Rapid and robust analytical method for the determination of copper content in commercial pesticides and antifouling biocides

doi:10.21203/rs.3.rs-2238493/v1

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Rapid and robust analytical method for the determination of copper content in commercial pesticides and antifouling biocides

https://doi.org/10.21203/rs.3.rs-2238493/v1

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published 11 May, 2023

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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. A new analytical method has been developed and is proposed as it fast, efficient, and economical for the determination of copper content in antifouling Product Type 21 (PT-21) biocidal products as well as in copper containing pesticides. The samples were oxidized by microwave-assisted acid digestion method and the concentration of copper was determined by flame atomic absorption spectrometry technique. The method performance presented high recoveries, low relative standard deviation, and sufficient Limit of Quantification (LOQ) for the scope of the present analysis. As a result, it is concluded that the method is easily applicable and transferable, with reasonable consumption of reagents, characterized by 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. 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 easy, with reasonable consumption of reagents, characterized by 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.

copper

antifouling

biocides

pesticides

determination

method

atomic absorption

A new rapid, robust and quick analytical method for copper determination in agrochemicals and biocides is introduced
The method presents excellent QA/QC and cam be easily adopted in every laboratory
It supersedes the commonly used titration technique proposed by CIPAC and used in several EU laboratories for market control

Copper-based compounds are used for a long time as fungicides to control fungi and bacterial diseases in vineyards, pome and stone fruit orchards, besides 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, copper oxide and metallic copper. According to IUPAC, copper hydroxide is the common name for copper (II) hydroxide (or copper(2+) hydroxide or cupric hydroxide), copper oxychloride is the common name for dicopper(II) chloride trihydroxide and copper(I) oxide or cuprous oxide is the common name for copper(I) oxide (or copper(1+) oxide or cuprous oxide) (EFSA 2018).

Copper is extensively allocated in biological tissues, where it occurs mainly in the form of organic complexes, countless of which are metalloproteins and 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 complex protein of connective tissues of the skeleton and blood vessels (EFSA 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 TBT-based products, copper in the form of cuprous oxide (Cu₂O), 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). A content of 30–40% Cu₂O (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 (Lagerstrom and Ytreberg 2021).

It is also a crucial micronutrient used in countless metabolic processes, hence low concentration of it is considered as necessary. However, higher copper concentrations are toxic to humans and other organisms (Lindgren et al. 2018).

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 and 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 in the commercial formulations.

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 (Turner 2010) 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.

Additionally, copper-based compounds 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). The most common copper products used in agriculture are copper oxychloride, copper oxide, copper hydroxide and metallic copper.

A great variety of analytical techniques have been employed in the determination of copper content in the commercially available formulations. CIPAC suggested an electro gravimetric method and redox titration using thiosulphate (CIPAC 1993). Additionally, flame atomic absorption method, inductively coupled plasma optical emission spectroscopy (ICP-OES), UV-visible spectrophotometry and voltammetry have also been used (Balduini et al. 1999; CIPAC 1993; Ferreira et al. 2014; Gallart-Mateu et al. 2015; Isildak et al. 1999; Shrivas et al. 2013). However, the use of digestion along with atomic absorption spectrophotometry was proved that also provides 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 sea water non-target organisms. The first step in this policy is the quality control and the monitoring of plant protection products (PPPs) and biocides which is addressed within the European Union in the context of the adopted regulation 1107/2009 replacing directive 91/414/EEC, as well as Regulation 528/2012/EC concerning marketability of biocidal products (EC 1991; 2009; 2012). On the other hand, another recently introduced EU Directive 128/2009/EC (EC 2009) establishes regular monitoring programs of PPPs in Member States. Antifouling products belong to Product Type 21 sub-category of biocides.

The present study concerns the development, validation and application of a suitable spectroscopic method for the determination of copper in antifouling paints and fungicides with flame atomic absorption spectrometer, a commonly available method in most analytical laboratories. To the best of our knowledge, this is the first report with values regarding copper determination using atomic absorption spectroscopy, that constitutes a simple and economical, yet efficient analytical method.

2.1 Antifouling paints and copper fungicide samples

Antifouling paint samples of blue color used to cover wall surfaces and copper fungicides were provided by the Greek Market during December 2017. Thus, in total, 4 commercial antifouling paints and 2 fungicide agrochemical samples, were selected during December 2017 and analyzed to investigate the variability of copper composition. The agricultural fungicide samples were of a nominal concentration of 57.3% on weight basis as copper hydroxide, corresponding to a copper content of 40%, and the antifouling biocidal products presented in their label a nominal concentration of 47.78% on weight basis of cuprous oxide (Cu₂O), 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.

2.2 Materials

Metal-free chemicals and specifically water (H₂O), nitric acid (HNO₃) and sulphuric acid (H₂SO₄) were obtained from Fisher Scientific (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 1M metal-free grade HNO₃, then rinsed several times with metal-free water and dried at 40^oC in an oven before use. Copper standard solution of trace analysis grade at concentration 1000 µg mL^− 1 was also purchased from Fisher Scientific (UK).

2.3 Working standard preparation

The working standards solutions were prepared daily at four different concentrations (5, 10, 15 and 20 ppm), by appropriate dilutions of the stock solutions with 0.1M HNO₃. Acid-washed plastic vessels were used for preparing and storing solutions.

2.4 Instrumentation

The analysis of copper content in the samples was carried out using a flame atomic absorption spectrophotometer (model AA-6501F, Shimadzu, Germany) with deuterium background correction, equipped with multiple lamps turret and an autosampler (ASC-6000, Shimadzu, Germany). Hollow cathode lamp (Hamamatsu Photonics K.K., 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. 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 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, USA) microwave oven equipped with Omni XP-1500 tubes was used to digest the samples.

2.5 Sample preparation

All samples were mechanically shaken for 60 minutes in a horizontal shaker and homogenized immediately 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. Due to the viscosity of the antifouling samples, i.e., their paint-like texture, special attention was required at this stage and as such single use 150 mm Pasteur pipettes (Kimble, Germany) were used. Subsequently, 5 ml of concentrated metal-free HNO₃ and 5 ml of metal-free H₂SO₄ 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 were not ssufficientto decompose the antifouling paint samples matrix. Since it was prerequisite to develop a unique method both for pesticides and biocides, the combination of nitric acid and sulphuric acid was selected and the maximum solvents volume for the microwave vessels was used. This mixture was then digested for 10 min at a power of 1600 W at a temperature of 160^oC and for additional 5 min at 180^oC, following thus a slightly modified EPA-3052 preparation method for the digestion of siliceous and organically based matrices (US EPA 1996). After cooling and depressurizing, an exact aliquot (0.8 mL) was transferred to Falcons tubes 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.

2.6 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 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 plus standard 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 were determined according to the following equations:

$$LOD=3.33 X \left(\frac{s}{S}\right)$$

$$LOQ=10 X \left(\frac{s}{S}\right)$$

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.

2.7. 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 3 fortification levels and in quintuplicate, specifically 5%, 10%, and 15% above the respective formulated biocidal and pesticide products’ nominal concentration, following addition of appropriate aliquots of the stock standard solution. From the spiked samples’ analysis, the recoveries were found to range between 98.6% and 99.95% for the pesticide products and from 87.9–97.6% 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 (R²) of 0.998 (Fig. 2), while the current method LOQ was estimated to be 5 µg ml^− 1, thus the corresponding LOD was 1.52 µg ml^− 1.

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 & 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 and thus its use was banned (Tessier et al. 2011). Other biocides such as diuron and irgarol, have adverse environmental effect and consequently also banned. Since their ban in the early 1980s, copper has become again the predominant antifouling biocide in 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 (Cu²⁺), with the latter being the most toxic form as it easily migrates through cell membranes.

Application to commercial pesticides and biocidal products - Analysis results

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. The estimation of the tested samples concentration was performed using the following equations:

$$Content \left(\frac{g}{kg}\right)=\frac{Actual Instr. Conc. X 10 mL \left(initial acids dilution\right) X 250 mL \left(final dilution\right)}{0.8 mL \left(from digested solution\right) X Weight \left(mg\right)}$$

where:

$Actual Inst. Conc. = \frac{Sample Absorption-0.0458}{0.0404}$ (4), according to the calibration curve.

Table 1

Analytical concentrations in the examined copper containing products
Sample ID	Nominal Cu concentration (as presented in the product label and registration document)	Actual instrumental Concentration (µg/g) (mean of 5 replications)	Actual Copper content (%) (mean of 5 replications)
Marine AF A	42.44% expressed as Cu	14.03	42.95
Marine AF B		11.91	44.21
Marine AF C		11.85	43.04
Marine AF D		12.92	42.97
WG Fungicide A	40% expressed as Cu	13.08	40.71
WG Fungicide B	40% expressed as Cu	12.54	39.43

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. The examined biocidal antifouling products obtained from the Greek market, exhibited copper concentrations in the acceptable range according to the limits set by FAO ανδ WHO (2022).

Disclosure statement

All authors have no conflict of interest to declare.

Funding

No funding was obtained for the present research.

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No competing interests reported.

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You are reading this latest preprint version

Rapid and robust analytical method for the determination of copper content in commercial pesticides and antifouling biocides

Status:

Journal Publication

Version 1

Abstract

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Highlights

1. Introduction

2. Materials And Methods

2.1 Antifouling paints and copper fungicide samples

2.2 Materials

2.3 Working standard preparation

2.4 Instrumentation

2.5 Sample preparation

2.6 Quality control

2.7. Method performance

3. Results And Discussion

3. Conclusions

Declarations

References

Additional Declarations

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Journal Publication

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