in general, is possible to suppose that the nuclei of the macromolecule of HA, the aromaticity, the size, the weight were not disrupt by the temperature reached in this event fire, instead, the wildfire could have enough energy to produce a disruption onto the linkage which retain together the small fraction of the supramolecular structure and thus a disaggregation could take place; this behavior is shown through the increment of the absorbance values.

#### **3.4.3 Potentiometric titration: Acid base properties and charge evolution**

The charges-pH curves (-Q versus pH) of the HA isolated, between pH 3 and 11, obtained from potentiometric titration, corrected for blank solution and fitted with sixth degree polynomial according to Machesky (1993) and Campitelli & Ceppi (2008), are shown in the Figure 1a. This smoothing function was selected for their simplicity.

decarboxyilation which explain the progressive alteration in the colloidal properties of soil

UV-Visible spectra were recorded for both HA analyzed, the specific absorbance decreases steadily with increasing wavelength. The spectra are close to those presented in other studies related to the chemical nature of humic acids (Senesi et al., 1989; Fuentes et al., 2006). The absorption properties are conventional and versatile for the characterization and were used to evaluate the condensation degree of the humic aromatic nuclei. Various absorption wavelengths at 270, 280, 300, 400, 465 nm, among other, and their ratios have been cited for the spectral differentiation of humic substances (Sellami et al., 2008; Uyguner

By analyzing the absorption spectrum of UV-Visible, three important regions were observed at 280, 460 and 660 nm. The absorbance at 280 nm (E280) is related to lignin, aniline derivatives, polyenes and polycyclic aromatic hydrocarbon with two or more rings (Uyguner & Bekbolet, 2005). The absorbance at 460 nm (E460) is the result of organic macromolecules with a low polymerization degree, and the absorbance at 660 nm (E660) is characteristic of high oxygen content, aromatic compound, high size and molecular weight

The absorbance of the HA extracted from the burned soil is greater than the absorbance of the HA isolated from the unburned soil, similar to those obtained for Vergnoux et al.(2011a). This behavior indicate that the HA isolated from the soil exposed to high temperatures have greater content of different fraction of organic compounds. The increase of the absorption at 280 nm (Table 4) indicate the presence of fraction like lignin derivatives and compounds with aliphatic chains; the absorption at 460 (Table 4) suggest the increment of compounds with a low polymerization degree or less condensed structural domains and the increment of the absorption at 660 nm (Table 4) suggest the increase of aromatic compounds with great microbial and /or chemical resistance, structures that have refractory character (Vergnoux et

The growth observed in the content of all these fractions could be due through the incorporation of the compounds produced by an incomplete combustion of the vegetation, and therefore, a considerable amount of newly formed C forms were adding together to the thermal modified C forms previously existing in the ecosystem (Cofer et al., 1997; Gonzalez-Perez et al., 2004). Through the E4/E6 value for both HA, burned and unburned HA, (3.92 and 3.77 respectively), in general, is possible to suppose that the nuclei of the macromolecule of HA, the aromaticity, the size, the weight were not disrupt by the temperature reached in this event fire, instead, the wildfire could have enough energy to produce a disruption onto the linkage which retain together the small fraction of the supramolecular structure and thus a disaggregation could take place; this behavior is shown

The charges-pH curves (-Q versus pH) of the HA isolated, between pH 3 and 11, obtained from potentiometric titration, corrected for blank solution and fitted with sixth degree polynomial according to Machesky (1993) and Campitelli & Ceppi (2008), are shown in the

al. 2011a; Sellami et al., 2008; Santin et al 2008; Gonzalez-Perez et al., 2004).

**3.4.3 Potentiometric titration: Acid base properties and charge evolution** 

Figure 1a. This smoothing function was selected for their simplicity.

affected by fire (Gonzalez-Perez et al., 2004).

(Sellami et al., 2008; Uyguner et al., 2005).

through the increment of the absorbance values.

**3.4.2 Spectroscopic properties** 

et al., 2005).

Fig. 1. (a): Charge-pH curves of humic acids extracted from burned (HA-BS) and unburned soil (HA-UBS). The charge developments were calculated on the basis of the sixth polynomial equation (with R2 values exceeding 0.999 in all cases). (Charge development were calculated taking into account the ash content); (b): Apparent proton-affinity distribution of humic acids extracted from from burned (HA-BS) and unburned soil (HA-UBS) obtained from the first derivatives through charge-pH curves [d(−Q)/d (pH)] smoothing with sixth degree polynomial equation through the experimental data in the range of 3–10

The charge development of HA isolated from the burned soil (HA-BS) is greater than for the humic acids extracted from unburned soil (HA-UBS) in the region of pH 6 to 11 and lower at the more acidic region (3 to 6). Total acidity is about 60% greater in the HA extracted from burned soil than those of the unburned soil. In the acidic pH region (3 to 6) the lower charge development for HA isolated from burned soil could be due to the loss of strong acidic sites produced by the disruption of the supramolecular structure.

Fire Impact on Several Chemical and Physicochemical Parameters in a Forest Soil 79

in this way the carboxylic groups that remains in the surface are those with very strong acidic characteristics, probably those in the aromatic structures, like *o*-COOOH or in greater fractions, and the phenolic groups are those in the small fraction produced by the disaggregation (Table 5) (Knicker et al., 2007; Sharma et al., 2004). For both type of acidic groups (*o*-COOH and OH-Phenolic), the contribution could be from the partial combustion of vegetation and then extracted with the alkaline media, without discrimination (Adani et

*Humic acids* o-COOH pKaap -COOH pKaap phenolic-OH pKaap *HA-UBS* 320a 2.3 473 5.6 567a 11.2 *HA-BS* 588b 3.5 --- --- 1318b 10.8 Acidic groups: cmol kg-1 Different letters (a–b) in the same column indicate significant differences

Table 5. Acidic functional groups content (*o*-carboxylic, carboxilic and phenolic) content calculated by integration of the area under each maximum of the curves (d-Q/dpH) obtained through the first derivative of smoothed experimental data. The pKaap values

The fire event altered the concentration of acidic sites (Table 5) and therefore the buffer capacity. For the burned soil, the buffer capacity of HA was neglectable at soil pH value around 6 (Table 1) and for pH value ranging between ≈ 3 – 7. This can be attributed to the great heterogeneity of HA in this pH range and to the lost of carboxylic groups with pKaap

In the zone up to pH 8 (weak acidic sites) the buffer capacity is greater than that observed for HA from unburned soil, but this groups, in both cases, are not dissociated at soil pH values, thus they have not a significant contribution to the soil buffer capacity. The fire event produced important changes in the acid-base properties, principally in the buffer capacity of

The loss of carboxylic groups onto this HA structure produced by fire event (Table 5), i.e. the decrease of negative charge development below pH 6, cause a deficiency of charged site to make linkage between the inorganic and organic fraction through cation-bound; and thus, the formation of soil aggregates. In this way, this characteristic could be the key factor promoting soil erosion (Mill & Fey 2004). The fire event could generate important

At the lowest pH measured (sites domains below 4), the HA-UBS shows a developing peak (Fig 1b) indicating that very acidic sites could be present in the macromolecule, in HA-BS it seems that this sites are the only present (Table 5). The minimum around pH 4, which could be considered as a separation of both type of acidic sites (like COOH) from the very acidic sites (like o-COOH), is clearer in the HA extracted from unburned soil (HA-UBS) than in the HA from the burned soil (HA-BS), this indicate, also, the heterogeneity of the acidic groups present in the HA extracted from soil exposed to high temperatures, due to the

The main characteristics of HA are the occurrence of acidic site with different strength, the principal groups are the strong (carboxilic groups) and weak (phenolic groups) acidic site.

disaggregation produced by the temperature developed during the wildfire.

al., 2004).

(p<0.05) according to Tukey test.

values around 5.

the HA.

correspond to the maximum of each peak

modification in the physicochemical properties of the HA

**3.4.4 Capillary zone electrophoresis** 

The disaggregation produced by temperature could be the reason for the increment of the negative charge development up to pH 6, because the negative charged groups increase as the size of the fractions decrease (Tombacz, 1999). This behavior is in agreement with that observed through the spectroscopic analysis.

Through the first derivative of the –Q versus pH curves (-dQ/dpH) obtained from the titration curves smoothed with the polynomial equation (Figure 1b), is possible: i) to obtain the average of apparent proton-dissociation constant (pKaap) of each set of acidic groups, ii) to analyze the chemical heterogeneity of each class of acidic group present in the HA macromolecule, iii) to estimate the concentration of each set of acidic groups by the calculus of the area under each peak and iv) to estimate the buffer capacity developed by each class of acidic site (Nederlof et al., 1994; Koopal et al., 2005; Campitelli et al., 2006; Campitelli & Ceppi, 2008). In this way, is possible to follow how the acid-base characteristics, i.e, the evolution in quantity and quality for the principal acidic groups (carboxylic and phenolic), were changed for the fire event.

The number of site classes (set of acidic groups) is then equal to the number of peaks and the peak position could be used as an average of the apparent dissociation constant (pKaap)(de Wit et al.,1993)

The samples of HA extracted from unburned soil (HA-UBS) show two main peaks, the first would be assigned to the carboxylic groups (strong acidic sites) and the second to the phenolic groups (weak acidic sites). For the HA isolated from burned soil (HA-BS) the first peak is only a shoulder and the second peak is well defined. In both HA samples (HA-BS and HA-UBS), also is observed, a small or developing peaks at more acidic pH values (≤ 4), indicating, probably, a presence of stronger acidic sites; this behavior is more clear in HA-BS. This is in agreement with previous results obtained studying HA extracted from soil (Campitelli et al., 2006; Campitelli & Ceppi, 2008).

 HA isolated from burned soil (HA-BS) presents the first peaks or shoulder, not well defined, with the maxima at around pH 3.5 and the second with a maximum at pH 10.8; the first could be assigned to strong acid sites (carboxylic groups) and the second to weak acidic sites (phenolic groups). The peak at pH=3.5 was wider than the peak at pH=10.8. The minimum was not well defined, and the partial overlapping of peaks indicate that there is no significant differences among the acidic sites in the surface, in terms of proton dissociation strength. This results suggest a large chemical heterogeneity on the HA present or the production of small organic compounds during the fire event.

These small organic compounds could be produced by the incomplete combustion of the vegetation present; Knicker et al. (2007) suggested that around 250 0C new molecular structures are produced; the principal structures could be aliphatic C; phenol and/or furan C; Sharma et al. (2004) suggested that some decarboxylation could occur at higher temperatures (>2500C) but the aromatic rings still remain essentially intact. This behavior could justify the decrease in the negative charge development at pH values lower than 6 and their increase at higher pH values (pH > 6).

HA isolated from unburned soil (HA-UBS) have two well defined peaks, the first with the maximum at pH 5.6 and the second at pH 11.2, these values are similar to other obtained for soil derived humic acids (Campitelli et al., 2006; Campitelli & Ceppi 2008)

The pKaap for the carboxylic and phenolic groups in the HA derived from the burned soil (HA-BS) are lower than the corresponding for HA extracted from unburned soil (HA-UBS), this could be due to the disruption of the supramolecular structure of the humic acids, and

The disaggregation produced by temperature could be the reason for the increment of the negative charge development up to pH 6, because the negative charged groups increase as the size of the fractions decrease (Tombacz, 1999). This behavior is in agreement with that

Through the first derivative of the –Q versus pH curves (-dQ/dpH) obtained from the titration curves smoothed with the polynomial equation (Figure 1b), is possible: i) to obtain the average of apparent proton-dissociation constant (pKaap) of each set of acidic groups, ii) to analyze the chemical heterogeneity of each class of acidic group present in the HA macromolecule, iii) to estimate the concentration of each set of acidic groups by the calculus of the area under each peak and iv) to estimate the buffer capacity developed by each class of acidic site (Nederlof et al., 1994; Koopal et al., 2005; Campitelli et al., 2006; Campitelli & Ceppi, 2008). In this way, is possible to follow how the acid-base characteristics, i.e, the evolution in quantity and quality for the principal acidic groups (carboxylic and phenolic),

The number of site classes (set of acidic groups) is then equal to the number of peaks and the peak position could be used as an average of the apparent dissociation constant (pKaap)(de

The samples of HA extracted from unburned soil (HA-UBS) show two main peaks, the first would be assigned to the carboxylic groups (strong acidic sites) and the second to the phenolic groups (weak acidic sites). For the HA isolated from burned soil (HA-BS) the first peak is only a shoulder and the second peak is well defined. In both HA samples (HA-BS and HA-UBS), also is observed, a small or developing peaks at more acidic pH values (≤ 4), indicating, probably, a presence of stronger acidic sites; this behavior is more clear in HA-BS. This is in agreement with previous results obtained studying HA extracted from soil

 HA isolated from burned soil (HA-BS) presents the first peaks or shoulder, not well defined, with the maxima at around pH 3.5 and the second with a maximum at pH 10.8; the first could be assigned to strong acid sites (carboxylic groups) and the second to weak acidic sites (phenolic groups). The peak at pH=3.5 was wider than the peak at pH=10.8. The minimum was not well defined, and the partial overlapping of peaks indicate that there is no significant differences among the acidic sites in the surface, in terms of proton dissociation strength. This results suggest a large chemical heterogeneity on the HA present

These small organic compounds could be produced by the incomplete combustion of the vegetation present; Knicker et al. (2007) suggested that around 250 0C new molecular structures are produced; the principal structures could be aliphatic C; phenol and/or furan C; Sharma et al. (2004) suggested that some decarboxylation could occur at higher temperatures (>2500C) but the aromatic rings still remain essentially intact. This behavior could justify the decrease in the negative charge development at pH values lower than 6 and

HA isolated from unburned soil (HA-UBS) have two well defined peaks, the first with the maximum at pH 5.6 and the second at pH 11.2, these values are similar to other obtained for

The pKaap for the carboxylic and phenolic groups in the HA derived from the burned soil (HA-BS) are lower than the corresponding for HA extracted from unburned soil (HA-UBS), this could be due to the disruption of the supramolecular structure of the humic acids, and

observed through the spectroscopic analysis.

(Campitelli et al., 2006; Campitelli & Ceppi, 2008).

their increase at higher pH values (pH > 6).

or the production of small organic compounds during the fire event.

soil derived humic acids (Campitelli et al., 2006; Campitelli & Ceppi 2008)

were changed for the fire event.

Wit et al.,1993)

in this way the carboxylic groups that remains in the surface are those with very strong acidic characteristics, probably those in the aromatic structures, like *o*-COOOH or in greater fractions, and the phenolic groups are those in the small fraction produced by the disaggregation (Table 5) (Knicker et al., 2007; Sharma et al., 2004). For both type of acidic groups (*o*-COOH and OH-Phenolic), the contribution could be from the partial combustion of vegetation and then extracted with the alkaline media, without discrimination (Adani et al., 2004).


Acidic groups: cmol kg-1 Different letters (a–b) in the same column indicate significant differences (p<0.05) according to Tukey test.

Table 5. Acidic functional groups content (*o*-carboxylic, carboxilic and phenolic) content calculated by integration of the area under each maximum of the curves (d-Q/dpH) obtained through the first derivative of smoothed experimental data. The pKaap values correspond to the maximum of each peak

The fire event altered the concentration of acidic sites (Table 5) and therefore the buffer capacity. For the burned soil, the buffer capacity of HA was neglectable at soil pH value around 6 (Table 1) and for pH value ranging between ≈ 3 – 7. This can be attributed to the great heterogeneity of HA in this pH range and to the lost of carboxylic groups with pKaap values around 5.

In the zone up to pH 8 (weak acidic sites) the buffer capacity is greater than that observed for HA from unburned soil, but this groups, in both cases, are not dissociated at soil pH values, thus they have not a significant contribution to the soil buffer capacity. The fire event produced important changes in the acid-base properties, principally in the buffer capacity of the HA.

The loss of carboxylic groups onto this HA structure produced by fire event (Table 5), i.e. the decrease of negative charge development below pH 6, cause a deficiency of charged site to make linkage between the inorganic and organic fraction through cation-bound; and thus, the formation of soil aggregates. In this way, this characteristic could be the key factor promoting soil erosion (Mill & Fey 2004). The fire event could generate important modification in the physicochemical properties of the HA

At the lowest pH measured (sites domains below 4), the HA-UBS shows a developing peak (Fig 1b) indicating that very acidic sites could be present in the macromolecule, in HA-BS it seems that this sites are the only present (Table 5). The minimum around pH 4, which could be considered as a separation of both type of acidic sites (like COOH) from the very acidic sites (like o-COOH), is clearer in the HA extracted from unburned soil (HA-UBS) than in the HA from the burned soil (HA-BS), this indicate, also, the heterogeneity of the acidic groups present in the HA extracted from soil exposed to high temperatures, due to the disaggregation produced by the temperature developed during the wildfire.

#### **3.4.4 Capillary zone electrophoresis**

The main characteristics of HA are the occurrence of acidic site with different strength, the principal groups are the strong (carboxilic groups) and weak (phenolic groups) acidic site.

Fire Impact on Several Chemical and Physicochemical Parameters in a Forest Soil 81

The electropherogram of HA isolated from burned soil (HA-BS) presents the main peak at lower time migration (8.40 min) than in HA of unburned soil (HA-UBS), and several peaks are detected before and after the main peak (fig 2); at migration time higher than 25 min no

The peak at 11.73 min observed in the electropherogram corresponding to the HA-UBS, could indicate that in these experimental conditions the macromolecule migrate as a unbroken entity, the tailing observed at lower time migration, could be assigned to some structure with low mass/charge ratio difficult to be separated; i.e. the macromolecule is not easy to be separated in subfraction with different electrokinetic properties, similar behavior

The different time migration for the principal peak of the HA from burned soil (HA-BS) and the peaks detected at both side of the peak at 8.40 min could indicate changes in the

The BGE, borate, could react with phenols, phenols carboxylic, polycarboxilic acids, dihidroxy or perihydroxy groups present in the solution and thus the separation of each fraction would be improved ( Fetsch & Havel 1998). The phenolic groups present in HA isolated from burned soil (HA-BS) is greater than that quantified in HA extracted from unburned soil (HA-UBS), this characteristic could produce the interaction between the BGE

The electropherogram profile of the HA extracted from burned soil (AH-BS) indicates the presence of distinct subfraction, which could be produced by the disaggregation of the macromolecule of HA and/or the formation of newly small carbon compounds after heating, suggesting that the temperature reached during the fire event, breaks, disaggregates or creates new structure, with lower and higher mass/charge ratio and diverse electrokinetic mobility. This behavior confirms the large heterogeneity, the disaggregation and the new carbon compound produced for the wildfire and are in agreement with those observed through the other different analytical techniques used to

The temperature reached in the fire event was enough to produce several changes in the organic matter characteristics, i.e. changes in the quantity and/or quality of their fraction:

The fire event produced important changes in the structure of the macromolecule of humic acids, like break and/or disaggregation which generate compound with lower size, weight, mass/charge ratio and/or newly formed carbon compounds originated by the incomplete

The fire event could generate important modification in the physicochemical and acid-base

The amount of acidic functional group was changed: the COOH sites were decreased and the OH phenolic sites were increased by the fire event. The pKaap values were modified, in general, the acidic site are stronger after fire than in the unburned soil. The COOH groups with pKaap value about 5 were lost after fire. The buffer capacity is lower or practically

The negative charge development decrease significantly at field pH (≈ 6) after the fire event, producing a deficiency on sites to make linkage between the organic and inorganic soil

was observed for Fetch & Havel (1998); Pokorna et al.(2000); Peuravouri et al.(2004).

macromolecule structure and the presence of subfraction.

and this acidic groups and enhance the separation.

light fraction, humic acids, fulvic acids, free lipidic fraction.

peaks were distinguished.

study these HA.

**4. Conclusions** 

properties of the HA.

combustion of the vegetal materials.

missing at soil pH (≈ 6) after fire.

For these HA analyzed the average Pkaap value are around 3.5 – 5.5 for the carboxylic groups and 10 – 11 for the phenolic groups (Table 5).

Fig. 2. Electropherograms of acids extracted from burned (HA-BS) and unburned soil (HA-UBS) in buffer borate 20 mmol L-1 (pH=9.3), temperature 250C, the concentration of the AH solutions were 1000 ppm. CZE conditions: voltage of -30kV, injection hydrodynamic 5000 Pa for 20 s, detection at 260 nm, fused-silica capillary, 73 cm total length, 75 µm i. d. (effective length 64.5 cm). Total run time 30 min (for time migration higher than 30 min no significant peak were observed)

At the experimental condition (pH ≈ 9) all of the strong acidic groups and approximately, the half of the weak acidic groups of HA are deprotonated (negatively charged). The presence of negative charges permit to separate HA by electrophoresis in an electrical field (+) to (-) in which the EOF (electro osmotic flow) is responsible for the movement of the analyte (Peuravouri et al., 2004).

The electropherogram of HA extracted from unburned soil (HA-UBS) (fig 2) shows a principal and well defined peak at time migration 11.73 min and the characteristic hump at time migration around 7 – 8 min, just before the main peak, is shown as a tail; at migration time higher than 12 min no peaks were distinguished.

The electropherogram of HA isolated from burned soil (HA-BS) presents the main peak at lower time migration (8.40 min) than in HA of unburned soil (HA-UBS), and several peaks are detected before and after the main peak (fig 2); at migration time higher than 25 min no peaks were distinguished.

The peak at 11.73 min observed in the electropherogram corresponding to the HA-UBS, could indicate that in these experimental conditions the macromolecule migrate as a unbroken entity, the tailing observed at lower time migration, could be assigned to some structure with low mass/charge ratio difficult to be separated; i.e. the macromolecule is not easy to be separated in subfraction with different electrokinetic properties, similar behavior was observed for Fetch & Havel (1998); Pokorna et al.(2000); Peuravouri et al.(2004).

The different time migration for the principal peak of the HA from burned soil (HA-BS) and the peaks detected at both side of the peak at 8.40 min could indicate changes in the macromolecule structure and the presence of subfraction.

The BGE, borate, could react with phenols, phenols carboxylic, polycarboxilic acids, dihidroxy or perihydroxy groups present in the solution and thus the separation of each fraction would be improved ( Fetsch & Havel 1998). The phenolic groups present in HA isolated from burned soil (HA-BS) is greater than that quantified in HA extracted from unburned soil (HA-UBS), this characteristic could produce the interaction between the BGE and this acidic groups and enhance the separation.

The electropherogram profile of the HA extracted from burned soil (AH-BS) indicates the presence of distinct subfraction, which could be produced by the disaggregation of the macromolecule of HA and/or the formation of newly small carbon compounds after heating, suggesting that the temperature reached during the fire event, breaks, disaggregates or creates new structure, with lower and higher mass/charge ratio and diverse electrokinetic mobility. This behavior confirms the large heterogeneity, the disaggregation and the new carbon compound produced for the wildfire and are in agreement with those observed through the other different analytical techniques used to study these HA.

## **4. Conclusions**

80 Soil Health and Land Use Management

For these HA analyzed the average Pkaap value are around 3.5 – 5.5 for the carboxylic

Fig. 2. Electropherograms of acids extracted from burned (HA-BS) and unburned soil (HA-UBS) in buffer borate 20 mmol L-1 (pH=9.3), temperature 250C, the concentration of the AH solutions were 1000 ppm. CZE conditions: voltage of -30kV, injection hydrodynamic 5000 Pa for 20 s, detection at 260 nm, fused-silica capillary, 73 cm total length, 75 µm i. d. (effective length 64.5 cm). Total run time 30 min (for time migration higher than 30 min no

At the experimental condition (pH ≈ 9) all of the strong acidic groups and approximately, the half of the weak acidic groups of HA are deprotonated (negatively charged). The presence of negative charges permit to separate HA by electrophoresis in an electrical field (+) to (-) in which the EOF (electro osmotic flow) is responsible for the movement of the

The electropherogram of HA extracted from unburned soil (HA-UBS) (fig 2) shows a principal and well defined peak at time migration 11.73 min and the characteristic hump at time migration around 7 – 8 min, just before the main peak, is shown as a tail; at migration

groups and 10 – 11 for the phenolic groups (Table 5).

significant peak were observed)

analyte (Peuravouri et al., 2004).

time higher than 12 min no peaks were distinguished.

The temperature reached in the fire event was enough to produce several changes in the organic matter characteristics, i.e. changes in the quantity and/or quality of their fraction: light fraction, humic acids, fulvic acids, free lipidic fraction.

The fire event produced important changes in the structure of the macromolecule of humic acids, like break and/or disaggregation which generate compound with lower size, weight, mass/charge ratio and/or newly formed carbon compounds originated by the incomplete combustion of the vegetal materials.

The fire event could generate important modification in the physicochemical and acid-base properties of the HA.

The amount of acidic functional group was changed: the COOH sites were decreased and the OH phenolic sites were increased by the fire event. The pKaap values were modified, in general, the acidic site are stronger after fire than in the unburned soil. The COOH groups with pKaap value about 5 were lost after fire. The buffer capacity is lower or practically missing at soil pH (≈ 6) after fire.

The negative charge development decrease significantly at field pH (≈ 6) after the fire event, producing a deficiency on sites to make linkage between the organic and inorganic soil

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**Part 3** 

**Soil Fertility and Irrigation**

