Monitoring of 2,4-dichlorophenoxyacetic Acid in Bee Products by Solid Surface Fluorescence

Summary

This work proposes the development of a new alternative methodology to traditional techniques for the control and monitoring of 2,4-dichlorophenoxycarboxylic acid in bee product samples from central and northern Argentina.

Abstract

Bee health is essential for honey production and crop pollination. Honey production can be negatively affected by the use of herbicides, as bees can come into contact with these chemicals when collecting nectar and pollen from treated plants, which can lead to honey contamination with these toxic residues. While 2,4-dichlorophenoxyacetic acid (2,4-D) is designed to control broad-leaf weeds, it can reach flowers and contaminate bees' production, potentially affecting bees' health and quality of life. For these reasons, it is important to analyze honey periodically, in order to detect the presence of herbicide residues and, if necessary, take corrective measures. This work proposes the development of a new alternative methodology to traditional techniques for the control and monitoring of 2,4-D in bee product samples from central and northern Argentina. Samples were conditioned at pH = 7.0 in the presence of the anionic surfactant sodium dodecyl sulfate (SDS), filtering the systems through blue-band filter paper as a solid support before determination by solid phase fluorescence. Under optimal working conditions, detection and quantification limits of 0.33 ng/L and 0.90 ng/L, respectively, and a linearity range of 0.90 x 10³ ng/L to 1.13 x 10³ ng/L were achieved. Among the advantages of the new method are the use of inexpensive instruments and eco-friendly solvents, low waste generation, and, consequently, its safeguarding of some principles of green chemistry.

Introduction

Bee health is essential for honey production and crop pollination. A healthy colony depends on comprehensive health management that includes proper nutrition, hygiene measures, and disease prevention and treatment. Honey production, if carried out responsibly, does not harm bees, as beekeepers only extract a small portion of the total honey, leaving reserves for the colony^1,2.

Honey production can be negatively affected by the use of herbicides, as bees can come into contact with these chemicals when collecting nectar and pollen from treated plants, which can lead to honey contamination with pesticide residues. Furthermore, some herbicides, such as glyphosate, can directly affect bee development and behavior, reducing their foraging ability and physiological development^3,4,5,6. Although 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide has been designed to control broadleaf weeds, it can reach flowers and contaminate honey, potentially impacting bee health and honey quality^7,8,9.

Domestic and international trade in honey and other bee products has shown significant and sustained growth in recent years, as reflected in increased production^10,11,12. According to data from the Food and Agriculture Organization of the United Nations (FAO), there are five major honey-producing countries in the world: China, Argentina, Turkey, the United States, and Ukraine¹³. Beekeeping production is of great importance in Argentina and is constantly growing due to the export market opportunities that have emerged in recent years. Argentina's environmental conditions (climate, flora, etc.) and the technology invested in production have allowed the country to position itself in an important position worldwide. On the other hand, the presence of xenobiotics constitutes an issue of concern and requires surveillance, as in other countries, since it affects both the marketing of honey and the health of consumers due to its toxic effects^14,15.

2,4-D is a widely used, selective systemic herbicide that effectively controls broadleaf weeds by acting as a synthetic auxin, causing their uncontrolled growth and death. It is used in agriculture, horticulture, and forestry, and is particularly useful for controlling weeds in crops such as wheat, corn, and rice, as it does not harm grass or cereals¹⁶. 2,4-D can also be used as a plant growth regulator and is available in different formulations, including amine and ester salts, to suit a variety of applications. 2,4-D functions are influenced by the dose administered and the susceptibility of particular species and tissue types^17,18. For instance, 2,4-D contact has been implicated in adverse reproductive outcomes and significant genetic alterations in mice, indicating a notable genotoxic effect¹⁹.

Honeybees, as key pollinators and model organisms for studying eusociality, learning, and memory, are highly vulnerable to direct poisoning from agrochemicals used in fields²⁰. Aerial application of herbicides and insecticides during bloom can lead to significant mortality among honeybees and drastically reduce honey production. Pesticide mixtures, even at sub-lethal doses, can disorient foraging bees, impair their memory, and diminish foraging efficiency. This, in turn, weakens colonies by reducing pollen and nectar collection, resulting in nutritional deficiencies. Additionally, 2,4-D has been detected in honey samples, and contaminated pollen and nectar may be spread among hive mates²¹.

It is important to periodically analyze honey for the presence of herbicide residues and, if necessary, take corrective measures. For the detection and quantification of food contaminants such as herbicides, in this particular case 2,4-D, the most commonly used instrumental methods are chromatography, namely high-performance liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS/MS), and gas chromatography tandem mass spectrometry (GC-MS/MS)^{22,23,24,25,26}. However, researchers periodically present new monitoring methods for 2,4-D that offer advantages over conventional quantification methods^27,28,29, for example: use of less expensive instruments, operational simplicity, use of fewer solvents, application to more complex samples, and others.

Solid-phase fluorescence is a versatile technique that, by combining molecular fluorescence with solid-phase extraction methods, enhances the already high sensitivity inherent to fluorescence instrumentation. It also improves the linearity range and selectivity by reducing or eliminating matrix effects^30,31.

In this research, a new analytical methodology for monitoring and quantifying 2,4-D is proposed and applied to honey and other bee samples from central and northern Argentina. The samples contain unknown amounts of 2,4-D. What is known are the concentrations of the super-added levels, which are the concentration provided by the sample plus the concentration of 2,4-D that we added. This latter value is known and allows us to calculate the recovery. The new methodology is based on the direct determination of the analyte using solid-phase fluorescence, demonstrating multiple advantages in terms of instrumentation savings, lower operating costs, and increased environmental protection.

Protocol

This manuscript does not contain any studies with human participants or animals performed by any of the authors.

Apparatus used
Spectrofluorimetric measurements were performed using a PC-based spectrofluorometer equipped with a 150 W xenon lamp. A sample holder was used for solid surface fluorescence (SSF) measurements. The parameters used for 2,4-D quantification were as follows: λ_em= 580 nm, using λ_ext= 555 nm (Slits 3-3), using a solid sample holder.

Sampling and sample treatment
This study on the production of honey samples produced in 2025 was conducted in San Luis, San Juan, Jujuy, and Salta provinces in the north-central region of Argentina. The samples analyzed were: four multifloral honeys, two propolis honeys, pure honey candies, and candies made from a mixture of honey, coca, and propolis, purchased from beekeepers in the regions. The honeys studied were fresh, extracted from the hives within less than 1 week of production, in order to avoid possible degradation of 2,4-D by different mechanisms. All samples were collected in new, sterile vessels and immediately transported to the laboratory. They were stored at 4-8 °C in a dark place until analysis. The solid caramel was homogenized with a mortar and pestle; its contents were diluted in 5 mL of ultrapure water, and 0.5 µL was taken from that solution.

Methodology
Aliquot of 0.5 µL or 1 µL of 2,4-D (1.23 ng/L and 3.49 ng/L), 100 µL of sample, 250 µL of SDS (1 x 10^-4 mol/L), and 100 µL of phosphate buffer (1 x 10^-4 mol/L, pH=7) were added, and the mixture volume was made to 3 mL by adding double-distilled water. The mixture was filtered through the solid support (paper filter; see Table of Materials for details). Solid supports were dried at room temperature, and then solid surface fluorescence (SSF) was measured at λ_em= 580 nm, using λ_ext= 555 nm (Slits 3-3) with a solid sample holder.

The above describes the general procedure of the developed methodology, in which each parameter was studied and optimized, as shown in the Results section.

Effect of pH and buffer
pH optimization was performed by adjusting the systems to the pH under study using hydrochloric acid or sodium hydroxide to bring them to the required value (range tested pH 5-7). Subsequently, once the most appropriate pH range for achieving a suitable analytical signal was obtained, the buffer to be used was selected.

The buffers tested were phosphate, Tris, and borax, which were prepared at a concentration of 1 x 10^-4 mol/L. Their volume was varied to obtain the best fluorescence intensity signal. The results for the only buffer phosphate that improved the fluorescence intensity are shown, along with its respective optimal concentration. Here, the selected buffer was phosphate, and the pH = 7.

Surfactant concentration
The use of different surfactants in molecular fluorescence offers advantages that improve the determination of the analyte under study. Micellar media are used to minimize intermolecular interactions between the analyte and the components of the sample matrix. Furthermore, the photophysical properties of fluorescent solutes can be modified in the micellar medium, thus improving fluorescence sensitivity. The effect of different surfactants (SDS and HTAB) on the quantification of 2,4-D using solid surface fluorescence (SSF) was studied. It was found that the anionic surfactant SDS, at a concentration of 8.3 x 10⁻⁶ mol/L, increased the fluorescence intensity of the herbicide under study.

Solid support
Given that the planar configuration is energetically preferred in the excited fluorescence state, the retention of the herbicide on solid supports was explored. The systems were filtered through various types of membranes, including nylon, cellulose acetate, mixed esters, and Blue-Ribbon filter paper. The filtered solutions were collected in separate, clean containers, and the membranes were dried at room temperature. Subsequently, the membranes were placed in a solid sample holder, and solid-phase fluorescence (SSF) was recorded. Adequate and selective retention was observed on filter paper, so this support was selected for the solid-phase fluorescence determination. The filtered solutions were also analyzed by molecular fluorescence. The absence of the 2,4-D complex was evident, demonstrating the retention of the herbicide on the filter paper.

Recovery study
2,4-D was added to an appropriate volume of each sample studied (0.5 µL was used for the honey samples and 1 µL for the other samples analyzed), gradually increasing its concentration to 1.23 ng/L and 3.49 ng/L. Analyte concentrations were determined using the methodology, as the average of six replicates (n = 6).

Precision study
Method repeatability and intra-day precision were studied by repeated testing of samples (n = 6) containing 1.23 ng/L and 3.49 ng/L of 2,4-D and determining the analytical content using the methodology. In addition, the reproducibility of inter-day precision was evaluated over 7 days for the same systems.

Interference study
Different amounts of common ions were added to the test solution containing 3.49 ng/L of 2,4-D, and the methodology was applied. The following potential interferents were tested:

Concentration of Interferent/2,4-D mole ratio = 1000:1:00 for Na⁺, K⁺, Cl^-, Fe³⁺, Cu²⁺, Cd²⁺, Sb³⁺, Mn²⁺, As³⁺, CO₃^2-, SO₄^2-, Ca²⁺, Mg²⁺, NO^3-, Ni²⁺, fructose, glucose, sucrose, maltose, 2,4,5-T, sulfometuron-methyl, glyphosate, and atrazine.

Concentration of Interferent/2,4-D mole ratio = 500:1:00 for Zn²⁺, Co²⁺, Al³⁺, chlorsulfuron, bensulfuron-methyl, and triasulfuron.

Calculation of analytical quality parameters
The analytical quality parameters are the limit of detection (LOD) and the limit of quantification (LOQ). These were calculated by applying the following steps. The background noise was measured by measuring the response of 15 blank samples (samples without the analyte) to obtain a dataset of background noise. The standard deviation of the noise was calculated. This is accepted as a LOD value. The detection limits are based on 3.3x the standard deviations of the blank (N = 15). The quantification limits are based on 10x the standard deviations of the blank (N = 15).

Results

The results presented below indicate how the study of each of the variables that influence the general procedure, its optimization, and the optimal working conditions that were reached was approached step by step.

2,4-D spectrum characterization
2,4-D characterization was carried out by UV-Vis spectroscopy and molecular fluorescence, observing fluorescent intensity maxima at λ_em= 580 nm, using λ_ext= 555 nm (Slits 3-3; Figure 1

Discussion

The intensive use of herbicides has increased exponentially in Argentina and the rest of the world due to the need to meet the food demands of a growing population. If the use of such products is monitored appropriately, rationally, and periodically, it would not compromise the expected benefits or have negative effects on the environment as a whole. 2,4-D has been widely used worldwide, and many studies have shown that this herbicide induces alterations in non-target organisms. Therefore, continuing to evaluate the risk...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2,4-D	Sigma-Aldrich, St. Louis, USA	49083
Acetic acid/acetate	Mallinckrodt Chemical Works
Blue Ribbon filter papers	Sigma-Aldrich, St. Louis, USA	WHA1001929	2-5 μm pore size and 12.5 cm diameter
Cellulose acetate membrane	Sigma-Aldrich, St. Louis, USA		0.45 μm pore size and 47 mm
Hexadecyltrimethylammonium bromide (HTAB)	Sigma-Aldrich, St. Louis, USA	H5882
hydrochloric acid	Sigma-Aldrich, St. Louis, USA	1.09063
Immobilon (+) membranes	Millipore, Sao Paulo, Brazil	HATF04700	0.45 μm pore size and 47 mm
Mixed esters membranes	Sigma-Aldrich, St. Louis, USA		0.45 μm pore size and 47 mm
Nylon membranes	Millipore, Sao Paulo, Brazil	Z290793	0.45 μm pore size and 47 mm diameter
pHmeter (Orion Expandable Ion Analyzer, )	Orion Research, Cambridge, MA, USA	Model EA 94.
Potassium dihydrophosphate	Biopack, Buenos Aires, Argentina	2000168900
Potassium phthalate acid	Merk &Co., Inc
Sodium Dodecyl Sulfate	Sigma-Aldrich, St. Louis, USA	11667289001
Sodium hydroxide	Sigma-Aldrich, St. Louis, USA	S2770
Sodium tetraborate	Sigma-Aldrich, St. Louis, USA	221732
Spectrofluorimetric	Shimadzu	RF-5301	equipped with a 150 W Xenon lamp and 1.00 cm quartz cells
Tris-(hydroxymethyl)-aminomethane	Sigma-Aldrich, St. Louis, USA	77-86-1