Name: determination of the absorption, translocation, and distribution of imidacloprid in wheat
Uploaded: 2023-04-28T14:00:00
Description: Watch this Scientific Journal Video about Determination of the Absorption, Translocation, and Distribution of Imidacloprid in Wheat at JoVE.com

This work was supported by the National Natural Science Foundation of China (No. 42277039).

1. Germination of wheat seeds
<ol>
	<li>Select 1,000 wheat seeds (Jimai 20) with complete granules, intact embryos, and uniform size (length: 6 mm &#177; 0.5 mm).</li>
	<li>Transfer 333.3 mL of 30% H2O2 solution to a 1 L volumetric flask and dilute with deionized water to prepare 1 L of 10% H2O2 solution. Immerse the wheat seeds in 10% H2O2 solution for 15 min to disinfect the seed surface (Figure 1).</li>
	...

<ol>
	<li>Lin, P. C., Lin, H. J., Liao, Y. Y., Guo, H. R., Chen, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+poisoning+with+neonicotinoid+insecticides:+A+case+report+and+literature+review.">Acute poisoning with neonicotinoid insecticides: A case report and literature review.</a> Basic &amp; Clinical Pharmacology &amp; Toxicology. 112 (4), 282-286 (2013).</li><li>North, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Value+of+neonicotinoid+insecticide+seed+treatments+in+Mid-South+corn+(Zea+mays)+production+systems.">Value of neonicotinoid insecticide seed treatments in Mid-South corn (Zea mays) production systems.</a> Journal of Economic Entomology. 111 (1), 187-192 (2018).</li><li>Simon-Delso, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+insecticides+(neonicotinoids+and+fipronil):+Trends,+uses,+mode+of+action+and+metabolites.">Systemic insecticides (neonicotinoids and fipronil): Trends, uses, mode of action and metabolites.</a> Environmental Science and Pollution Research. 22 (1), 5-34 (2015).</li><li>Bass, C., Denholm, I., Williamson, M. S., Nauen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+status+of+insect+resistance+to+neonicotinoid+insecticides.">The global status of insect resistance to neonicotinoid insecticides.</a> Pesticide Biochemistry and Physiology. 121, 78-87 (2015).</li><li>Craddock, H. A., Huang, D., Turner, P. C., Quiros-Alcala, L., Payne-Sturges, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+neonicotinoid+pesticide+residues+in+food+and+water+in+the+United+States,+1999-2015.">Trends in neonicotinoid pesticide residues in food and water in the United States, 1999-2015.</a> Environmental Health. 18 (1), 7 (2019).</li><li>Shao, X. S., Liu, Z. W., Xu, X. Y., Li, Z., Qian, X. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overall+status+of+neonicotinoid+insecticides+in+China:+Production,+application+and+innovation.">Overall status of neonicotinoid insecticides in China: Production, application and innovation.</a> Journal of Pesticide Science. 38 (1-2), 1-9 (2013).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+neonicotinoid+insecticides+in+children+from+South+China:+Concentrations,+profiles+and+influencing+factors.">Urinary neonicotinoid insecticides in children from South China: Concentrations, profiles and influencing factors.</a> Chemosphere. 291, 132937 (2022).</li><li>Kurwadkar, S., Evans, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonicotinoids:+Systemic+insecticides+and+systematic+failure.">Neonicotinoids: Systemic insecticides and systematic failure.</a> Bulletin of Environmental Contamination and Toxicology. 97 (6), 745-748 (2016).</li><li>Sadaria, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passage+of+fiproles+and+imidacloprid+from+urban+pest+control+uses+through+wastewater+treatment+plants+in+northern+California,+USA.">Passage of fiproles and imidacloprid from urban pest control uses through wastewater treatment plants in northern California, USA.</a> Environmental Toxicology and Chemistry. 36 (6), 1473-1482 (2017).</li><li>Damalas, C. A., Eleftherohorinos, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pesticide+exposure,+safety+issues,+and+risk+assessment+indicators.">Pesticide exposure, safety issues, and risk assessment indicators.</a> International Journal of Environmental Research and Public Health. 8 (5), 1402-1419 (2011).</li><li>Hayes, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demasculinization+and+feminization+of+male+gonads+by+atrazine:+Consistent+effects+across+vertebrate+classes.">Demasculinization and feminization of male gonads by atrazine: Consistent effects across vertebrate classes.</a> Journal of Steroid Biochemistry and Molecular Biology. 127 (1-2), 64-73 (2011).</li><li>Rani, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extensive+review+on+the+consequences+of+chemical+pesticides+on+human+health+and+environment.">An extensive review on the consequences of chemical pesticides on human health and environment.</a> Journal of Cleaner Production. 283, 124657 (2021).</li><li>Xu, Y. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecotoxicity+evaluation+of+azoxystrobin+on+Eisenia+fetida+in+different+soils.">Ecotoxicity evaluation of azoxystrobin on Eisenia fetida in different soils.</a> Environmental Research. 194, 110705 (2021).</li><li>Yavari, S., Malakahmad, A., Sapari, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochar+efficiency+in+pesticides+sorption+as+a+function+of+production+variables-A+review.">Biochar efficiency in pesticides sorption as a function of production variables-A review.</a> Environmental Science and Pollution Research. 22 (18), 13824-13841 (2015).</li><li>Delcour, I., Spanoghe, P., Uyttendaele, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Literature+review:+Impact+of+climate+change+on+pesticide+use.">Literature review: Impact of climate change on pesticide use.</a> Food Research International. 68, 7-15 (2015).</li><li>Zhang, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+the+QuEChERS+methodology+in+the+determination+of+antibiotics+in+food:+A+review.">The application of the QuEChERS methodology in the determination of antibiotics in food: A review.</a> TrAC-Trends in Analytical Chemistry. 118, 517-537 (2019).</li><li>Wiilkowska, A., Biziuk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+pesticide+residues+in+food+matrices+using+the+QuEChERS+methodology.">Determination of pesticide residues in food matrices using the QuEChERS methodology.</a> Food Chemistry. 125 (3), 803-812 (2011).</li><li>Ishii, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPLC+determination+of+the+new+insecticide+imidacloprid+and+its+behavior+in+rice+and+cucumber.">HPLC determination of the new insecticide imidacloprid and its behavior in rice and cucumber.</a> Journal of Agricultural and Food Chemistry. 42 (12), 2917-2921 (1994).</li><li>Ko, A. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simple+extraction+and+oxidation+procedure+for+the+residue+analysis+of+imidacloprid+and+its+metabolites+in+lettuce+using+gas+chromatography.">Development of a simple extraction and oxidation procedure for the residue analysis of imidacloprid and its metabolites in lettuce using gas chromatography.</a> Food Chemistry. 148, 402-409 (2014).</li><li>Yuan, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+imidacloprid+controlled-release+granules+to+enhance+the+utilization+rate+and+control+wheat+aphid+on+winter+wheat.">Application of imidacloprid controlled-release granules to enhance the utilization rate and control wheat aphid on winter wheat.</a> Journal of Integrative Agriculture. 19 (12), 3045-3053 (2020).</li><li>Phugare, S. S., Kalyani, D. C., Gaikwad, Y. B., Jadhav, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+degradation+of+imidacloprid+and+toxicological+analysis+of+its+biodegradation+metabolites+in+silkworm+(Bombyx+mori).">Microbial degradation of imidacloprid and toxicological analysis of its biodegradation metabolites in silkworm (Bombyx mori).</a> Chemical Engineering Journal. 230, 27-35 (2013).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake,+translocation+and+accumulation+of+imidacloprid+in+six+leafy+vegetables+at+three+growth+stages.">Uptake, translocation and accumulation of imidacloprid in six leafy vegetables at three growth stages.</a> Ecotoxicology and Environmental Safety. 164, 690-695 (2018).</li><li>Badawy, M. E. I., Ismail, A. M. E., Ibrahim, A. I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+acetamiprid+and+imidacloprid+residues+in+tomato+fruits+under+greenhouse+conditions.">Quantitative analysis of acetamiprid and imidacloprid residues in tomato fruits under greenhouse conditions.</a> Journal of Environmental Science and Health Part B-Pesticides Food Contaminants and Agricultural Wastes. 54 (11), 898-905 (2019).</li><li>Zhai, R. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residue,+dissipation+pattern,+and+dietary+risk+assessment+of+imidacloprid+in+Chinese+chives.">Residue, dissipation pattern, and dietary risk assessment of imidacloprid in Chinese chives.</a> Frontiers in Nutrition. 9, 846333 (2022).</li><li>Aria, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+and+translocation+monitoring+of+imidacloprid+to+chili+and+tomato+plants+by+molecularly+imprinting+extraction+-+ion+mobility+spectrometry.">Uptake and translocation monitoring of imidacloprid to chili and tomato plants by molecularly imprinting extraction - ion mobility spectrometry.</a> Microchemical Journal. 144, 195-202 (2019).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+and+metabolism+of+imidacloprid+in+cabbage:+Application+of+C-14-labelling+and+LC-QTOF-MS.">Translocation and metabolism of imidacloprid in cabbage: Application of C-14-labelling and LC-QTOF-MS.</a> Chemosphere. 263, 127928 (2021).</li><li>Wild, S., Jones, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+chemicals+entering+agricultural+soils+in+sewage+sludges:+Screening+for+their+potential+to+transfer+to+crop+plants+and+livestock.">Organic chemicals entering agricultural soils in sewage sludges: Screening for their potential to transfer to crop plants and livestock.</a> Science of the Total Environment. 119, 85-119 (1992).</li><li>Gong, W. 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In recent years, methods for the pretreatment and detection of residues of the pesticide imidacloprid have been frequently reported. Badawy et al.23 used high-performance liquid chromatography to determine the content of imidacloprid in tomato fruit grown under greenhouse conditions and reported good linearity for imidacloprid in the range 0.0125-0.15 &#181;g/mL. Zhai et al.24 used LC-MS-MS to study the residue of imidacloprid in Chinese chives. In the present study, the Qu...

In present day agronomy, the use of pesticides is essential to increase crop yield. Neonicotinoid insecticides alter the membrane potential balance by controlling nicotinic acetylcholine receptors in the insect nervous system, thereby inhibiting the normal conduction of the insect central nervous system, leading to the paralysis and death of the insects1. Compared with traditional insecticides, neonicotinoids have advantages such as novel modes of action, high insecticidal activity, and strong root absorption, making them highly successful in the pesticide market2,3. The sales volume of neonicotinoids was reported to account for 27% of the world pesticide market in 2014. The average annual growth rate of neonicotinoids was 11.4% from 2005 to 2010, of which about 7% was registered in China4,5,6. From the end of 2016 to the first half of 2017, the sales of pesticides in China began to rebound after falling, and the prices of pesticides continued to rise, among which neonicotinoid insecticides showed a significant price increase7. So far, three generations of neonicotinoid insecticides have been developed, each containing pyridine chloride, thiazolyl, and tetrahydrofuran groups of nicotine, respectively8.
Imidacloprid represents the first generation of neonicotinoid insecticides, whose molecular formula is C9H10ClN5O2, and is a colorless crystal. Imidacloprid is used mainly to control pests, such as aphids, planthoppers, mealworms, and thrips9 and can be applied to crops such as rice, wheat, corn, cotton, and vegetables such as potatoes, as well as fruit trees. Due to the long-term, substantial, and continual application of pesticides, both beneficial insects and the natural enemies of pests have been rapidly reduced, and some agricultural pests have become resistant to pesticides, resulting in a vicious circle of applying continual and increasing amounts of pesticides10. In addition, the extensive application of pesticides has led to the deterioration of soil quality, persistent pesticide residues in agricultural products, and other ecological problems, which not only cause significant damage to the agricultural ecological environment11 but also pose a serious threat to human health12. Pesticide spraying severely impacts the growth and quality of soil microbes and soil animals13. The unreasonable or excessive use of pesticides has caused significant security risks to the soil and water environment, animals and plants, and even human life14. In recent years, the problem of excessive pesticide residues in crops has become more severe with the extensive application of pesticides. When imidacloprid was used to increase vegetable yield, the absorption rate of imidacloprid in the vegetables increased with the increase in the amount and residue of imidacloprid15. As a major food crop, both the production and safety of wheat are critical. Therefore, the residue and distribution policies of pesticides used for wheat need to be clarified.
In recent years, many methods have been developed to extract imidacloprid residues from water, soil, and plants. The QuEChERS method (quick, easy, cheap, effective, rugged, and safe) is a new method that combines solid-phase microextraction technology and dispersed solid-phase extraction technology and involves the use of acetonitrile as the extraction solvent and the removal of mixed impurities and water in the sample using NaCl and anhydrous MgSO4, respectively16. The QuEChERS method requires minimal glassware and has simple experimental steps, making it one of the most popular pesticide extraction methods17. For the detection of imidacloprid, a detection limit as low as 1 &#215; 10&#8722;9 g18 has been achieved with liquid chromatography (LC), and 1 &#215; 10&#8722;11 g19 has been achieved with gas chromatography (GC). Due to their high resolution and sensitivity, LC-MS and GC-MS have shown even lower imidacloprid detection limits of 1 &#215; 10-13 to 1 &#215; 10-14 g20,21; these techniques are, therefore, well suited for the analysis of trace imidacloprid residues.
In the present study, imidacloprid was chosen as the target pollutant, and wheat was selected as the test crop to study the distribution of imidacloprid residues in wheat. This protocol details a method for the comprehensive analysis of the enrichment and transfer of the pesticide imidacloprid in wheat by exploring the absorption and storage of imidacloprid in different parts of wheat plants grown under hydroponic conditions. The present study aims to provide a theoretical basis for the risk assessment of pesticide residues in wheat, guide the rational application of pesticides in agricultural production activities to reduce pesticide residues, and improve the safety of crop production.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich (Shanghai) Trading Co. Ltd.</td>
			<td>01-06-1995</td>
			<td>Suitable for HPLC, gradient grade, &#62;99.9%</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Sartorius Lab Instruments Co.Ltd.</td>
			<td>GL124-1SCN</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial climate incubator&#160;&#160;</td>
			<td>Shanghai Badian Instrument Equipment Co. Ltd.</td>
			<td>HK320</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf China Co. Ltd.</td>
			<td>Centrifuge5804</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable syringe</td>
			<td>Sigma-Aldrich (Shanghai) Trading Co. Ltd.</td>
			<td>Z116866</td>
			<td>Capacity 5 mL, graduated 0.2 mL, non-sterile</td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Sigma-Aldrich (Shanghai) Trading Co. Ltd.</td>
			<td>Y0001970</td>
			<td>European pharmacopoeia reference standard</td>
		</tr>
		<tr>
			<td>Graphitized carbon black (GCB)</td>
			<td>Sigma-Aldrich (Shanghai) Trading Co. Ltd.</td>
			<td>V900058</td>
			<td>45 &#956;m</td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma-Aldrich (Shanghai) Trading Co.Ltd.</td>
			<td>31642</td>
			<td>30% (w/w)</td>
		</tr>
		<tr>
			<td>Hoagland&#8217;s Basal Salt Mixture</td>
			<td>Shanghai Yu Bo Biotech Co. Ltd.</td>
			<td>NS1011</td>
			<td>Anhydrous, reagent grade</td>
		</tr>
		<tr>
			<td>Hydroponic equipment</td>
			<td>Jiangsu Rongcheng Agricultural Science and Technology Development Co.Ltd.</td>
			<td>SDZ04BD</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypersil BDS C18 column</td>
			<td>Thermo Fisher Scientific (China) Co. Ltd.</td>
			<td>28103-102130</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidacloprid</td>
			<td>Sigma-Aldrich (Shanghai) Trading Co. Ltd.</td>
			<td>Y0002028</td>
			<td>European pharmacopoeia reference standard</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich (Shanghai) Trading Co. Ltd.</td>
			<td>208094</td>
			<td>Anhydrous, reagent grade, &#62;97%</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich (Shanghai) Trading Co.Ltd.</td>
			<td>S9888</td>
			<td>Reagent grade, 99%</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Shanghai Thunder Magnetic Instrument Factory</td>
			<td>PHSJ-3F</td>
			<td></td>
		</tr>
		<tr>
			<td>Phytotron box</td>
			<td>Harbin Donglian Electronic Technology Co. Ltd.</td>
			<td>HPG-280B</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Eppendorf China Co. Ltd.</td>
			<td>Research plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Sigma-Aldrich (Shanghai) Trading Co.Ltd.</td>
			<td>SLGV033N</td>
			<td>Nylon, 0.22 &#181;m pore size, 33 mm, non-sterile</td>
		</tr>
		<tr>
			<td>Ultra performance liquid chromatography tandem triple quadrupole mass spectrometry</td>
			<td>Thermo Fisher Scientific (China) Co. Ltd.</td>
			<td>UltiMate 3000</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>TSQ Quantum Access MAX</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Shanghai Yetuo Technology Co. Ltd.</td>
			<td>Vortex-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat seed</td>
			<td>LuKe seed industry</td>
			<td></td>
			<td>Jimai 20</td>
		</tr>
	</tbody>
</table>

determination of the absorption, translocation, and distribution of imidacloprid in wheat

Neonicotinoids, a class of insecticides, are widely used because of their novel modes of action, high insecticidal activity, and strong root uptake. Imidacloprid, the most widely used insecticide worldwide, is a representative first-generation neonicotinoid and is used in pest control for crops, vegetables, and fruit trees. With such a broad application of imidacloprid, its residue in crops has attracted increasing scrutiny. In the present study, 15 wheat seedlings were placed in a culture medium containing 0.5 mg/L or 5 mg/L imidacloprid for hydroculture. The content of imidacloprid in the wheat roots and leaves was determined after 1 day, 2 days, and 3 days of hydroculture to explore the migration and distribution of imidacloprid in wheat. The results showed that imidacloprid was detected both in the roots and leaves of the wheat plant, and the content of imidacloprid in the roots was higher than that in the leaves. Furthermore, the imidacloprid concentration in the wheat increased with increasing exposure time. After 3 days of exposure, the roots and leaves of the wheat in the 0.5 mg/L treatment group contained 4.55 mg/kg &plusmn; 1.45 mg/kg and 1.30 mg/kg &plusmn; 0.08 mg/kg imidacloprid, respectively, while the roots and leaves of the 5 mg/L treatment group contained 42.5 mg/kg &plusmn; 0.62 mg/kg and 8.71 mg/kg &plusmn; 0.14 mg/kg imidacloprid, respectively. The results from the present study allow for a better understanding of pesticide residues in crops and provide a data reference for the environmental risk assessment of pesticides.

The instrument limit of detection (LOD) of imidacloprid was 5.76 &#215; 10&#8722;14 g, and the method&#39;s LOD of imidacloprid in the wheat root or leaf was 0.01 &#181;g/kg; no matrix effect was observed. The recovery yields of imidacloprid in wheat are shown in Table 2. The recovery yields of imidacloprid from the wheat roots exposed to imidacloprid concentrations of 0.5 mg/L and 5 mg/L were 94.0%-97.6% and 98.8%-99.2%, respectively; the coefficients of variation were 1.92% and 0.20%, respec...

Watch this Scientific Journal Video about Determination of the Absorption, Translocation, and Distribution of Imidacloprid in Wheat at JoVE.com

Determination of the Absorption, Translocation, and Distribution of Imidacloprid in Wheat

Presented here is a protocol for the determination of the absorption, translocation, and distribution of imidacloprid in wheat under hydroponic conditions using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The results showed that imidacloprid can be absorbed by wheat, and imidacloprid was detected in both the wheat roots and leaves.

Neonicotinoids, a class of insecticides, are widely used because of their novel modes of action, high insecticidal activity, and strong root uptake. ...

This method helps to answer how Imidacloprid is absorbed, transported, and distributed in wheat. The main advantage of this technique is the high sensitivity of imidacloprid detection, which can be widely used to determine imidacloprid in soil, water, plant, and other samples. To germinate the wheat seeds, select 1000 suitable seeds having complete granules, intact embryos, and uniform size.Immerse the seeds in 10%hydrogen peroxide solution for 15 minutes to disinfect their surfaces. Then rinse the seeds five times by running sterile water for 10 seconds each time. Now, spread the wheat seeds evenly with the embryos pointing up in a glass Petri dish containing moist, sterile filter paper.Place the Petri dish in an artificial climate incubator maintained at 30 degrees Celsius and 80%relative humidity. Culture the wheat seeds in the dark for three days until they germinate and develop roots. After the wheat seeds have germinated, place 15 wheat seedlings in a hydroponic apparatus containing 100 milliliters of half Hoaglands nutrient solution.Then, place the entire hydroponic setup in an artificial climate incubator at 25 degrees Celsius and 80%relative humidity for seven days. To set up the pesticide exposure experiments with two different imidacloprid concentrations, prepare hydroponic devices containing half Hoaglands nutrient solution with either 0.5 milligrams per liter or five milligrams per liter of imidacloprid. Set up 15 hydroponic devices for each imidacloprid concentration group to ensure adequate samples during sampling.Now, transplant 15 wheat plants post their seven-day hydroponic period into each prepared hydroponic device. Place the entire hydroponic setup in an artificial climate incubator for three days at 25 degrees Celsius and 80%relative humidity. Throughout the imidacloprid exposure period, collect roots and leaves from the plants daily.For each imidacloprid concentration, integrate the wheat samples from every fifth of the 15 hydroponic devices as a parallel group. To extract imidacloprid from wheat roots, shred the wheat roots using scissors into approximately one centimeter pieces. Weigh 10 grams of the shredded wheat roots and place them in a 50 milliliter centrifuge tube.Then, add 10 milliliters of acetyl nitrile to the centrifuge tube and vortex the tube for one minute before adding four grams of anhydrous magnesium sulfate and 1.5 grams of sodium chloride to the tube. Immediately vortex the tube for 30 seconds. Next, centrifuge the tube at 6, 000 G for five minutes.Aspirate the supernatant with a disposable syringe and pass it through a 0.22 micron syringe filter to obtain the sample. To extract imidacloprid from wheat leaves, shred the fresh leaves using scissors into pieces of approximately one centimeter. Weigh 10 grams of the shredded wheat leaves and place them in a 50 milliliter centrifuge tube.Add 10 milliliters of acetonitrile to 10 grams of the shredded wheat leaves in a 50 milliliter centrifuge tube and vortex for one minute. Then treat the mixture with anhydrous magnesium sulfate and sodium chloride, and centrifuge as demonstrated previously. To remove pigmentation and moisture from the sample, take two milliliters of the supernatant obtained after the centrifugation and add it to a five milliliter centrifuge tube containing graphitized carbon black and anhydrous magnesium sulfate.After 30 seconds of vortex mixing, centrifuge the tube at 6, 000 G for five minutes, followed by aspirating the supernatant with a disposable syringe and passing it through a 0.22 micron syringe filter to obtain the sample. Use liquid chromatography tandem mass spectrometry to quantify the amount of imidacloprid from the wheat samples as described in the manuscript. The recovery yields of imidacloprid from wheat samples showed that the average imidacloprid recoveries from wheat roots and leaves exposed to 0.5 milligrams per liter of imidacloprid or lesser compared to the average recoveries from samples exposed to five milligrams per liter of imidacloprid.Imidacloprid was detected in both roots and leaves of wheat plants even after one day of exposure, indicating its rapid absorbance and conductance into the plants. The imidacloprid content was higher on day three of exposure than on day one, and the imidacloprid concentration was consistently higher in the roots than in the leaves. No apparent inhibition of plant growth was observed, even after three days of imidacloprid exposure in plants exposed to imidacloprid as compared to the controls.The root concentration factor, or RCF, in the treated samples was greater than one, indicating wheat has an enrichment effect on imidacloprid. However, the translocation factor, or TF, for leaves was less than one, suggesting that the pesticide was not easily transferred from the wheat roots to the leaves. The most important steps are the extraction and purification of imidacloprid.Ensure that imidacloprid is extracted without loss during these steps.

determination-absorption-translocation-distribution-imidacloprid

Research

JoVE Journal

Environment

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female&#39;s seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George&#39;s Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

Fishery-induced changes to exploited crustacean fisheries, such as the American lobster fishery, could potentially influence their reproductive dynamics, leading to a reduction in mating success. This study&#39;s goal was to develop a noninvasive method for ascertaining the mating success of female lobsters that may be physiologically or functionally mature.

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American ...

Determination of Inorganic Arsenic in a Wide Range of Food Matrices using Hydride Generation - Atomic Absorption Spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to inorganic arsenic through diet, information about distribution of arsenic species in various food types must be generated. A method, previously validated in a collaborative trial, has been applied to determine inorganic arsenic in a wide variety of food matrices, covering grains, mushrooms and food of marine origin (31 samples in total). The method is based on detection by flow injection-hydride generation-atomic absorption spectrometry of the iAs selectively extracted into chloroform after digestion of the proteins with concentrated HCl. The method is characterized by a limit of quantification of 10 &#181;g/kg dry weight, which allowed quantification of inorganic arsenic in a large amount of food matrices. Information is provided about performance scores given to results obtained with this method and which were reported by different laboratories in several proficiency tests. The percentage of satisfactory results obtained with the discussed method is higher than that of the results obtained with other analytical approaches.

The usefulness of an analytical method to determine inorganic arsenic in a wide range of food matrices is demonstrated. The method consists of selective extraction of inorganic arsenic into chloroform with a final determination by hydride generation-atomic absorption spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to ...

Determination of the Settling Rate of Clay/Cyanobacterial Floccules

The mechanisms underpinning the deposition of fine-grained, organic-rich sediments are still largely debated. Specifically, the impact of the interaction of clay particles with reactive, planktonic cyanobacterial cells to the sedimentary record is under studied. This interaction is a potentially major contributor to shale depositional models. Within a lab setting, the flocculation and sedimentation rates of these materials can be examined and measured in a controlled environment. Here, we detail a protocol for measuring the sedimentation rate of cyanobacterial/clay mixtures. This methodology is demonstrated through the description of two sample experiments: the first uses kaolin (a dehydrated form of kaolinite) and Synechococcus sp. PCC 7002 (a marine coccoid cyanobacteria), and the second uses kaolin and Synechocystis sp. PCC 6803 (a freshwater coccoid cyanobacteria). Cyanobacterial cultures are mixed with varying amounts of clay within a specially designed tank apparatus optimized to allow continuous, real-time video and photographic recording. The sampling procedures are detailed as well as a post-collection protocol for precise measurement of chlorophyll a from which the concentration of cyanobacterial cells remaining in suspension can be determined. Through experimental replication, a profile is constructed that displays sedimentation rate.

The interaction and sedimentation of the clay and bacterial cells within the marine realm, observed in natural environments, can be best investigated in a controlled lab environment. Here, we describe a detailed protocol, which outlines a novel method for measuring the sedimentation rate of clay and cyanobacterial floccules.

The mechanisms underpinning the deposition of fine-grained, organic-rich sediments are still largely debated. Specifically, the impact of the interaction ...

Wastewater Irrigation Impacts on Soil Hydraulic Conductivity: Coupled Field Sampling and Laboratory Determination of Saturated Hydraulic Conductivity

Since the early 1960s, an alternative wastewater discharge practice at The Pennsylvania State University has been researched and its impacts monitored. Rather than discharging treated wastewater to a stream, and thereby directly impacting the stream quality, the effluent is applied to forested and cropped land managed by the University. Concerns related to reductions in soil hydraulic conductivity occur when considering wastewater reuse. The methodology described in this manuscript, matching soil sample size with the size of the laboratory-based hydraulic conductivity measurement apparatus, provides the benefits of a relatively rapid collection of samples with the benefits of controlled laboratory boundary conditions. The results suggest that there may have been some impact of wastewater reuse on the soil&#39;s ability to transmit water at deeper depths in the depressional areas of the site. Most of the reductions in the soil hydraulic conductivity in the depressions appear to be related to the depth from which the sample was collected, and by inference, associated with the soil structural and textural differences.

Here we present a methodology which matches a soil sample size and a hydraulic conductivity measurement device to prevent the so-called wall flow along the inside of the soil container from being erroneously included in water flow measurements. Its use is demonstrated with samples collected from a wastewater irrigation site.

Since the early 1960s, an alternative wastewater discharge practice at The Pennsylvania State University has been researched and its impacts monitored. ...

Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) coupled with synchrotron X-radiation. A cube-shaped sample assembly is prepared and compressed, at room temperature, by a set of four X-ray transparent sintered diamond anvils and two tungsten carbide anvils, in the lateral and the vertical planes, respectively. All six anvils are housed within a 250-ton hydraulic press and driven inward simultaneously by two wedged guide blocks. A horizontal energy dispersive X-ray beam is projected through and diffracted by the sample assembly. The beam is commonly in the mode of either white or monochromatic X-ray. In the case of white X-ray, the diffracted X-rays are detected by a solid-state detector array that collects the resulting energy dispersive diffraction pattern. In the case of monochromatic X-ray, the diffracted pattern is recorded using a two-dimensional (2-D) detector, such as an imaging plate or a charge-coupled device (CCD) detector. The 2-D diffraction patterns are analyzed to derive lattice spacings. The elastic strains of the sample are derived from the atomic lattice spacing within grains. The stress is then calculated using the predetermined elastic modulus and the elastic strain. Furthermore, the stress distribution in two-dimensions allow for understanding how stress is distributed in different orientations. In addition, a scintillator in the X-ray path yields a visible light image of the sample environment, which allows for the precise measurement of sample length changes during the experiment, yielding a direct measurement of volume strain on the sample. This type of experiment can quantify the stress distribution within geomaterials, which can ultimately shed light on the mechanism responsible for compaction. Such knowledge has the potential to significantly improve our understanding of key processes in rock mechanics, geotechnical engineering, mineral physics, and material science applications where compactive processes are important.

We report detailed procedures for compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus coupled with synchrotron X-radiation. Such experiments allow quantification of the stress distribution within samples, that ultimately sheds light on compaction processes in geomaterials.

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water distribution in plant tissues in combination with freeze fixation techniques using liquid nitrogen (LN2). For woody species, however, preparations for observing the xylem transverse-cut surface involve some difficulties due to the orientation of wood fibers. Additionally, higher tension in the water column in xylem conduits can occasionally cause artifactual changes in water distribution, especially during sample fixation and collection. In this study, we demonstrate an efficient procedure to observe the water distribution within the xylem of woody plants in situ&#160;by using a cryostat and cryo-SEM. At first, during sample collection, measuring the xylem water potential should determine whether high tension is present in the xylem conduits. When the xylem water potential is low (&#60; ca. &#8722;0.5 MPa), a tension relaxation procedure is needed to facilitate better preservation of the water status in xylem conduits during sample freeze fixation. Next, a watertight collar is attached around the tree stem and filled with LN2&#160;for freeze fixation of the water status of xylem. After harvesting, care should be taken to ensure that the sample is preserved frozen while completing the procedures of sample preparation for observation. A cryostat is employed to clearly expose the xylem transverse-cut surface. In cryo-SEM observations, time adjustment for freeze-etching is required to remove frost dust and accentuate the edge of the cell walls on the viewing surface. Our results demonstrate the applicability of cryo-SEM techniques for the observation of water distribution within xylem at cellular and subcellular levels. The combination of cryo-SEM with non-destructive in situ observation techniques will profoundly improve the exploration of woody plant water flow dynamics.

Observing the water distribution within the xylem provides significant information regarding water flow dynamics in woody plants. In this study, we demonstrate the practical approach to observe xylem water distribution in situ&#160;by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation.

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water ...