**7. Analysis of herbicides**

Our research has focused mainly on the determination of herbicides in olives destined for production of olive oil and olive oil. However, at this moment, we are continuing with this research, analyzing herbicide residues in table olives. On average, 5 kg of olives are required for the production of 1 L of oil. Taking into account that most pesticides (including herbicides) are lipophilic, a concentration effect could occur when obtaining the olive oil. Thus, MRL for herbicides have been set by the European Union in both olives and olive oil. For this reason, our research group has developed analytical methods that allow the analysis of the selected herbicides in olive oil and olives. In this section we will describe the analytical procedures that have been employed for this purpose as well as the results obtained.

For olive oil samples (Guardia-Rubio et al., 2006b), the preparation procedure included a liquid-liquid extraction followed by GPC clean-up step: 1) Two grams of the olive oil sample were dissolved in n-hexane saturated in acetonitrile. The solution was transferred to a separation funnel where it was extracted three times with acetonitrile saturated in n-hexane. The extracts were combined in a round-bottomed flask and were concentrated to dryness in a rotary evaporator. 2) The residue was dissolved in the GPC mobile phase (ethyl acetatecyclohexane, 1:1 (v:v)) and injected into the GPC column. The collected eluate fraction was transferred to a round-bottomed flask and concentrated to dryness in a rotary evaporator. 3) The residue was redissolved in cyclohexane and analyzed by GC-MS.

For olive samples (Guardia-Rubio et al., 2007c), approximately 130 g of olives (including the seeds) were first crushed by means of a hammer mill. Afterwards, a 100 g portion was weighed in a glass tube and 50 g of anhydrous sodium sulphate were added. The sample was then extracted twice with light petroleum by homogenization with Ultra-Turrax (a high flow mixing tool) and the extracts evaporated using a vacuum rotary evaporator. The solid residue was dissolved in n-hexane saturated in acetonitrile and the same liquid-liquid extraction and GPC clean-up procedure employed for olive oil were performed before the GC-MS analysis.

The following Table shows the retention times (tR) and analytical parameters obtained for each selected herbicide, including the detection limit (DL). The procedures previously detailed were applied to the determination of the cited herbicides in olives and olive oil samples.

As it was commented in Section 3, there are different olive harvesting methods. Depending on this, the olive fruits can be grouped into three categories: a) fruits picked directly from the tree without any contact with the soil (flight olives); b) fruits picked from the ground (soil olives); and c) fruits that are not separated before the elaboration process (nonseparated olives). The separation of flight and soil olive fruits before olive oil elaboration is critical in order to obtain appropriate results (high-quality virgin olive oil). If both fruits are not separated, the quality of the oil and the percentage of extra virgin olive oil obtained decrease. In addition, the harvesting method may be very important for the presence of herbicide residues in olives, because they remain concentrated in the top 5-15 cm of soil, even after several months since their application. 94 and 33 samples of olives and olive oil, respectively, were analyzed. Diuron and terbuthylazine were found in many of these samples, 79 of olives (specially soil olives) and 31 of olive oil. In four of the soil olives,

Our research has focused mainly on the determination of herbicides in olives destined for production of olive oil and olive oil. However, at this moment, we are continuing with this research, analyzing herbicide residues in table olives. On average, 5 kg of olives are required for the production of 1 L of oil. Taking into account that most pesticides (including herbicides) are lipophilic, a concentration effect could occur when obtaining the olive oil. Thus, MRL for herbicides have been set by the European Union in both olives and olive oil. For this reason, our research group has developed analytical methods that allow the analysis of the selected herbicides in olive oil and olives. In this section we will describe the analytical procedures that have been employed for this purpose as well as the results

For olive oil samples (Guardia-Rubio et al., 2006b), the preparation procedure included a liquid-liquid extraction followed by GPC clean-up step: 1) Two grams of the olive oil sample were dissolved in n-hexane saturated in acetonitrile. The solution was transferred to a separation funnel where it was extracted three times with acetonitrile saturated in n-hexane. The extracts were combined in a round-bottomed flask and were concentrated to dryness in a rotary evaporator. 2) The residue was dissolved in the GPC mobile phase (ethyl acetatecyclohexane, 1:1 (v:v)) and injected into the GPC column. The collected eluate fraction was transferred to a round-bottomed flask and concentrated to dryness in a rotary evaporator. 3)

For olive samples (Guardia-Rubio et al., 2007c), approximately 130 g of olives (including the seeds) were first crushed by means of a hammer mill. Afterwards, a 100 g portion was weighed in a glass tube and 50 g of anhydrous sodium sulphate were added. The sample was then extracted twice with light petroleum by homogenization with Ultra-Turrax (a high flow mixing tool) and the extracts evaporated using a vacuum rotary evaporator. The solid residue was dissolved in n-hexane saturated in acetonitrile and the same liquid-liquid extraction and GPC clean-up procedure employed for olive oil were performed before the

The following Table shows the retention times (tR) and analytical parameters obtained for each selected herbicide, including the detection limit (DL). The procedures previously detailed were applied to the determination of the cited herbicides in olives and olive oil

As it was commented in Section 3, there are different olive harvesting methods. Depending on this, the olive fruits can be grouped into three categories: a) fruits picked directly from the tree without any contact with the soil (flight olives); b) fruits picked from the ground (soil olives); and c) fruits that are not separated before the elaboration process (nonseparated olives). The separation of flight and soil olive fruits before olive oil elaboration is critical in order to obtain appropriate results (high-quality virgin olive oil). If both fruits are not separated, the quality of the oil and the percentage of extra virgin olive oil obtained decrease. In addition, the harvesting method may be very important for the presence of herbicide residues in olives, because they remain concentrated in the top 5-15 cm of soil, even after several months since their application. 94 and 33 samples of olives and olive oil, respectively, were analyzed. Diuron and terbuthylazine were found in many of these samples, 79 of olives (specially soil olives) and 31 of olive oil. In four of the soil olives,

The residue was redissolved in cyclohexane and analyzed by GC-MS.

**7. Analysis of herbicides** 

obtained.

GC-MS analysis.

samples.

diuron levels were higher than the MRL established by the European Union (0.2 mg kg-1); however, none of the olive oil samples presented levels higher than the corresponding MRL (0.8 mg kg-1). Terbuthylazine was also quantified in many samples with values over the MRL in some of them, being the established MRLs 0.05 and 0.2 mg kg-1 for olives and olive oil, respectively. Although other herbicides were also detected, in most cases the levels were below the quantification limit of the system. In general, the levels of herbicides found in soil olives have been significantly higher than those ones found in flight olives, which have not been in contact with the soil. These results suggest that herbicide residues are mainly caused by the contamination of the olives when they come to contact with the soil after falling down (Guardia-Rubio et al., 2006b; Guardia-Rubio et al., 2006c).


Table 2. Analytical parameters for olives and olive oil determination.

Evaluation of the Contamination by Herbicides in Olive Groves 223

olives meant an increase in herbicide residues. These results confirmed that the waters were being continuously contaminated with herbicides and a decontamination process would be required in the middle of the day (Guardia-Rubio et al., 2008); b) with respect to mud samples, 18 samples were analyzed. Diuron and terbuthylazine were found in nearly all the analyzed samples. Diuron appeared in all samples at concentration levels that ranged between 2.8 and 401.3 ng g−1. Terbuthylazine was detected in 16 samples at concentration levels between 7 and 1031.4 ng g−1. Simazine, prohibited in olive farming in the European Community but a very persistent pollutant, was detected in four samples, although in three of them the concentration was below the quantification limit (Guardia Rubio et al., 2006a). In general, the analysis of mud and waters from the washing device showed that, although the washing process eliminate a high percentage of the herbicide residues, a control over the re-

The production of virgin olive oil has increased in recent years and it is being exported to different countries, representing an important part of the economy in some Mediterranean countries. As we described in this chapter, it poses a lot of beneficial properties for the human health. However, it is important to carry out exhaustive quality controls in order to maintain its high standards. One of the most important ones is the analysis of residues of pesticides (including herbicides), which can be very dangerous for the human body, presenting different adverse effects over the organism depending on their toxicity. Here, we have described the methods developed in our research group for the analysis of herbicides in olives and olive oil. It has been shown that diuron and terbuthylazine are commonly found in these samples, although usually at levels below the established MRLs. In addition, the separation of flight and soil olives, together with the washing process in the olive mills, is a critical step to control the levels of herbicides. However, the efficiency of the washing step decreases over the time and the washing waters need to be replaced in order to keep the process being useful along the whole working day. Although the studies here presented have focused on the analysis of olive oil and olives destined to oil production, further research is currently being performed for the analysis of processed

Calatayud, J.M., De Ascenção, J.G. & Albert-García, J.R. (2006). FIA-fluorimetric

Catalá-Icardo, M., López-Paz, J.L. & Peña-Bádena, A. (2011). FI-photoinduced

Cicerale, S., Lucas, L. & Keast, R. (2010). Biological activities of phenolic compounds present

*Sciences*, Vol. 27, No. 3, (March 2011), pp. 291-296, ISSN: 0910-6340

No. 1, (January 2006), pp. 61-67, ISSN: 1053-0509

2010), pp. 458-479, ISSN: 1422-0067

determination of the pesticide 3-indolyl acetic acid. *Journal of Fluorescence*, Vol. 16,

chemiluminescence method for diuron determination in water samples. *Analytical* 

in virgin olive oil. *International Journal of Molecular Sciences*, Vol. 11, No. 2, (February

used washing water needs to be performed.

**8. Conclusions** 

table olives.

**9. References** 

The fruit goes into the olive-oil mill with a great quantity of impurities, leaves, branches, mud, etc, which are necessary to eliminate. Therefore, for this purpose, cleaners and washing devices are employed to eliminate impurities (Section 3), especially present in soil olives. It would be interesting to evaluate what fraction of the herbicides could be eliminated from the olives after the washing process in the olive mill, previous selection of the herbicides that frequently appear in these samples. The selected plaguicides were diuron and terbuthylazine. Our research group presented the first exhaustive study of the influence of the olives washing in the mills over the concentration of these herbicides (Guardia Rubio et al., 2006a; Guardia Rubio et al., 2007a; Guardia-Rubio et al., 2007b; Guardia-Rubio et al., 2008). Olive samples were collected before and after the washing process in the mill and were analyzed by GC-MS.

The most outstanding conclusion from the obtained results was the drastic reduction in the levels of herbicides in soil olives after the washing process when compared to the same olive samples before the washing step. The washing process significantly diminished the levels of residues of herbicides in soil olives, while the influence of the washing step was not clearly appreciated in flight olives. In the case of non-separated olives, it is interesting to remark that some of the washed olives presented higher levels of herbicides than the non-washed ones. This can be due to the contamination of herbicides-free olives during the washing process. The washing machines for the olives are cleaned and filled with fresh water at the starting of the day. With this water, up to 160000 kg of olives can be washed before the water is changed next day. The water can be contaminated after the washing of soil olives, therefore contaminating following herbicides-free olives in the washing process. Therefore, the water and mud from the olive washing devices were analyzed to confirm this theory. The procedures employed for both type of analyses follow:


From the analyses carried out over mud and washing water samples, the following results were obtained: a) regarding water analysis, the waters from an olive mill were collected at different times during the same day, and it was observed that the concentration of herbicides increased continuously along the day. An increase in the amount of washed olives meant an increase in herbicide residues. These results confirmed that the waters were being continuously contaminated with herbicides and a decontamination process would be required in the middle of the day (Guardia-Rubio et al., 2008); b) with respect to mud samples, 18 samples were analyzed. Diuron and terbuthylazine were found in nearly all the analyzed samples. Diuron appeared in all samples at concentration levels that ranged between 2.8 and 401.3 ng g−1. Terbuthylazine was detected in 16 samples at concentration levels between 7 and 1031.4 ng g−1. Simazine, prohibited in olive farming in the European Community but a very persistent pollutant, was detected in four samples, although in three of them the concentration was below the quantification limit (Guardia Rubio et al., 2006a). In general, the analysis of mud and waters from the washing device showed that, although the washing process eliminate a high percentage of the herbicide residues, a control over the reused washing water needs to be performed.

#### **8. Conclusions**

222 Herbicides – Properties, Synthesis and Control of Weeds

The fruit goes into the olive-oil mill with a great quantity of impurities, leaves, branches, mud, etc, which are necessary to eliminate. Therefore, for this purpose, cleaners and washing devices are employed to eliminate impurities (Section 3), especially present in soil olives. It would be interesting to evaluate what fraction of the herbicides could be eliminated from the olives after the washing process in the olive mill, previous selection of the herbicides that frequently appear in these samples. The selected plaguicides were diuron and terbuthylazine. Our research group presented the first exhaustive study of the influence of the olives washing in the mills over the concentration of these herbicides (Guardia Rubio et al., 2006a; Guardia Rubio et al., 2007a; Guardia-Rubio et al., 2007b; Guardia-Rubio et al., 2008). Olive samples were collected before and after the washing process in the mill and

The most outstanding conclusion from the obtained results was the drastic reduction in the levels of herbicides in soil olives after the washing process when compared to the same olive samples before the washing step. The washing process significantly diminished the levels of residues of herbicides in soil olives, while the influence of the washing step was not clearly appreciated in flight olives. In the case of non-separated olives, it is interesting to remark that some of the washed olives presented higher levels of herbicides than the non-washed ones. This can be due to the contamination of herbicides-free olives during the washing process. The washing machines for the olives are cleaned and filled with fresh water at the starting of the day. With this water, up to 160000 kg of olives can be washed before the water is changed next day. The water can be contaminated after the washing of soil olives, therefore contaminating following herbicides-free olives in the washing process. Therefore, the water and mud from the olive washing devices were analyzed to confirm this theory.

a. In the case of washing water samples, they were first filtered and then slowly passed through a SPE cartridge packed with C18 using a 12-port SPE vacuum manifold. The retained herbicides were then eluted from the solid phase with dichloromethane. The eluate, filtered and dried with anhydrous Na2SO4, was evaporated to dryness and the

b. In the case of mud samples, a solid-liquid extraction was carried out with a mixture of cyclohexane/acetone (3:1) in an ultrasonic water bath. The extracts were then filtered to eliminate particulate material, dried with anhydrous Na2SO4 and evaporated to dryness by means of a rotary evaporator. After that, an additional clean-up step was necessary in order to remove any remaining fat in the extract after the extraction procedure. This step involved the use of a chromatographic column that was packed with activated alumina suspended in cyclohexane. Once the extract was applied to the column, a mixture of cyclohexane/acetone (3:1, v:v) followed by dichloromethane was used to elute the herbicides. Once the eluate was evaporated and redissolved in cyclohexane,

From the analyses carried out over mud and washing water samples, the following results were obtained: a) regarding water analysis, the waters from an olive mill were collected at different times during the same day, and it was observed that the concentration of herbicides increased continuously along the day. An increase in the amount of washed

The procedures employed for both type of analyses follow:

residue was dissolved in cyclohexane for GC-MS analysis.

the sample could be analyzed by GC with ECD or TSD detection.

were analyzed by GC-MS.

The production of virgin olive oil has increased in recent years and it is being exported to different countries, representing an important part of the economy in some Mediterranean countries. As we described in this chapter, it poses a lot of beneficial properties for the human health. However, it is important to carry out exhaustive quality controls in order to maintain its high standards. One of the most important ones is the analysis of residues of pesticides (including herbicides), which can be very dangerous for the human body, presenting different adverse effects over the organism depending on their toxicity. Here, we have described the methods developed in our research group for the analysis of herbicides in olives and olive oil. It has been shown that diuron and terbuthylazine are commonly found in these samples, although usually at levels below the established MRLs. In addition, the separation of flight and soil olives, together with the washing process in the olive mills, is a critical step to control the levels of herbicides. However, the efficiency of the washing step decreases over the time and the washing waters need to be replaced in order to keep the process being useful along the whole working day. Although the studies here presented have focused on the analysis of olive oil and olives destined to oil production, further research is currently being performed for the analysis of processed table olives.

#### **9. References**


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**Prediction of Herbicides Concentration** 

Natural and anthropogenic variables of stream drainage basins such as hydrogeologic parameters (permeability, porosity etc.), amount of agricultural chemicals applied, or percentage of land planted affect agricultural chemical concentration and mass transport in streams. The use of herbicides, pesticides, and other chemicals in agricultural fields increase the concentration of chemicals in streams which severely affects the health of human and environment. The transport of chemical pollutants into river or streams is not straight forward but complex function of applied chemicals and land use patterns in a given river or stream basin. The factors responsible for transport of chemicals may be considered as inputs and chemical concentration measurements in streams as outputs. Each of these inputs and outputs may contain measurement errors. Present work exploited characteristics of fuzzy sets to address uncertainties in inputs by incorporating overlapping membership functions for each of inputs even for limited data availability situations. Soft computing methods such as the fuzzy rule based and ANN (Artificial Neural Networks) is used for characterization of herbicides concentration in streams. The fuzzy c-means (FCM) algorithm is used for the optimization of membership functions of fuzzy rule based models for the estimation of diffuse pollution concentration in streams. The general methodology based on fuzzy, ANN and FCM for estimation of diffuse pollution in streams is presented. The application of the proposed methodology is illustrated with real data to estimate the diffuse pollution concentration in a stream system due to application of a typical herbicide, atrazine, in corn fields with limited data availability. Solution results establish that developed fuzzy rule base model with FCM outperform fuzzy or ANN and capable for the estimation of diffuse

pollution concentration values in water matrices with sparse data situations.

Application of pesticides, insecticides and herbicides, cause diffuse pollution, commonly referred to as non-point source pollution in river or streams. Diffuse pollution from agricultural activities is a major cause of concern for the health of human and environment. Diffuse (non-dot, dispersed) pollution generally arises from land-use activities (urban and rural) that are dispersed across a catchment or subcatchment, where as point sources of pollution arise as a process industrial effluent, municipal sewage effluent, deep mine or farm effluent discharge (Novotny 2003, based on CIWEM (D'Arcy et al., 2000)). Potential point sources of pollution is characterised by its location, magnitude and duration of activity; and the sources of pollution is characterized when these parameters are identified

**1. Introduction** 

*Department of Civil Engineering, MNNIT Allahabad,* 

**in Streams** 

Raj Mohan Singh

*India* 

Walters, S.M. (1990). Clean-up techniques for pesticides in fatty foods. *Analytica Chimica Acta*, Vol. 236, pp. 77-82, ISSN: 0003-2670 **13** 
