Research and Advances: Environmental Sciences

ISSN: 2652-3655

Research Article

Residue Dynamics of Hydrogen Cyanamide and Ethephon in Apple

Sapna Katna1, Jatiender Kumar Dubey1*, Surender Kumar Patyal1, Nisha Devi1, Ashraf AlamWani2, Gagandeep Singh1, Gaganpreet Singh Brar1 and Ajay Sharma1

1Department of Entomology, Dr Y.S. Parmar University of Horticulture and Forestry, India

2Research Centre for Residue and Quality Analysis (RCRQA) Laboratory of SKAUST, India

Received: 12 July 2019

Accepted: 23 August 2019

Version of Record Online: 10 September 2019


Katna S, Dubey JK, Patyal SK, Devi N, AlamWani A, et al. (2019) Residue Dynamics of Hydrogen Cyanamide and Ethephon in Apple. Res Adv Environ Sci2019(1): 61-68.

Correspondence should be addressed to
Jatiender Kumar Dubey, India



Copyright © 2019 Jatiender Kumar Dubey et al. This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and work is properly cited.


In the present study, supervised field trials were conducted in Royal delicious variety of apple trees to evaluate the persistence of hydrogen cyanamide and ethephon during 2015-16 at different locations of Himachal Pradesh and Jammu & Kashmir. Standardised analytical methods simulated to the lab conditions were validated and adopted to find out the recoveries of hydrogen cyanamide and ethephon in apple fruits. Residues of hydrogen cyanamide were determined by HPLC-PDA with mobile phase comprising of methanol and water (2:8) following the application of Dormex 49% AS at the rate of 0.5% a.i., 1.0 % a.i. and 2.0 % a.i. during dormant stage of apple trees. Formulation of ethrel 39% SL was applied on fruit bearing trees close to harvest stage were determined as ethylene by head space analysis. Hydrogen Cyanamide residues were not detected in any sample right from walnut stage of apple after 85-111 days of treatment to the final harvesting between 179-209 days irrespective of any location.It has been observed that spray application of ethephon caused early fruit drop therefore, samples from all the four locations were analysed up to 10 days. Initial deposits of ethephon as ethylene on apple fruits at Matiana and Thanedhar ranges between 2.044 - 2.147 mg kg-1and 4.036- 4.133 mg kg-1 which reduces between 0.056-0.057 and 0.083-0.088 mg kg-1on day 10 due to the single and double dose application, respectively. Harvest time residues of ethephon in Youngora and Lassipors locations of J&K were 0.100 and 0.099 and 0.143 and 0.137 mg kg-1 at single and double dose, respectively. A safe waiting period of 5 days is suggested between ethephon application and fruit harvest to safeguard the consumer’s health. No residues of hydrogen cyanamide were detected in fruits and hence, does not pose any threat to the consumers.


Dormex; Ethephon; Harvest; Hydrogen Cyanamide; Residues


Apple (Malus domestica) is commercially cultivated in temperate zone of the world and India is fourth largest producer of apple after banana, orange and grape fruits [1]. Like other plants, growth and development of apple plant is regulated by Plant Growth Regulators (PGRs) which ultimately affect fruit quality and quantity[2]. Ethephon and hydrogen cyanamide are plant growth regulators whose titer affect the fruit yield. Apple is a deciduous plant which stops growing, lose their leaves and enter in a state of dormancy thereby provide protection to the plant from freezing temperature during winter. Deciduous fruit tree does not resume normal growth, including flowering and fruit set, until it has experienced an amount of cold equal to its minimum chilling requirement [3]. Fruit trees grown in areas where temperature during winter islacking to satisfy the variety’s chilling requirement, then blooming and foliation can be delayed which results in erratic fruit set and poor fruit quality besides reduction in fruit yield[4]. Grapes grown in warmer climates are reported to receive inadequate chilling, resulting in delayed and erratic bud break. Insufficient chilling requirement can be overcome by exogenous application of Dormex® as a commercial formulation having hydrogen cyanamide (H2CN2) as an active ingredient. Hydrogen cyanamide is generally applied to grapevines after pruning, about four to eight weeks before bud break [5]. Most of the researchersin the past have agreed upon the effectiveness of hydrogen cyanamide as bud breaking agent [4,6-11]. Hydrogen cyanamide had been used frequently to break the dormancy in grapevine floral buds [12,13] besides, its use in other fruit species like kiwi, peach and other stone fruits[14,15]. Hydrogen cyanamide alone and in combination with mineral oil has shown positive results in apple trees [16,17] and enhanced fruit yield.

The fluctuations in weather conditions due to various contributing factors leading to rise in temperature from 1.8 to 4.8°C in the past few decades reflected in decrease of effective chilling hours during winter[18] and early rise in temperature during summer. The increase in minimum temperature was more for the low altitude areas (below 700 m amsl) as compared to higher altitude areas [19]. Fruit colour development is poor in apple orchards situated in warmer and lower (below 1800 m) elevations which decline the market acceptability and crop quality value. Ethephon has been found to be effective in improving the colour of fruits 7-10 days before harvesting [20] to attract customer attention and thus fetching higher price in the market.

However, the plant growth regulators hydrogen cyanamide and ethephon are classified as pesticides and are being regulated for sale purpose under Insecticide Act 1968 in India. Impact of Dormex® at 2% on red Fuji apple gave the benefits of compressing or advancing flowering, depending on the time of application, without any detrimental effects[10]. Preliminary work in Brazil [21] has shown that application of hydrogen cyanamide can break the winter dormancy in apple trees and increase the fruit weight as a result of earlier bud break. Application of hydrogen cyanamide to increase the fruit size due to early bud breakand use of ethephon close to harvest accelerates the ripening process by slowly releasing ethylene, which stimulates anthocyanin accumulation (reddening) and subsequently leads to enhancement of colour as well asearly ripening may be subjected to harmful residues from consumer’s interest point of view. The available literature on the persistence of hydrogen cyanamide and ethephon is mainly confined to grapes [22,23]. However, from risk assessment point of view in apple fruitsneither such studies in India and nor in abroad have been experimented. Therefore, the present investigations have been carried out to assess the residue status of hydrogen cyanamide and ethephon used to increase the fruit size and for early ripening in apple fruits for fetching remunerative price in the market with the aim to safeguard consumer’s health as well as utilization of data by CIBRC (Central Insecticide Board & Registration Committee) for registration in India.

Material and Methods

Reagents and chemicals

The reference analytical standard of hydrogen cyanamide (purity 99.5%) and commercial formulation Dormex 49% AS were supplied by M/s PNP & Associates Pvt. Ltd, Faridabad, India. The ethephon reference standard of purity 98.2% and its commercial formulation, Ethrel 36 % SL was provided by M/s Bayer CropScience Ltd., Maharashtra, India. HPLC grade water, methanol and all other analytical grade chemicals like acetone, Potassium Hydroxide (KOH), Hydrochloric Acid (HCl), ammonium formateand acetic acid were purchased from Merck (Mumbai, India).

Preparation of standard solutions

The stock solution of hydrogen cyanamide was prepared by dissolving 15mg reference material into 250 mL HPLC grade water and the stock solution of ethephon was prepared by accurately weighing 100 mg of reference ethephon into 100 ml volumetric flask, diluted to volume with 0.1N HCl. Working standard solutions were prepared by serial dilution in respective solvent mixture and used for sample spiking. All standard solutions were stored below -4°C before use.

Field trial layout and sampling

Supervised field experiments were laid out in a Randomized Block Design (RBD) to study the persistence of hydrogen cyanamide and ethephon on apple variety Royal delicious during 2015-16. Each treatment comprised of three replications and two trees per replication. Hence, 6 trees in eachtreatment.

Persistence of hydrogen cyanamide was studied on apple at four locations of Himachal Pradesh viz., Mashobra (31.12870N, 77.22740E, 2312 m amsl), Bajaura (31.99550N, 77.13980E, 1553 m amsl), Sharboo (31.53900N, 78.26750E, 2400 m amsl) and Rohru (31.20450N, 77.75230E, 1591 m amsl). Hydrogen cyanamide (Dormex 49% AS) was sprayed at three different concentrations i.e., 0.5, 1.0 and 2.0 % a.i. during dormant stage on apple trees by using a high-volume power sprayer. Control apple trees were sprayed with water to observe the impact. Apple fruit samples (1kg) were collected at three sampling intervals. The first fruit sampling was done at 85-111 days (walnut stage), second sampling at 150-163 days and third sampling at 279-209 days (at harvest) of application irrespective of locations to observe for residues dynamic study.

Persistence of ethephon was experimented in two locations of Himachal Pradesh viz., Matiana (31.21020 N, 77.40560E, 2100 m amsl) and Thanedhar (31.31830N, 77.44580E, 2300 m amsl) and in two locations of Jammu & Kashmir (J&K) viz., Youngora (34.21640N, 74.77190E, 1600 amsl) and Lassipors (33.94730 N, 74.55250 E, 2104 m amsl). Ethephon (Ethrel 39% SL) in apple was applied on bearing apple trees close to physiological immature un-ripenedfruits at X-dose of 585 g a.i./ha and 2-X dose of 1170 g a.i./hausing a high-volume power sprayer. Apple fruit samples (1 kg) from Matiana and Thanedhar werecollected upto 10 days after application i.e., at 0 (2 hr), 1, 3, 5, 7- and 10-days interval whereas fruit samples from Youngora and Lassipors were collected on10thday after foliar application. Control apple trees were sprayed with water only. Weather parameters existing during the experimental period is shown in figure 1.

Residue Dynamics of Hydrogen Cyanamide and Ethephon in Apple

Figure 1: Average weather parameters of locations during the experimental period.

Sample preparation and instrumentation

Apple fruit samples was homogenized in Robot Coupe high volume homogenizerand processed for respective analysis.

Hydrogen cyanamide: Fruit samples were analysed for hydrogen cyanamideresidues as per the method described by Pramanik et al., [23]. Homogenized 12.5 g sample was transferred into centrifuge tube containing 25 mL water. The tube was centrifuged at 1200 rpm for 5 minutes, 4 mL supernatant was taken in a test tube and evaporated to dryness under air current in Turbovap concentrator at 40°C. The residues were re-dissolved in 1.5mL water, added 0.5 mL of 0.1% acetic acid, mixed thoroughly by shaking and centrifuged at 10,000 rpm for 5 minutes. The obtained supernatant was finally filtered through 13 mm nylon filter (0.45µm pore size) and injected 20 µl into Liquid chromatograph SHIMADZU LC-20AT equipped with DGU-20A5 degasser, autoinjectorSIL-20 AHT, SPD-M20A Photodiode Array Detector (PDA) connected with Waters µ BONDAPAKTM RP C18 column (2.0 mm X 30 cm).The mobile phase comprising of methanol and water (2:8) having 5 mM ammonium formate was run in an isocratic mode at flow rate of 1 ml/min. Hydrogen cyanamide was detected at 200 nm wavelength.

Ethephon: Ethephon residues were determined in terms of ethylene based on the principle of alkaline hydrolysis to ethylene at high temperature [24] (Figure 2) and its determination on gas chromatograph equipped with Flame Ionization Detector (FID) as per the procedure described by Tseng et al.,[25]. Homogenized 5 g fruit sample was weighed in 24 mL headspace amber coloured vial. A mixture of 4 mL of water, 1 mL of acetone and 1 mL of 30% KOH solution was transferred to the vial and sealed immediately with Teflon septum. The vials were placed in water bath at 60°C for 3 h and shaken after every 15 minutes. The vials wereallowed to cool at ambient temperature; liberated ethylene gas was drawn (1 mL) from the headspace of the vials with a 10 ml headspace constant volume syringe and injected in to SHIMADZU GC 2010 equipped with FID and GS-Q column (30 m x 0.53 mm i.d., J&W Scientific, CA, USA). Initial oven temperature of GC was maintained at 40°C for 5 min and subsequently programmed to 120°C at 30°C/min. The temperature of injection port and FID was kept at 250°C. The flow rate of carrier nitrogen gas was kept at 5 mL/min, hydrogen gas flow rate was at 70 mL/min and zero air flow rate was maintained at 700 mL/min.

Residue Dynamics of Hydrogen Cyanamide and Ethephon in Apple

Figure 2:The reaction equation showing the conversion of ethephon into ethylene.

Validation of analytical methodology

It is important to establish that if the method we are using is optimum enough for usage or not? The validity of method is determined by the agreement between the true value of analyte in the sample and the value obtained by its analysis. Measure of accuracy is expressed as a percentage of the analyte recovered of the true value of the amount of spiking [26]. To establish efficiency of the method validity and to record the linearity of the pesticides, recovery studies were conducted by fortifying apple fruit samples with hydrogen cyanamide at four different levels viz.,0.05, 0.25, 0.50 and 1.00 mg kg-1and ethephon in apple fruit at six levels viz.,0.05, 0.25, 0.50, 1.00, 2.00 and 5.00 mg/kg. Each level was replicated five times to work out the precision of the method expressed as per cent Relative Standard Deviation (% RSD) for replicate analysis of the matrix matched spiked samples (Figure 3a,b).

Residue Dynamics of Hydrogen Cyanamide and Ethephon in Apple

Figure 3: Matrix matched response of (a) hydrogen cyanamide and (b) ethephon as ethylene at different concentrations.

Data analysis

The residue data obtained from ethephon persistence studies were subjected to statistical analysis according to Hoskins [27] to compute the residue half-life. Log residues were regressed on time interval to half-life ofresidues. Residue half-life (RL50) was calculated as T1/2 =Log2/b, whereas T1/2 = Residue half-life (RL50) in days; b = slope.

The time lapsed between last application of insecticide on the crop and harvest of the produce is referred as the Pre Harvest Interval (PHI) that is calculated as

Where, Tsitime taken in days by the insecticide to reach tolerance limit; log k2 : log of initial deposit ; log tol: log of proposed tolerance limit ; k1 (b): slope of regression equation.

The PHI is calculated on the basis of MRL of 0.8 mg kg-1fixed by Codex Alimentarius Commissionon18 September 2017) for ethephon in apples.

Results and Discussion

Method validation

The analytical method was validated in terms of Limit of Quantitation (LOQ), linearity, precision and recovery. The data presented in table 1depicts reliability of analytical method tested by spiking of untreated apple fruit samples at different concentrations. In our investigations, the average recovery of hydrogen cyanamide at the respective levels was 90.00 - 100.60 per cent with RSD ranged from 0.52 - 9.94% in apple fruitswith the LOQ of 0.05 mg kg-1 were satisfactory. The recovery of ethephon in terms of ethylene was in the range of 88.00 - 99.22 per cent at the respective levels with the precision of the analytical method RSD rangedfrom 1.56 - 10.00 % (Table 1). Ethephon residues as ethylene were rapidly monitored in agricultural commodity at pH values more than 12-14 omitting complex method of determination [28-30]The LOQ of ethephon was observed to be at 0.05 mg/kg, the lowest spiking level. The reproducibility of ethephon analytical headspace method, following the cleanup procedure described by Tseng et al.,[25]was found to be efficient enough and as such no significant matrix effect was observed.

Spiked Level (mg kg-1)

*Per cent recovery (% RSD)

Hydrogen cyanamide


Hydrogen cyanamide

Ethephon (Ethylene)



90.00 (4.55)

94.00 (10.00)



92.80 (6.37)

88.00 (9.24)



94.00 (9.94)

95.00 (9.60)



100.60 (0.52)

97.80 (3.83)




98.35 (4.69)




99.22 (1.56)

Table 1: Recovery of hydrogen cyanamide and ethephon from fortified apple fruit.

*Mean of five replications 

Persistence and residues

Hydrogen cyanamide: No residues of hydrogen cyanamide were detected in apple fruit samples, collected at different day intervals and at harvest following different treatments viz. 0.5, 1.0 and 2.0 per cent a.i. irrespective of locations. In our experiments, the interval between the last application and sampling was 179 to 209 days in all the locations, which supportthe work of Sharma et al., [31] and Pramanik et al., [23] that hydrogen cyanamide degraded during the period of exposure to weathering. As per the literature [32] breakdown of hydrogen cyanamide in aerobic soil leads to CO2 and dicynodiamide with an effective half-life of 3 days. Dicyanodiamide metabolitecleavage with a half-life of 4 days to the commonly used fertilizers like urea and ammonia. Hydrogen cyanamide and its metabolites are potentially mobile in soilas they highly solubilized in water. However, the use of hydrogen cyanamide does not pose specific threat in ground water since the parent compound and its by-products are non-persistent, short lived, ubiquitous, low molecular weight organic nitrogen compounds that can be readily utilized into the metabolism by microbes and higher plants.

Ethephon:Ethephon used in apple rapidly degrades to phosphate, ethylene and chloride[33], and releases ethylene gas as a major metabolite produced in plant parts. Conspicuously, early fruit drop was observed after 10 days of ethephon treatments as fruit abscised from the branches and fell on the ground and subsequently deteriorates the quality of fruits. Therefore, the residue experiment could not be extended beyond 10 days. The initial deposits of ethephon on apple fruits at Matiana and Thanedhar were 2.044-2.147 and 4.036-4.133mg/kg due to the application of ethephon at recommended dose (585ga.i./ha) and double the recommended dose (1170 ga.i./ha), respectively. These deposits reduced rapidly below 96-98 % in 10 days tothe tune of 0.057 to 0.083 and 0.056 to 0.088 mg kg-1 at respective applications (Table 2). The dissipation pattern in our investigation was found to be parallel despite almost double the rate of application as compared to Cochrane et al., [34] who observed ethephon residues on zero day to the tune of 1.60 ppm, 0.75 ppm on 10th day and 0.4 ppm after 13 days at the application rate of 300 ppm on apple. However, in grapes, Ugareet al., [22] observed around 51% and 49% dislodging of initial deposits on day 15 at 97.5 and 195g a.i/ha of ethephon. The residues of ethephon on apple fruits at Youngora and Lassipors ranged between 0.099 -0.100 and 0.137-0.143 mg kg-1after 10 days.



Ethephon* residues expressed as ethylene in mg/kg



X dose±SE

2-X dose±SE

X dose±SE

2-X dose±SE


















0.931±0. 006


















Table 2: Persistence of ethephon on apple fruits at Matiana (Location I) and Thanedhar (Location II).

ND = Not detected; *Mean of three replication

The kinetic equation, half-lives, and correlation coefficient (R2) calculated from the dissipation data of ethephon at two different locations of Himachal Pradesh is summarized in table 3. The data revealed that the dissipation of ethephon in apple followed first order kinetics with the half-life values calculated to be 2.1 and 1.9 days at X and 2X dose level, respectively. On the basis of ethephon dissipation behaviourin different locations based on the codex MRL of 0.8 mg/kg, 3.42 - 4.8 days PHI was worked out. Hence, a safe waiting period of 5 days is suggested between ethephon application and fruit harvest to safeguard the consumer’s health.


(g a.i./ha)


Regression Equation

Correlation Coefficient (R2)

Half Life (days)

PHI (days)























Table 3: Dissipation kinetics of ethephon in apple at two locations.

PHI= Pre Harvest Interval


Hydrogen cyanamide and ethephon are commonly used PGRs in apple by the growers to increase productivity. Since, no residues of hydrogen cyanamide were detected in our investigations at various intervals irrespective of dose;therefore, it will not pose any threat of residual toxicity in harvested apple fruit to the consumers. A safe waiting period of 5 days is suggested between ethephon application and fruit harvest to safeguard the consumer’s health.


Authors are thankful to M/s Bayer CropScience, Maharashtra India and PNP & Associates Pvt. Ltd, Faridabad, India for the financial assistance and providing the analytical standards and formulated products.


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