Rapid and Robust Analytical Method for the Determination of Copper Content in Commercial Pesticides and Antifouling Biocides

The quality control of the agrochemicals and biocidal products in the market requires valid determination methods for the active ingredient content and is of utmost interest to ensure environmental protection, human health, and successful pest control. Copper has been used as fungicide for centuries, and today is still in the market in hundreds of products for various uses and is applied in very high application rates, both in pesticides and biocides. The aim of the present study was to develop a new fast, efficient, and inexpensive analytical method for the determination of copper content in antifouling Product Type 21 (PT-21) biocides as well as in copper containing pesticides. The samples were oxidized by microwave-assisted acid digestion method and copper content was determined by flame atomic absorption spectrometry technique. The recoveries of the method ranged from 87.9% to 97.6% for antifouling paints, and 98.6% to 99.95% for pesticides, while the percentage Relative Standard Deviation (%RSD) was lower than 6% in all cases. The validated method Limit of Quantification (LOQ) was 5 μg mL−1 that was sufficient for the present analysis needs. As a result, it is concluded that the method is easily applicable and transferable, with reasonable consumption of reagents, characterized by high reliability and sensitivity; therefore, it is suitable for monitoring the levels of copper in antifouling products as well as pesticides containing copper as active substance. A new rapid and robust analytical method for fungicidal copper analysis is introduced The method is applicable for all types of copper containing biocides and pesticides The method presents excellent QA/QC and can be easily adopted in every laboratory It can efficiently supersede the commonly used titration technique proposed by CIPAC A new rapid and robust analytical method for fungicidal copper analysis is introduced The method is applicable for all types of copper containing biocides and pesticides The method presents excellent QA/QC and can be easily adopted in every laboratory It can efficiently supersede the commonly used titration technique proposed by CIPAC


Introduction
Copper-based compounds have been used for a long time as fungicides to control fungi and bacterial diseases in vineyards, pome, and stone fruit orchards, as well as for vegetable crops (Merry et al. 1983). They are used alone or in combination with other fungicides (Gharieb et al. 2004;Gallart-Mateu et al. 2016). The most common copper products used in agriculture are copper oxychloride, copper oxide, copper hydroxide and metallic copper. According to IUPAC, copper hydroxide is the common name for copper (II) hydroxide (or copper (2 +) hydroxide or cupric hydroxide) and has the chemical formula Cu(OH) 2 , copper oxychloride is the common name for dicopper(II) chloride trihydroxide with chemical formula Cu 2 (OH) 3 Cl, and cuprous oxide is the common name for copper(I) oxide (or copper(1 +) oxide) with its respective chemical formula to be Cu 2 O (EFSA 2018). These copper-based products are extensively used alone or in combination with other active ingredients in agriculture due to their antifungal properties in crop management (Gallart-Mateu et al. 2016).
Copper is extensively allocated in biological tissues, where it occurs mainly in the form of organic complexes, countless of which are metalloproteins which function as enzymes, thus are involved in a variation of metabolic reactions, such as the utilisation of oxygen during cell respiration and energy utilisation. Additionally, they are involved in the synthesis of essential compounds, such as neurotransmitters and proteins of connective tissues of the skeleton and blood vessels in mammals (EFSA 2018). It is also a crucial micronutrient used in numerous metabolic processes, hence certain concentration of copper is needed for most organisms, yet high copper concentrations can be potentially toxic (Lindgren et al. 2018).
Copper-based compounds belong also to the group of biocides due to their use as antifouling (AF) paints to combat marine fouling. Over the years various biocides have been used as antifouling agents, however, due to the restriction of tributyltin (TBT) and TBTbased products, copper in the form of cuprous oxide (Cu 2 O), copper thiocyanate (CuSCN) or metallic copper are currently utilized as the major biocide (Perez et al. 2015). Cuprous oxide was the first biocide commenced for large scale industrial production of antifouling paints (Cima and Ballarin 2012;Okamura et al. 2022). A content of 30-40% Cu 2 O (w/w) is typical in antifouling coatings, making Cu one of the major constituents of the paint films as it exhibits antifouling activity against organisms such as barnacles, tube worms and most of algal fouling species (Lagerström and Ytreberg 2021).
High copper concentrations (cuprous and/or cupric cations; free and/or complexed forms) in seawater, sediments and biological tissues have been detected close to marinas and harbors worldwide, and particularly in high-traffic ports (Katranitsas et al. 2003;Batista-Andrade et al. 2016). For that purpose, increasing pressure on the quality control of copper containing paints have caused strengthened research into the development and validation of appropriate methods for their analysis both in environmental samples and copper-containing commercial formulations (Singh and Turner 2009a, b;Ytreberg et al. 2015;Adeleye et al. 2016;Gallart-Mateu et al. 2016;Squissato et al. 2019;Soroldoni et al. 2020;Lagerström et al. 2020;Viana et al. 2020).
At the same time, materials submerged in seawater are quickly covered by a macromolecular film, which then favors settlement of bacteria (prokaryotes), microalgae, protozoans (unicellular) as well as barnacles, mussels and tubeworms (multicellular eukaryotes) (Perez et al. 2015). This phenomenon is called biofouling and can be defined as the accumulation of micro-and macro-organisms on surfaces submerged in the sea. Biofouling signifies a major annoyance for the maritime industries, particularly for shipping, as biofouling on ship hulls increases the boat mass then inducing over-consumption of fuel and increased maintenance costs (Dafforn et al. 2011;Yebra et al. 2004;Schultz et al. 2011). Additional effects on ship hulls comprise frictional resistance due to caused harshness and potential speed reduction and loss of maneuverability as well as increase of the frequency of dry-docking operations and corrosion routes, which escort to the creation of large amounts of toxic wastes (Yebra et al. 2004). To avoid and diminish impacts from biofouling, antifouling measures should be taken, which comprise the use of coatings to boat hulls and other submerged structures such as fishing nets and marine structures (Singh and Turner 2009b;Okamura et al. 2022) to maximize their effects against biofouling organisms. The most effective method to prevent fouling attachment has elaborated coating ship hulls with metal-containing antifouling paints, mainly including copper.
A great variety of analytical techniques have been employed in the determination of copper content in the commercially available formulations. The Collaborative International Pesticides Analytical Council (CIPAC 1993) suggested the following analytical techniques for the determination of copper involving electrogravimetric method and redox titration using thiosulphate. In the international scientific literature, alternative analytical methods for copper include flame atomic absorption method, inductively coupled plasma-optical emission spectroscopy (ICP-OES), UV-visible spectrophotometry and voltammetry (Buldini et al. 1999;CIPAC 1993;Singh and Turner 2009b;Ferreira et al. 2014;Gallart-Mateu et al. 2015;Isildak et al. 1999;Shrivas and Jaiswal 2013;Silva et al. 2016;Soroldoni et al. 2017;Schneider et al. 2019). However, the use of digestion along with atomic absorption spectrophotometry was proved to also provide respectable results (Gallart-Mateu et al. 2016).
Plant protection and biocides policy at both European and National level in member states, aims to decrease risks associated with their use especially in the case of antifouling paints which are in direct contact with sea water and aquatic non-target organisms. The first step in this policy is the quality control of plant protection products (PPPs) and biocides which is addressed within the European Union legislation (EC 1991(EC , 2009a(EC , b, 2012. Monitoring of PPPs and biocides is addressed within the European Union in the framework of the adopted regulation 1107/2009/EC (EC 2009a, b), which regulates the placing of PPPs and biocides in the market. Additionally, the EU Directive 128/2009/EC (EC 2009b) establishes regular monitoring programs of PPPs and biocides in member states.
This work sought to develop a spectrophotometric method for the determination of copper in antifouling paints and in agricultural fungicides, as an alternative to CIPAC (1993) methods which are rather complicated and outdated. Using appropriate sample preparation procedures achieves rapid and robust analysis. The method has been properly validated for linearity, precision, and accuracy. One of the greatest advantages of the suggested method is that small portions of solvents (acids) are required in the sample preparation step. This is extremely costeffective since waste disposal is not only expensive but also environmentally unsound. To the best of our knowledge, this is the first report providing a thorough extraction and determination procedure for fungicide copper products, using atomic absorption spectroscopy, that constitutes a simple, fast, and inexpensive, yet efficient analytical method, commonly available in most laboratories.

Antifouling Paints and Copper Fungicide Samples
Antifouling paint samples of blue color used to cover wall surfaces and copper fungicides were obtained from the Greek market during December 2017. Thus, in total, four commercial antifouling paints and two fungicide agrochemicals were analyzed to investigate the copper content. The agricultural fungicide samples were of a nominal concentration of 57.3% w/w as copper hydroxide, corresponding to a copper content of 40%, and the antifouling biocidal products presented a nominal concentration of 47.78% w/w of cuprous oxide (Cu 2 O), corresponding to 42.44% of copper. The pesticides were in the form of Wettable Granules (WG) and the biocides were in Liquid (LIQ) form. The analytical methodology was validated both using an agricultural pesticide and one of the available biocides, and was thereafter applied in all samples.

Materials
Metal-free grade solvents and specifically water (H 2 O), nitric acid (HNO 3 ) and sulphuric acid (H 2 SO 4 ) were obtained from Fisher Scientific (Leicestershire, UK) and were used for the whole experimental procedure. All glassware and plastic materials used in the present study were treated for 24 h in an acid bath containing 1 M metal-free grade HNO 3 , then rinsed several times with metal-free water and dried at 40 °C in an oven before use. Copper standard solution of trace analysis grade at concentration 1000 μg mL −1 was also purchased from Fisher Scientific (Leicestershire, UK) and was used for preparing the working standard solutions at concentration 5, 10, 15 and 20 μg mL −1 .

Working Standard Preparation
The working standards solutions were prepared daily at four different concentrations (5, 10, 15 and 20 μg mL −1 ) by appropriate dilutions of the copper standard solution (1000 μg mL −1 ) with 0.1 M HNO 3 . The latter was prepared by concentrated nitric acid (70%) and dilution with metal free water obtained from Fisher Scientific (Leicestershire, UK). Acid-washed plastic vessels were used for preparing and storing solutions.

Instrumentation
The analysis of copper content in the samples was carried out using a flame atomic absorption spectrophotometer (model AA-6501F, Shimadzu, Duisburg, Germany) with deuterium background correction, equipped with multiple lamps turret and an autosampler (ASC-6000, Shimadzu, Duisburg, Germany). Hollow cathode lamp (Hamamatsu Photonics K.K., Shizuoka, Japan) was used as radiation source. The flame type in all cases was Air-Acetylene at a 14 cm burner head. Deuterium lamp (D2) correction was automatically applied by the atomic absorption spectrophotometer. System control and data analysis were carried out using AA WizAArd (version 2.20) software (Shimadzu, Duisburg, Germany). All instrumental settings for the flame atomic absorption spectrometer were those recommended by the manufacturer. In more detail, the lamp current was set at 10 mA, its operating wavelength was 324.8 nm, the silt width was 0.5 nm, the burner height was set at 7 mm and the fuel gas flow was 1.8 L min −1 .
As regards the sample preparation prior analysis, a CEM/MARS 5 model MD 9132 (Matthews, NC, USA) digestion oven equipped with Omni XP-1500 tubes was used to digest the samples. The digestion conditions are presented in the sample preparation section below.

Sample Preparation
Biocide samples, which were in the viscous liquid form, were mechanically shaken for 60 min in a horizontal orbital shaker (GFL 3017, Burgwedel, Germany) to be homogenous before being weighed. To eliminate organic solvents, an accurate mass of approximately 100 mg from each sample was weighted in quintuplicate into separate polytetrafluoroethylene (PTFE) microwave reaction vessels. The microwave assisted extraction method requires only a small portion of solvents (acids) for sample digestion (compared to typical digestion methods), whereas the whole procedure is run under controlled conditions without any harmful fumes or vapours released to the environment. Due to the viscosity of the antifouling samples, special attention was required at this stage and as such single-use 150 mm Pasteur pipettes (Kimble, Mainz, Germany) were used as these helped to control flow during weighing. Subsequently, 5 mL of concentrated metal-free HNO 3 and 5 mL of metal-free H 2 SO 4 were added in the vessels. Alternative preparations using the exact proposals of EPA 3052 method (US EPA 1996) (with nitric acid and hydrogen peroxide) as well as previous studies in the literature that used only nitric acid (e.g., Bentlin et al. 2007) were tested; however, they were not sufficient to decompose the antifouling paint samples matrix. Since it was prerequisite to develop a unique method both for pesticides and biocides, the combination of nitric and sulphuric acid was selected and the maximum solvent volume for the microwave vessels was used. The EPA 3052 digestion program includes rise of samples to 180 ± 5 ºC in approximately 5.5 min, and then, remain at 180 ± 5 ºC for 9.5 min, yet it was found that it was not sufficient. Thus, the mixture was finally digested for 10 min at a power of 1600 W reaching a temperature of 180 °C and for additional 5 min at 200 °C. The ramp time for each temperature was less than one minute in both cases. After cooling and depressurizing, an exact aliquot (0.8 mL) was transferred to falcon tubes (Isolab, Eschau, Germany) and diluted with metal free water adjusting the final volume at 250 mL in a volumetric flask. Copper content was quantified with atomic absorption. The whole procedure is schematically presented in Fig. 1.

Quality Control
A reagent blank (solvents without sample) was carried out throughout the whole procedure. Copper concentration was obtained directly from calibration graphs after correction of the Fig. 1 Sample preparation procedure schematically absorbance of the signal obtained from an appropriate reagent blank. Due to the absence of certified materials for the antifouling paint samples, the accuracy of the whole procedure was proved using standard addition for determining the recovery. For the standard addition tests, an aliquot of copper solution was added to the sample before decomposition using the proposed method to obtain a final concentration of 5 mg L −1 of copper. The sample with the standard addition was subjected to the same process described above. One sample of antifouling paint and one of the selected fungicides were selected for the test.
Method accuracy (percentage recoveries) was estimated by recovery experiments from spiked samples, i.e., as the ratio of the difference of observed concentrations between the fortified and real samples to the spiked value. Linearity, limit of detection (LOD), limit of quantification (LOQ), sensitivity and accuracy were validated before the analytical assays. Precision was obtained from every sample determination. The average of reagent blank signal was subtracted from the respective signal of samples before interpolation on the calibration curve. Standard solutions were run between each sample analysis to control the adequate performance of the procedures and apparatus. The LOD and LOQ (μg mL −1 ) were determined according to the following equations: where s is the standard deviation of the measures referring to the blank (n = 5) and S is the inclination-slope of the analytical curve.
The estimation of the tested sample concentration was performed using the following equations: where: according to the calibration curve.

Results and Discussion
Copper-based AF paints have been used to fight marine fouling since the past century; however, concerns have been raised recently for the effect of these products on the marine environment due to the widespread use of toxicants (Srinivasan and Swain 2007). Since then, copper was an effective and extensively used biocide; nevertheless, its effectiveness was comparatively short-lived, and thus, paint reapplication was often required (Tessier et al. 2011). The discovery of the antifouling efficacy of trialkyltins, with tributyltin (TBT) being the most important representative of this chemical group, was thought to solve this problem. Though the use of TBT was proved to be associated with reduced oyster spatfall in the marine environment, anomalies in larval development and shell malformation resulted in its banning (Tessier et al. 2011). Other biocides such as diuron and irgarol, have adverse environmental effects and consequently also are banned. As a result, since their ban in the early 1980s, copper has become again the predominant antifouling biocide in marine AF paints. The use of copper-based AF paints has a main advantage, as copper is a naturally occurring element. Furthermore, copper in low concentrations is considered an essential micronutrient taken up by plants and animals. Copper occurs in nature in two valence states, cuprous (Cu + ) and cupric (Cu 2+ ), with the latter being the most toxic form as it easily migrates through cell membranes of living organisms.
In the present study, understanding the necessity of a simple, yet efficient analytical technique for copper content determination of AF products, an analytical method based on acidic extraction and atomic absorption technique was developed, validated, and applied in real samples. The major advantage of this combination in comparison with other available techniques, is the speed, the simplicity, the reduced hazard from the necessary extraction as well as the reduced cost, as a minimum amount of reagents is needed and FAAS is a common analytical instrument available in most laboratories, which also has very low maintenance costs. At the same time, a microwave-assisted extraction apparatus is most usually present in laboratories dealing with trace metals analysis on organic matrices; yet, if this equipment is not available, then "open-air" digestion is deemed necessary, increasing thus the preparation time.
Previous studies have used sequential acidic-basic extraction followed by NIR spectrometry which though requires previously well-characterized samples (Gallart-Mateu et al. 2016), as well as inductively coupled plasma optical emission spectroscopy (ICP-OES) (dos Santos et al. 2023;Gallart-Mateu et al. 2015) which is though difficult to adapt to the high concentrations of the formulated products. Other methods available in the literature propose copper determination via voltammetry (Buldini et al. 1999), UV-visible spectrophotometry (Isildak et al. 1999), and derivative chronopotentiometric stripping analysis (La Pera et al. 2008), all of them presenting lower accuracies and repeatability, and in addition not being applied to formulated product matrices. Accordingly, the CIPAC manual that applies for copper-containing pesticides proposes an electrolytic method and a volumetric thiosulfate method, which present lower accuracy and repeatability compared to the spectrophotometry technique presented in this study, whereas they also require higher reagents quantities (CIPAC 1993).

Method Performance
The method has been fully validated to be used for regulatory quality control of pesticides and biocidal PT-21 antifouling products containing copper compounds and commercial copper-containing pesticides.
Recoveries were performed at three fortification levels and in quintuplicate, specifically 5%, 10%, and 15% above the respective formulated biocidal and pesticide product nominal concentration, following addition of appropriate aliquots of the stock standard solution. From the analysis of the spiked samples, the recoveries were found to range between 98.6% and 99.95% for the pesticide products and from 87.9% to 97.6% for the biocidal product, with the %RSD between the separate determinations to be less than 0.92% and 3.74%, for the pesticide and biocide samples, respectively. The calibration curve was y = 0.0404x + 0.0458 (where y stands for absorption and x for concentration) and exhibited a coefficient of regression (R 2 ) of 0.998, which proves the high linearity of the method for variable concentration levels as it can be also observed in Fig. 2. The current method LOQ was estimated to be 5 μg mL −1 , which was also the lowest point in the calibration curve (Fig. 2); thus the corresponding LOD was 1.52 μg mL −1 .
It shall be remarked that, even though linearity was obtainable at higher concentrations, these were not considered for the development of the method, since based on the FAAS fundamentals the maximum acceptable absorption shall not exceed 0.9, in order to achieve high reliability and repeatability. At the same rationale, lower detection and linearity levels could be obtained based on the preliminary tests conducted; however, these were not taken into account for being non-representative for either agricultural or biocidal copper products and could thus create bias to the calibration curve characteristics.

Application to Commercial Pesticides and Biocidal Products
After validation of the method, the two agricultural pesticides and the four PT-21 antifouling biocides of the same active ingredient content were analyzed to assess the copper concentration in each one. This procedure was considered as crucial to examine the method efficiency, under actual quality control procedures of marketed pesticides and biocides, run in the national pesticides control laboratory. The analyses results are presented in detail in Table 1. The nominal concentration of the marine antifouling products was 42.44% of Cu, with the respective findings from our analyses to range from 42.95 to 44.24%. At the same rationale, agricultural copper fungicides contained 40% of Cu, and the respective measured values were 39.43% and 40.71%, respectively. All the examined products were in the acceptable range according to the limits set by FAO and WHO (2022), which is ± 5% for products with declared content between 250 and 500 g kg −1 of active ingredient.

Conclusions
A simple and efficient extraction and analytical determination method for the determination of copper content in antifouling biocidal products was developed and applied. The method was evaluated through a series of comprehensive reliability testing and exhibited good sensitivity, high repeatability, linearity, precision, and acceptable recoveries, and as such can serve the needs for copper-containing biocides quality control, whereas it may also be applied to pesticides as demonstrated by our analyses. The proposed method is preferable compared to other currently available techniques as it provides robust and Fig. 2 The FAAS calibration curve used for samples quantification Table 1 Analytical concentrations in the examined copper containing products *The commercial names of the products used are concealed Sample ID* Nominal Cu concentration (as presented in the product label and legal registration document) Actual instrumental concentration (μg/g) (mean of 5 replications) reliable results with minimal preparation, hazard, and cost, thus it can be easily adapted in quality control laboratories for the copper content control for monitoring commercial products quality, aiming to secure environmental and human safety. Apart from that, it can be easily adapted to serve other organic matrices that require digestion in order to obtain their metal content. Finally, the examined antifouling and agrochemical products obtained from the Greek market, exhibited copper concentrations corresponding to the nominal content declared in their label.
Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by George Pavlidis, Helen Karasali and George P. Balayiannis. The first draft of the manuscript was written by George Pavlidis and Helen Karasali and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.