**4. Pesticides: definition and classification**

The denomination pesticides (or plaguicides) include a wide variety of products that are very different in their chemical composition and characteristics. A plaguicide can be described as a substance (or formulation containing at least one of them) that presents any of the following uses:


212 Herbicides – Properties, Synthesis and Control of Weeds

The FFA limit in olive oil for direct consumption is 2%. Virgin olive oils with higher content are called "lampantes" and must be refined prior to consumption. The components to be removed are all those ones that are detrimental to the flavor, color and stability of the oil, mainly FFA, phosphoacylglycerols, pigments, volatiles and contaminants. The standard processes used are chemical and physical refining. The main difference between both processes is that chemical refining procedure includes caustic soda treatment to neutralize the oil while, following physical refining, FFA are eliminated by distillation during deodorization. Physical refining reduces the loss of neutral oil, minimizes pollution and enables recovery of high quality FFA. Nevertheless, not all oils can be physically refined.

 "Table olives" means the product: a) prepared from the sound fruit of varieties of the cultivated olive tree (*Olea europaea L*.) that are chosen for their production of olives whose volume, shape, flesh-to-stone ratio, fine flesh taste, firmness and ease of detachment from the stone make them particularly suitable for processing; b) treated to remove its bitterness and preserved by natural fermentation or by heat treatment, with or without the addition of preservatives; c) packed with or without covering liquid (International Olive Oil Council,

Table olives are classified in one of the following types according to the degree of ripeness




To prevent olive damage, fruits destined for table olives production are picked by hand and carefully placed in special padded basket that are hung by their neck. Olive transportation is carried out in perforated plastic containers, which have plastic netting as walls (supported by an iron structure). The perforated walls permit the aeration of the fruits and the reduced weight also contributes to minimizing the damage of the fruits. Sometimes, olives are also transported in bulk, although this transportation system is not recommended due to the

Once the olives have been transported, their processing takes place. In general, any processing method aims to remove the natural bitterness of the fruit, caused by the glucoside oleuropein. The bitterness may be removed by alkaline treatment, by immersion in a liquid to dilute the bitter compound, or by biological processes. The product so obtained may be preserved in brine according to its specific characteristics, in dry salt, in a modified atmosphere, by heat treatment, my preservatives, or by acidifying agents. The most


**3.2 Table olives** 

of the fresh fruits:

reached.

brine".

at colour change.

they have reached normal size.

increased risk of damaging the olives.

common trade preparations are (Sánchez et al., 2006):

2004).


Taking into account the specific action of the plaguicides, different classifications can be made, being the decimal classification one of the most frequently used:


Laboratory studies show that pesticides can cause health problems, such as birth defects, nerve damage, cancer, and other effects that might occur over a long period of time. However, these effects depend on how toxic the pesticide is and how much of it is

Evaluation of the Contamination by Herbicides in Olive Groves 215

N

N

N

Cl

N

NH NH

NH Cl

HN

N

N

N

Cl N

NH

N

Cl NH

NH

N

HN NH

S

NH

O

Cl NO2

N

O

N

F

F F

N

Cl

Cl

O CH3

N

N

**(mg L-1)** 

**KO/W 25ºC** 

1.3 2.10

33 2.50

20 3.34

8.5 3.21

22 3.65

36.4 2.85

0.12 4.86

**Plaguicide Structure W.S.** 

Simazine

Atrazine

Terbutrine

Diuron

Oxyfluorfen

Table 1. Herbicides in olive groves.

Trietazine <sup>N</sup>

Terbuthylazine <sup>N</sup>

consumed. Some pesticides also pose unique health risks to children. For these reasons, the governments carefully regulate pesticides to ensure that their use does not pose unreasonable risks to human health or the environment.

The mechanisms of action of the plaguicides over the organism are very different depending on the chemical composition. These mechanisms are well-known for some pesticides, even at the molecular level. However, they are completely unknown for some others. Even among pesticides from the same family, some of them can be classified as scarcely toxic while others are very toxic. As a result, it is difficult to establish general rules when dealing with the toxicity of plaguicides.

Herbicides are required to control the growth of weeds. The absence of weeds in the soil around the olive tree presents two major benefits: the weeds do not waste water resources for the olive trees and the harvest of olives from the soil is easier when they naturally fall down from the tree.

Depending on the period of application, herbicides can be classified as:


In Table 1, the herbicides that are usually employed in the Spanish olive groves are shown. Simazine, which is forbidden, is also present because it is very persistent and has to be analyzed in order to ensure the absence of residues. The herbicides comprise triazines (simazine, atrazine, trietazine, terbuthylazine and terbutrine), phenylurea diuron and phenylether oxyfluorphen.

The water solubility (W.S.) at 25 ºC and the KO/W value are also presented in Table 1. The KO/W value is the partition coefficient between octanol and water, and its logarithm is an indication of fat solubility of the pesticide. A high value of the coefficient KO/W for a particular plaguicide indicates that its solubility in water is low; hence, this plaguicide would be fat-soluble and there would be risks of bio-accumulation in fatty tissues. It has been demonstrated that fat-soluble pesticides tend to concentrate in olive oil during its production and extraction from the olives. For this reason, higher concentration levels of fatsoluble plaguicides are expected in the oil than in the olives from which the oil was produced. On the other hand, polar pesticides do not tend to preconcentrate in olive oil and their concentration is lower.

Monitoring herbicide residues in olive oil and table olives is of great interest to ensure food safety related to their use. The development of multi-residue methods is required in this case in order to determine all the herbicides in the same analysis. These methods need to present a high sensitivity in order to be able to analyze the samples at the legislated MRLs.

The analysis of herbicides in these samples is very challenging, because of the inherent complexity of the matrix. As a result, it is necessary to extract the pesticide fraction from the whole matrix to isolate the compounds that will be analyzed. Taking into account that some herbicides are fat-soluble, it is difficult to completely separate them from the matrix. Hence, a clean-up step is required after the extraction procedure.

consumed. Some pesticides also pose unique health risks to children. For these reasons, the governments carefully regulate pesticides to ensure that their use does not pose

The mechanisms of action of the plaguicides over the organism are very different depending on the chemical composition. These mechanisms are well-known for some pesticides, even at the molecular level. However, they are completely unknown for some others. Even among pesticides from the same family, some of them can be classified as scarcely toxic while others are very toxic. As a result, it is difficult to establish general rules when dealing

Herbicides are required to control the growth of weeds. The absence of weeds in the soil around the olive tree presents two major benefits: the weeds do not waste water resources for the olive trees and the harvest of olives from the soil is easier when they naturally fall

Depending on the period of application, herbicides can be classified as:



In Table 1, the herbicides that are usually employed in the Spanish olive groves are shown. Simazine, which is forbidden, is also present because it is very persistent and has to be analyzed in order to ensure the absence of residues. The herbicides comprise triazines (simazine, atrazine, trietazine, terbuthylazine and terbutrine), phenylurea diuron and

The water solubility (W.S.) at 25 ºC and the KO/W value are also presented in Table 1. The KO/W value is the partition coefficient between octanol and water, and its logarithm is an indication of fat solubility of the pesticide. A high value of the coefficient KO/W for a particular plaguicide indicates that its solubility in water is low; hence, this plaguicide would be fat-soluble and there would be risks of bio-accumulation in fatty tissues. It has been demonstrated that fat-soluble pesticides tend to concentrate in olive oil during its production and extraction from the olives. For this reason, higher concentration levels of fatsoluble plaguicides are expected in the oil than in the olives from which the oil was produced. On the other hand, polar pesticides do not tend to preconcentrate in olive oil and

Monitoring herbicide residues in olive oil and table olives is of great interest to ensure food safety related to their use. The development of multi-residue methods is required in this case in order to determine all the herbicides in the same analysis. These methods need to present a high sensitivity in order to be able to analyze the samples at the legislated MRLs. The analysis of herbicides in these samples is very challenging, because of the inherent complexity of the matrix. As a result, it is necessary to extract the pesticide fraction from the whole matrix to isolate the compounds that will be analyzed. Taking into account that some herbicides are fat-soluble, it is difficult to completely separate them from the matrix. Hence,


a clean-up step is required after the extraction procedure.

unreasonable risks to human health or the environment.

with the toxicity of plaguicides.

down from the tree.

phenylether oxyfluorphen.

their concentration is lower.


Table 1. Herbicides in olive groves.

Evaluation of the Contamination by Herbicides in Olive Groves 217

sample) and, after a mixing time, both phases (sample and extractant) are separated. During the mixing period, the analyte is distributed between both phases until it reaches the

The KO/W value is critical to decide the appropriate extractant. If this value is high, it indicates that the analyte tends to dissolve in the organic phase instead of in water. As a general rule, the organic solvents selected are volatile substances that present high affinity

It is probably the most extensively used technique for the analysis of pesticide residues in olive oil, usually after a liquid-liquid extraction. In this technique, an aliquot of an olive oil extract (obtained from the extraction step) is injected into the GPC system. The selected fraction is collected and, after a solvent-exchange step, the sample is analyzed by GC. A GPC system is composed of a chromatographic pump, a fraction collector and a detector. The columns are made from polymeric porous microspheres that enable the separation of compounds according to their molecular weights (which are related to the size of the compound). Using this principle, the herbicide fraction is separated from the triglyceride fraction, which presents higher molecular weight. In GPC, the compounds are eluted from

It is a separation process by which compounds that are dissolved or suspended in a liquid mixture are separated from other compounds in the mixture according to their physical and chemical properties. SPE is used to concentrate and purify samples for analysis and to

SPE uses the affinity of the analytes dissolved or suspended in a liquid (known as the mobile phase) for a solid through which the sample is passed (known as the stationary phase) to separate a mixture into desired and undesired components. The result is that either the desired analytes of interest or undesired impurities in the sample are retained on the stationary phase. The portion that passes through the stationary phase is collected or discarded, depending on whether it contains the desired analytes or undesired impurities. If the portion retained on the stationary phase includes the desired analytes, they can then be removed from the stationary phase for collection in an additional step, in which the

The stationary phase comes in the form of a packed syringe-shaped cartridge that can be mounted on its specific type of extraction manifold. The manifold allows multiple samples to be processed by holding several SPE media in place and allowing for an equal number of samples to pass through them simultaneously. A typical cartridge SPE manifold can accommodate up to 24 cartridges and is equipped with a vacuum port. Application of vacuum speeds up the extraction process by pulling the liquid sample through the stationary phase. The analytes are collected in sample tubes inside or below the manifold

for the analytes and are immiscible with the sample.

isolate the analytes of interest from a wide variety of matrices.

stationary phase is rinsed with an appropriate eluent.

after they pass through the stationary phase.

**5.3 Gel-permeation chromatography** 

higher to lower molecular weight.

**5.4 Solid-phase extraction** 

equilibrium.
