Qualitative detection of pesticide residues using mass spectral data based on convolutional neural network

Excessive pesticide residues in crops directly threaten human life and health, so rapid screening and effective measurements of agricultural pesticides residues have important application significance in the field of food safety. It is imperative to detect different pesticide residue types in actual complex crop samples cause mixture analysis can provide more information than individual components. However, the accuracy of mixture analysis can be obviously affected by the impurities and noise disturbances. Purification and denoising will cost a lot of algorithm time. In this work, we used the problem transformation method to convert pesticide residues prediction into multi-label classification problem. In addition, a new convolutional neural network structure Pesticide Residues Neural Network (PRNet) was proposed to solve the problem of multi-label organophosphate pesticide residue prediction. The method of binary correlation and label energy set was used to adapt 35 pesticide residues labels. The Cross Entropy were used as loss functions for PRNet. The comprehensive comparison performances (e.g. 97% optimal accuracy rate) of PRNet is better than the other four models. By comparing the ROC curves of the five models, PRNet performs the best. The PRNet can separate the independent mass spectrometry data by different collision energy applied to phosphorus pesticide compounds through a three-channel structure. No complicated data preprocessing is required, the PRNet can extract the characteristics of different compounds more efficiently and presents high detecting accuracy and good model performance of multi-label mass spectrometry data classification. By inputting MS data of different instruments and adding more offset MS data, the model will be more transplantable and could lay the foundation for the wide application of PRNet model in rapid, on-site, accurate and broad-spectrum screening of pesticide residues in the future.


Introduction
Pesticides are widely used in agricultural production. They play a significant role in preventing insects or diseases and increasing yields. However, unreasonable use of pesticides occurs occasionally, which could not only cause pesticide residues, but also pollute the environment severely [1]. There are many different types of pesticides based on their structure, including carbamate, organochlorine, organophosphorus, pyrethroids, heterocycles and amides, etc. Among those, organophosphorus pesticides are the most widely used. There are more than 100 organophosphorus pesticides. Most of them have the irreversible cholinesterase inhibitors for organisms to bring detrimental impacts on human healthy [2]. Therefore, it is a necessary and difficult problem to realize rapid detection and identification of organophosphorus pesticides in vegetables according to the detection limit, sensitivity and accuracy requirements. At present, mass spectrometry (MS), as a strong qualitative function, is one of the most broadly used confirmations in practice. It firstly dissociates the molecules of the substance to ions with different masses, and then uses the different motion behavior of ions in electric field or magnetic field to separate the ions according to the mass charge ratio (m/z) to obtain the mass spectrometry [3]. The qualitative and quantitative information of the samples can be obtained from the MS data. Since it is targeted at the molecular structure of the sample. The information obtained by MS is more accurate and reliable for qualitative analysis. In real life, different compounds are always mixed together. Due to the complexity of MS analysis, the amount of MS data is usually very large. Consequently, most MS peaks are meaningless for the components' identification. Considering that MS data is usually accompanied by some noise to conceal sample ion strength. The introduced confused information can not only make the identification algorithm-more computationally intensive, but also increase the possibility of random matching. It results in a decrease in the appraisal results reliability and an increase in the false positive or false negative identifications. Even though, some mathematical methods attempted to detect specific compounds from mixed samples MS data [4].
Over the past several years, many works on Artificial Neural Network (ANN) in the area of MS have been reported. MSnet, created by Curry et al. may be the first generation neural network for MS [5]. It involves a hierarchical system of several neural networks for MS data. The work of Werther et al. uses classical multi-dimensional numerical analysis techniques to compare the performance of ANNs [6]. A. Eghbaldar et al. presented a methodology for optimizing ANN to identify the compounds' structural features from MS data [7]. Ankita thakur uses ANN to mine MS data to detect ovarian cancer [8]. Ion mobility spectra can also be successfully classified by neural networks from the combination of drift time, number, intensity, and peak shape [9].
In general, the dimensionality of MS data inputs is always very large. Although, the performance of the neural network becomes better for the larger data set. Unfortunately, small number of samples and the large dimension of MS data inputs construct the typical dilemma for the realistic datasets. Deep learning can directly extract features from raw large dimension data by neural unit learning data characteristics. Hence, it was explored to predict molecular substructure in the mass spectral data. With the development of deep learning, some models based on recurrent neural network have shown amazing potential when processing sequence data. J. Liu proposed a material classification system based on short and short time memory (LSTM) [10]. Nevertheless, it is still difficult to detect a class similar molecular inside the mixture sample MS data.
Since the multi-label classification can study each example associated with a set of labels simultaneously. The performance of multi-label classification algorithm in the detection of organophosphorus pesticide residues was investigated in this work. The practical algorithm is based on convolutional neural network (CNN). CNN is the most widely used deep learning method to achieve the desirable classification performance in various classification problems. Compared with other algorithms, CNN can separate the independent MS contributed by different compounds, so as to accomplish better accuracy. In this study, we mimicked the CNN's architecture and use the three-channel architecture as input [11]. Compared with the traditional classification methods, the PRNet (Pesticide Residues Neural Network) can significantly improve the prediction accuracy and performance of mixture samples MS data. This model has a respectable potential in large target MS data analysis.

Chemical and materials
All high purity pesticide standards with purity greater than 97.0% were purchased from Dr. Ehrenstorfer (Augsburg, Germany) or LGC Standards (Teddington, UK). Stock solutions were prepared in acetonitrile at a concentration of 100 mg/L, stored at − 20 °C and restored to room temperature and diluted the stock solution to 100 µg/L before detection. Acetonitrile and methanol (Merck, German), formic acid (Fluka) and ammonium acetate (Sigma-Aldrich, Germany) were chromatographically pure. Anhydrous magnesium sulphate (MgSO 4 ), sodium chloride (NaCl), adsorbents: octadecyl chemically bonded phase silica gel (C18, 43-60 μm) and N-primary secondary amine (PSA, 40-60 μm) were all of analytical grade. A Milli-Q Advantage A10 ultrapure water system from Millipore (Milford, MA) was used to obtain the HPLC-grade water during the analyses. Three fruits or vegetables (grapes, apples and cabbage) were purchased from markets in Beijing.

Sample preparation and extraction
We precisely weighed 10 g crushed sample of each fruit or vegetable with the accuracy to 0.001 g. Then every sample was put in 50 mL polytetrafluoroethylene (PTEF) centrifuge tube and added 10 mL acetonitrile, extracted in 1 min through high-speed homogenate, added 4 g MgSO 4 and 0.5 g NaCl, vortexed in 1 min, centrifuged in 5 min under 5000 r/min. Then 6 mL upper acetonitrile phase solution was put into 15 mL centrifuge tube of containing 400 mg C18 + 400 mg PSA + 1200 mg MgSO 4 , vortexed in 1 min and centrifuged in 5 min under 5000 r/min; then the supernatant was filtrated with 0.22 μm organic filter membrane for the following determination.

Preparation of spiked samples
We precisely weighed 10 g targeted sample of grape, apple and cabbage with the accuracy to 0.001 g respectively. Then we added 10 µL monocrotophos and phoxim stock solutions to grape, 10 µL isazofos and methamidophos stock solutions to apples, and 10 µL dichlorvos and chlorpyrifos stock solutions to cabbage. After mixing each sample, we could prepare 0.1 mg/kg spiked samples according to the extraction procedure in above mentioned.

UPLC-Q-TOF/MS analysis
Ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS, Agilent 1290-G6545, USA) equipped with Zorbax Eclipse Plus-C18 column (150 mm (L) × 3 mm with 1.8 μm of particle size, Agilent, USA) was used for chromatographic separation. 2 mM of ammonium acetate and 0.05 % formic acid solution were used as mobile phrase A, and methanol aqueous solution which contained 0.05 % formic acid was used as mobile phrase B. The column was equilibrated with 90% of mobile phase A and 10% of mobile phase B for 30 min before injection. The amount of mobile phase A maintained unchanged at 90% from 0 to 0.5 min. From 0.5 to 3 min, the amount of mobile phase A decreased from 90 to 50%, from 3 to 20 min to 0%, then maintained for 4 min and at last go back to 90% at 24.1 min. The temperatures of column oven and auto sampler were set at 40 and 4 °C, respectively. The flow rate was 0.4 mL/min and the injection volume were 2 µL.
MS parameters: UPLC-Q-TOF/MS equipped with Dual AJS ESI source was operated in full-scan TOF mode and MS/MS spectra were acquired for further compound identification using auto MS/MS acquisition. MS detection was carried out in positive electro spray ionization mode (ESI + ). The following operating conditions were used: scan range, 50-1200 m/z; capillary voltage, 3500 V; fragmentor voltage, 120 V; skimmer voltage, 65 V. The temperature of the drying gas and sheath gas were 250 and 325 °C, and the flow rates of the drying gas and sheath gas were set to 7 L/min and 12 L/min. The nebulizer pressure was 35 psi, and the nozzle voltage was 300 V. Auto MS/MS acquisition conditions were set to: mass range, 50-1200 m/z; collision energy (CE) was set at 10 eV, 20 eV and 40 eV. The source and gas parameters used were the same as those used in full-scan TOF mode.

Preparation for dataset
The experiments were repeated with different pesticide solutions. MassHunter Acquisition B.03 and MassHunter Qualitative Analysis B.03 were used for the acquisition and treatment of data. In addition, Python 3.7 was used to create pkl file for the dataset of pesticides.
In this work, 35 kinds of common organophosphorus pesticides were mixed randomly, as listed in Table 1. The negative data came from the sample reagents that did not contain the 35 organophosphorus pesticide compounds. The positive data came from the sample reagents that added the mixture of organophosphorus pesticide compounds. There were 1-5 kinds of organophosphorus pesticides in each positive data. With the permission of experimental conditions, over 30,000 mixtures were prepared with more than 97 % purity. A typical mixture sample experimental MS data was shown in Fig. 1.
Additionally, in different conditions, the use of different instruments and experimental operations tend to produce different noises. In order to make experimental MS data closer to the real environment and make the training model more robust, three level of collision energy (10 eV, 20 eV, 40 eV) were chosen to generate the three-channel data besides the experimental MS data according to the assumption of the linear mixed model. The random noised was added in experimental MS data for training model, as demonstrated in Fig. 2. Based on linear composite hybrid approach, 10,000 noise simulation data were also generated to explore the noise impact on the performance of the experimental model eventually.
After finishing the dataset preparation, the total dataset was divided into 32,000 training sets, 4,000 validation sets and 4,000 test sets. For the data label, the Encoding label with one-hot Encoding for the specified compound was set as "1", and "0" for none specified compound. For the data label, we encode the output label, and set "1" as the existence of the specified compound, and "0" as the absence of the specified compound.

Support vector machine
Support Vector Machine (SVM) is a common classifier. The core idea of SVM is to find a linear classifier to separate hyperplane with the maximum interval in the feature space. Then SVM can classify the unknown sample set through the hyperplane. SVM uses inner product kernel function instead of nonlinear mapping to | https://doi.org/10.1007/s42452-021-04661-x high-dimensional space [12]. Due to the influence of noise, the classification effect is usually better on small sample set. Meanwhile, SVM uses quadratic programming to solve support vector (the calculation of m-order matrix, M is the number of samples). When the number of M is large, the storage and calculation of the matrix will consume a lot of machine memory and operation time [13]. Therefore, it is difficult to solve the multi classification problem with SVM.

Artificial neural network
Artificial Neural Network (ANN) is an abstract model for describing how the human brain organizes and operates. All neurons contained in a neural network record the weight corresponding to their inputs [14]. Corresponding to the three processing units, the neural network contains three levels: input layer, hidden layer and output layer [15]. By repeated learning and training of the input information data, the neural network can constantly improve the parameter values of the weights in the neural network to reach the closest conclusion, as represented in Fig. 3.

Extreme gradient boosting
extreme Gradient Boosting (XGBoost) is one of the Boosting algorithms. The idea of the Boosting algorithm is to integrate many weak classifiers together to form a strong classifier [16]. It corrects the residuals of all previous weak classifiers by continuously adding new weak classifiers. The final prediction is finally made by adding multiple classifiers. Thus, the accuracy rate will be higher than a single weak classifier. When adding a new model, the gradient boosting algorithm is used to minimize the loss. XGBoost has fast calculation speed and is widely used in small-scale classification tasks [17].

Long short-term memory
Long Short-Term Memory (LSTM) is a kind of time recurrent neural network. This model takes sequence data as input, recurses in the evolution direction of the sequence and connects all nodes in a chain way. The input of the hidden layer is not only related to the output of the input layer, but also related to the output of the hidden layer at the previous moment [10]. LSTM has two transmission states, the memory cell state and the hidden state. This strategy can effectively avoid the phenomenon of gradient disappearance and gradient explosion in the training process. It is very suitable for dealing with problems highly related to time series [18].

Convolution neural network
Convolution neural network (CNN) is a kind of deep neural network with convolution structure to reduce the memory occupation. Its three key operations are local receptive field, weight sharing, and pooling layer [19]. CNN can effectively reduce the number of network parameters and alleviate the over fitting problem of the model, as illustrated in Fig. 4. The differences between the CNN and the ANN were the usage of convolution layers and the dimension of input [20]. The convolutional layer reduces the number of parameters to be trained by receptive field and weight sharing. The convolutional layer is sometimes followed

Mass spectrum matrix
Assuming the MS data of a mixture are equal to the weighted sum of the MS of the individual compounds [23]. The MS data of mixtures can be written from the normalized intensities: where I i,j is the intensity of the i th mass in the j th mixture, n is the number of components in the mixture, a ik is the intensity of mass i in pure compound k , and s kj is the concentration of compound k in the source for mixture j .
Equation (1) can be simply expressed as: where I m is an i × j matrix, A is an i × k matrix and S is an k × j matrix; i , j , k represent the number of different mixtures, masses and pure compounds respectively and I , A , S represent overlapping MS data matrix, pure compound matrix and concentration matrix respectively. The I m here is the mixture MS data we used for training and testing, and (1) a ik s kj (2) I m = AS the information described by the m/z and intensity is the characteristics to be learned by the experimental model.

PRNet model training
Based on iteration algorithm, PRNet is an optimized multitarget detection CNN model for pesticide residue identification. The architecture of PRNet model is listed in Table 2. The mixture MS data of three energy (10 eV, 20 eV and 40 eV) were flatten as one-dimensional input. Based on the   [24]. Utilizing the twelve hundred m/z windows with 10 ppm interval, in range from 50 to 1200 m/z, the MS data of each energy could be flattened into one-dimensional matrix. With the character of input, one-dimensional convolution layers could further simplify the model. The information between different energy could be recognized by the convolution layers. Max pooling layers were applied to abstract the characteristics of the region and reduce the coupling degree of the model. Convolution layers and pooling layers were used for the feature extraction. The fully-connected layers were applied for the classification. In the last fully-connected layer, sigmoid activation was applied to output the probability of each compound presence. Usually the model threshold was set to 0.5. The threshold value also could be modified according to actual condition (If the goal is to screen for as many pesticide residues as possible, the threshold can be lower down). The operation flow is shown in Fig. 5.

Results and discussion
In the classification problem of machine learning, Sigmoid function is the commonly used activation function of the output layer of neural network. Since multiple classes can overlap each other. Compared with the Softmax, Sigmoid is more suitable for the multi-label classification task. In this work, Sigmoid function was applied as the activation function of the neural network. The binary cross entropy is the corresponding loss function of Sigmoid [25]. The smaller loss function represents the better model robustness. The loss function of this multilabel classification was utilized to estimate the difference between the predicted value and the true value from the model. This loss function can also make the training model as fast as possible and require less memory: x is the input sample, m is the total number of training data,y i is the output value of the ith data, and f i (x) is the predicted value of the ith model.
Because the output of each tag is assumed to be independent. The general configuration for multi-tag binary classification is the BCE and Sigmoid activation functions. Each category outputs a probability between 0 and 1. Each corresponds to a sigmoid function. The Adam algorithm was deployed as the optimizer to update the weights of the neural network iteratively according to the training data [26]. The Model PRNet refers a netural network model for pesticide residue prediction based on the convolutional neural network structure. All models run on an Ubuntu system that based Linux with 24   Table 3.
The comparison results show that these five machine learning models can detect multiple target compounds in overlapping samples well. They can effectively learn features from raw MS data. The results obtained by the neural network are usually classification results such as 0.5 and 0.8 instead of labels such as 0 or 1. Therefore, a threshold can be selected to diverge them. When the result is greater than the threshold, the predicted value can be judged as 1. On the contrast, the predicted value is judged to be 0. By increasing the threshold, more confidence can be obtained in the predicted results, thereby improving accuracy. But this will reduce recalls. Otherwise, the number of true cases missed by the model will decrease and the recall will increase. For those models, 0.5 was chosen as the threshold under comprehensive consideration. The ANN model only includes the full connection layers and batch normalization layers, it does not contain other additional structures. The performance effect of ANN is moderate. Due to the good adaptation and outlier processing, PRNet has the respectable feature extraction performance. For the larger mixture MS data predictions, PRNet always obtains higher accuracy than the other four models. Furthermore, for the target compounds detection, recall is an equally important indicator to reflect the proportion of correctly predicted (true) components in all predicted (true and true negative) components. Since the SVM model is difficult to predict a large number of samples with positive labels. The accuracy of SVM is much lower in performance than neural networks for higher number of detection targets. LSTM has a higher accuracy rate with a lower recall rate conversely. It requests a lower threshold. The effect of XGBoost is acceptable. But there is still a certain gap (accuracy and recall) between XGBoost and PRNet. For further observation, the receiver operating characteristic (ROC) [27] curves were arranged to evaluate the predictive ability of the five investigated models: The comparison of the five models' ROC curves is illustrated in Fig. 6; Table 4. The AUC (average area under curve) value is the area covered by the ROC curve. The better classification effect classifier has the larger AUC. The ROC curve is farther from the pure opportunity line (the dashed black line), the model has the stronger discrimination ability. Because of the mapping principle of ROC curve, the model based on neural network and other models have different curve shapes. According to Fig. 6, the AUC of PRNet is the largest to indicate the most robust classification performance among the five models.
The average precision (AP) score can also be summarized as a weighted average precision achieved at each threshold to evaluate the five models. In general, the average precision is the mean of the accuracy corresponding to all the values from 0 to 1 across the recall rate: This integral value is approximately the sum of the precision at each possible threshold multiplied by the change in recall rate: In multi-label classification, Mean Average Precision (MAP) is a commonly used evaluation method as well [28]. It measures the quality of the learned model in all categories by taking the average of all AP: From Table 5, the classifier performance of SVM and XGBOOST is also poor. Compared with the ROC curves in Fig. 6, the target detection performance of PRNet model for the mixture MS data is more stable than that of other models.   In general, the detection of pesticide residues involves more types of compounds. Consistent with the Table 3, when more organophosphorus pesticide compounds were added, the accuracy reduction of PRNet was less than that of other models. This indicates that PRNet is more suitable for large-sample detection.

Discussion
If the MS data is pre-processed in advance, such as smoothing, baseline correction, and peak picking [29]. The traditional machine learning algorithms, e.g. SVM, can also perform well, especially for single MS data classification. PRNet does not require much preprocessing or denoising, as demonstrated in Table 5.
In the 4000 testing MS data, about 10 % testing data are negative reagent data or other type compounds interference data (Multi-classification problems usually do not have negative samples, we treat categories other than 35 organophosphorus pesticides as negative samples). TP means that all compounds were detected. FP means that some compounds have been detected correctly. TN indicates the correct prediction of a negative reagent as the absence of the compound. FN means it falsely predict the presence of a pesticide compound that does not exist.
From Table 6, the performance of PRNet is better than ANN (the ANN is only composed of full connection layers and batch normalization layers), SVM, LSTM or XGBoost in multi-tag target detection for mixture MS data. In fact, SVM and others have a good effect on the double-label classification. As the detection of organophosphorus pesticides increased, SVM cannot work well in the multi-label classification. LSTM is more suitable for time series data analysis [18]. The molecular weight of organophosphorus compounds is usually not very large. So, the MS data of organophosphorus compounds may not reflect strong sequence characteristics, which may be the reason for the general performance of the LSTM model. In the case of more noise impurities, the classification problem with boosting method may appear overfitting. Compared with ANN, PRNet can effectively handle high-dimensional data processing due to the existence of convolution kernel and other structures. Compared with PRNet, the XGBoost model is less effective, which may be related to the characteristics of the MS data. The mixture MS data may have high variability, noise and high dimension. XGBoost is a kind of boosted trees model derived from ensemble learning [30]. Highdimensional sparse features may make the training efficiency of the tree model become extremely low and easy to overfit (Fig. 7).
If there are more MS data of different energies as input, PRNet based on the CNN can handle more relationships between different energies. Comparison with time series, MS energy is an easier controllable variable factor for the input channel of the CNN network structure. Once more MS data of different energies are added as inputs, especially continuous signals on the energy axis, the accuracy of PRNet will decline. Further research on the architecture of PRNet, including the depth of the network, alternating layer and filter size, will be launched to continuously improve the ability of learn and detect complex mixture target compounds.
The size of MS data set is a bottleneck to limit the performance of deep learning. Usually, when the number of detected targets increases, these models need large data sets to learn the characteristics of samples. Therefore, if the MS data set is very small, deep learning would be limited [30]. Our method based on the CNN can effectively realize the identification of multiple compounds from tandem mass spectrometry data. When the model was modeled with 35 organophosphorus pesticide compounds, the final mAP scores of the five models were 0.68, 0.78, 0.81, 0.84 and 0.95, respectively. This methodology can be applied to classify more pesticide compounds and even other types of compounds (Table 7).
The ★ is applied to compare the average training speeds of the five models in Table 7. In the case of small data samples, the training speed of SVM model is fast. But with the expansion of the training set and the increase of the types of phosphorus pesticides in the classification task, the training speed of SVM becomes very slow. Overall, the comprehensive training speed of SVM model is the slowest. Due to the large parameter space, the training speed of ANN is also slow. As the GPU speeds up and the batch normalization layer is added in the network, the training speed of ANN increases to some extent.
XGBoost has the fastest training speed, but XGBoost's parameter tuning is a little complicated. Thanks to GPU acceleration, the training speed of PRNet model is also moderated. The improvement of training speed obtained by LSTM through GPU acceleration is not obvious. Since LSTM is a kind of time series model, time t depends on the information at time t-1 and cannot be executed in parallel, its training speed is slower than the PRNet.
MS data sometimes have high variability, noise and high dimensionality [31], compared with traditional algorithms, deep learning is more universal and less vulnerable to MS data. The local receptive field of CNN can extract the subtle features of mass spectrometry data. CNN has the ability to learn low-level features from complex inputs. The CNN feature detection layer is learned from training data, avoiding explicit feature extraction. At the same time, due to the robustness of the filter, the model based on CNN is less affected by noise. In practice, the MS data may vary considerably depending on the operator, instrument, and laboratory environment. Controlling variables and capturing large amounts of data is crucial. The training model set by data obtained

Conclusions
In this paper, we propose a CNN-based method: PRNet. This model can directly detect a variety of organophosphorus pesticide compounds from mixture sample MS data obtained by UPLC-Q-TOF/MS. PRNet can directly extract features from the original high-dimensional MS data without complicated data preprocessing process. By evaluating different neural network structures, the average accuracy of the PRNet can reach 97 %. Compared with traditional machine methods such as SVM, ANN, XGBoost and LSTM, PRNet has the best performance in accuracy, recall rate and precision. The multi-component identification and detection of pesticide residues mass spectrometry data through convolutional neural networks would be an efficient way to help professionals and non-professionals detect pesticide residues quickly and correctly in the future. However, the presented PRNet model can only aimed at the existing pesticide species. If possible, the component concentration prediction would be considered in the future research and more conditions (e.g. spectral tilt) need to be explored to optimize quantitative analysis conditions. Benefit from the excellent transplantation and generalization of neural network, the model can be easily applied to mobile terminals for the pesticide residues detection. The model performance can be significantly improved through further study of input data and selection of structural components.
Author Contributions Jian Wei carried out the survey and the experimental operations and wrote the manuscript. Xuemei Wang contributed the sort of data set. Zhenyu Wang contributed the research ideas and the article composition. Jin Cao contributed to the article review. All authors discussed results and approved the final manuscript.

Fig. 7
Positive MS data were truly identified by the model (the selected MS data were marked by red dots) Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.