**Impact of Naturally Contaminated Substrates on** *Alphitobius diaperinus* **and** *Hermetia illucens***: Uptake and Excretion of Mycotoxins**

#### **Giulia Leni 1, Martina Cirlini 1,\*, Johan Jacobs 2, Stefaan Depraetere 2, Natasja Gianotten 3, Stefano Sforza 1 and Chiara Dall'Asta 1**

Received: 23 July 2019; Accepted: 16 August 2019; Published: 18 August 2019

**Abstract:** Insects are considered a suitable alternative feed for livestock production and their use is nowadays regulated in the European Union by the European Commission Regulation No. 893/2017. Insects have the ability to grow on a different spectrum of substrates, which could be naturally contaminated by mycotoxins. In the present work, the mycotoxin uptake and/or excretion in two different insect species, *Alphitobius diaperinus* (Lesser Mealworm, LM) and *Hermetia illucens* (Black Soldier Fly, BSF), grown on naturally contaminated substrates, was evaluated. Among all the substrates of growth tested, the *Fusarium* toxins deoxynivalenol (DON), fumonisin 1 and 2 (FB1 and FB2) and zearalenone (ZEN) were found in those based on wheat and/or corn. No mycotoxins were detected in BSF larvae, while quantifiable amount of DON and FB1 were found in LM larvae, although in lower concentration than those detected in the growing substrates and in the residual fractions. Mass balance calculations indicated that BSF and LM metabolized mycotoxins in forms not yet known, accumulating them in their body or excreting in the faeces. Further studies are required in this direction due to the future employment of insects as feedstuff.

**Keywords:** *Alphitobius diaperinus*; *Hermetia illucens*; edible insects; mycotoxin; uptake; excretion; feed safety

**Key Contribution:** This study demonstrates that *Alphitobius diaperinus* and *Hermetia illucens*; two common edible insects; reared on naturally contaminated substrates; did not accumulate DON; FBs and ZEN in their body; but seemed to metabolize them in forms not yet known; excreting them in the faeces also by the hydrolysis of masked forms.

#### **1. Introduction**

Given the large growth of the World population expected in the coming years, it has been estimated that the demand for food will raise of about the 60% in 2050 [1]. Along the food chain, the meat production represents the field with the most impact, with serious consequences on the demand of feed supply. Edible insects have been explored as an alternative to common livestock, and their use is encouraged for their sustainability and the minimal environmental impact applied in their breeding [2,3]. Furthermore, edible insects have been proposed as a promising alternative nutrient source due to the high content and quality of their macronutrients [4]. In general, they have a well-balanced nutrient profile, high in polyunsaturated fatty acids and essential amino acids which meet the requirement for humans and livestock, rich in micronutrients and vitamins [5].

In the European Union, the use of insects in the feed and food sector is nowadays regulated by a package of legislative texts. Insects are included in the category of Novel Foods and the European Food Safety Authority (EFSA) authorization is mandatory before their marketing in the European Union (EU) [6]. As alternative source for feedstock, they may substitute the sources of protein and fat normally added in animal feed, as soy, maize, grain and fishmeal [7]. Insects can be used as whole or processed. Whole insects, alive or dried, and fat fraction can be employed for livestock feed, except for aquaculture [8]. Whereas, the protein fraction isolated from insects can be used to feed pet and fur animals, but not for ruminants and monogastrics [9]. Recently, the European Commission (EC) has expanded also to the aquaculture sector, the addition of processed proteins from seven insect species: Black Soldier Fly (*Hermetia illucens*), Common Housefly (*Musca domestica*), Yellow Mealworm (*Tenebrio molitor*), Lesser Mealworm (*Alphitobius diaperinus*), House Cricket (*Acheta domesticus*), Banded Cricket (*Gryllodes sigillatus*) and Field Cricket (*Gryllus assimilis*) [8].

One of the limitations in the use of insects as feed and food is certainly linked to safety aspects. The potential hazards are related to exogenous and endogenous factors, which could be influenced also by harvesting and processing methods, and which could be achieved during all the insect life-cycle [4,10]. Exogenous factors may occur from the behavior and from the substrate of growth which, as feed, must meet the requirements of the current regulation that fixed the maximum limits of undesirable substances in feedstock [11,12]. In particular, the potential hazards could be heavy metal residues, pesticides and mycotoxins.

Mycotoxins are a wide range of different substances, produced by the secondary metabolism of various species of fungi, that can infect cereal or vegetable crops at pre- or post-harvest [13]. Mycotoxins are characterized by a large chemical diversity and may exert a broad spectrum of adverse effects in animals and humans [14,15]. In addition, they can undergo biotransformation in plants, microbes and animals [16] leading to the uptake of modified forms which may account for a similar toxicity compared to parent compounds [14].

Although mycotoxin occurrence in food and feed is extensively covered by regulation/guidelines at EU level, their presence at concentration levels not exceeding the EU limits cannot be ruled out in rearing substrates obtained from vegetable waste. Therefore, insects are potentially exposed to mycotoxins when reared on a contaminated substrate and, at least from a theoretical point of view, they could accumulate mycotoxins (or modified forms) at levels exceeding the legal limits.

However, despite a growing interest, only few papers have addressed the possible uptake and biotransformation of mycotoxins in insects so far [17–22]. Although information on the possible biotransformation and/or excretion mechanisms are still scattered, all the studies performed to date consistently demonstrated that parent mycotoxins are not bioaccumulated by insects grown in contaminated substrates.

However, many of these studies were done on substrates artificially contaminated with mycotoxins, possibly not exactly describing the situation arising in the case of a natural contamination.

The aim of this research, in the framework of the EU project InDIRECT, was to investigate the possible uptake and excretion of mycotoxins in two species of insect larvae, Lesser Mealworm (LM) and Black Soldier Fly (BSF), produced on different naturally contaminated substrates, obtained from vegetable and cereals waste.

#### **2. Results**

Different feed materials were selected for the experiments, in order to ensure optimal growth of the larvae on the basis of preliminary trials. These wastes derived from the processing of cereals, as wheat, corn, rice, while others were chosen among vegetables, as olive and apple pomace, rapeseed and chopped carrots (Table 1).

**Table 1.** Substrates used for feed formulations with results about their mycotoxin contamination (deoxynivalenol (DON), fumonisins 1 and 2 (FB1 and FB2), zearalenone (ZEN)). Results showed the detected mycotoxins and are reported as mean of two different replicates ± standard deviation.

Samples were analysed for all the regulated mycotoxins, according to the possible occurrence. In particular, samples were analysed for *Fusarium* toxins (deoxynivalenol (DON), fumonisins 1 and 2 (FB1 and FB2), zearalenone (ZEN)) as well as aflatoxins, while the possible presence of patulin was checked in vegetable-based samples. All samples underwent ochratoxin (OTA) analysis, in consideration of its possible synthetisation postharvest in all the considered feed materials. As shown in Table 1, only *Fusarium* toxins were found in three out of eight matrices, at concentration levels in agreement with the EU limits for cereal-based feed. While only DON (938 ± 100 μg/kg) was found in wheat middlings, the contemporary presence of DON, FB1, FB2 and ZEN was detected in corn wastes. In order to optimize and promote the insect growth, 15 feed formulations were obtained by mixing these substrates (originated from the same batch) in different percentages. In particular, corn distillation residues were mixed with chopped carrots, olive and apple pomace, while wheat middlings with corn gluten feed, rice bran and rapeseed, as indicated in Table 2.

According to the contamination pattern observed in raw waste materials, BSF and LM larvae were analysed for the target mycotoxins and results are reported in Table 2. No mycotoxin uptake was observed in BSF larvae, while DON was detected in six out of 13 LM larvae samples, being one contaminated by FB1 as well.

**Table 2.** Larvae of Black Soldier Fly (*Hermetia illucens*, BSF) and Lesser Mealworm (*Alphitobius diaperinus*, LM) reared on the feed formulations with results about the target mycotoxins occurrence. Results are the mean of two different replicates and are reported as mean ± standard deviation. Abbreviations: deoxynivalenol, DON; fumonisins 1 and 2, FB1 and FB2; zearalenone, ZEN; corn distillation residues, CDR; olive pomace, OP; apple pomace, AP; wheat middlings, WM; corn gluten feed, CG; rice bran, RB; rapeseed wastes, RW.

\* A small amount of chopped carrots was arbitrarily added in order to get the desired water content for optimal insect growth.

In particular, DON was detected in samples of LM larvae grown on substrates prepared with high percentages of wheat and/or corn residues, and the amount of contamination ranged between 416 ± 28 μg/kg of larvae produced on 100% of wheat wastes and 755 ± 134 μg/kg of insects cultivated on wheat (90%) added with rice (10%). Low concentrations of FB1 (127 ± 6 μg/kg) were detected in LM larvae grown on a substrate composed of 100% of corn gluten feed.

In order to evaluate the uptake and possible excretion of mycotoxins, the residual fractions were also analysed. All the results are listed in Table 3. Interestingly, the contemporary presence of DON, FB1, FB2 and ZEN was observed in the residual fraction obtained from BSF larvae grown on 100% corn residues, while DON and FB1 were detected in residual fractions from LM corn-based growing substrates. No residual contamination was observed in wheat-based residual fractions, in spite of the DON occurrence detected in wheat waste.

**Table 3.** Residual fractions harvested from insects resulted positive to the presence of target mycotoxins and results about their concentration level expresses as μg/kg. Results are the mean of two different replicates and are reported as mean ± standard deviation. Abbreviations: black soldier fly, BSF; lesser mealworm, LM; deoxynivalenol, DON; fumonisins 1 and 2, FB1 and FB2; zearalenone, ZEN; corn distillation residues, CDR; olive pomace, OP; apple pomace, AP; wheat middlings, WM; corn gluten feed, CG; chopped carrots, CC; rice bran, RB; rapeseed wastes, RW.

\* A small amount of chopped carrots was arbitrarily added in order to get the desired water content for optimal insect growth.

In order to better investigate the amount of mycotoxins and its distribution in the larvae and in the residual fraction, the mass balance was calculated for larvae which had been resulted positive to the presence of mycotoxins, or grown on contaminated substrates, as in the case of BSF. This calculation was performed on the basis of the concentration of contaminants found in feed, in larvae and in the respective residual fractions. The samples for which the amount of mycotoxins resulted below the LOD were considered as equal to LOD values. Results are represented in Figure 1.

**Figure 1.** Mass balance of deoxynivalenol (DON), fumonisins 1 and 2 (FB1 and FB2) in Lesser Mealworm (*Alphitobius diaperinus*, LM) (**A**) and Black Soldier Fly (*Hermetia illucens*, BSF) (**B**) treatments. Abbreviations: corn distillation residues, CDR; olive pomace, OP; apple pomace, AP; wheat middlings, WM; corn gluten feed, CG; rice bran, RB; rapeseed wastes, RW.

As far as DON concerned, the mass balance ranged from 2 to 81% in BSF, while it was found to be between 1% and 43% in LM. The average mass balance of fumonisins in BSF ranged between 4% and 72%, and it was ranged from 1% to 57% in LM for FB2 and FB1 respectively. Almost the total amount of ZEN resulted excreted in BSF (data not shown in figure).

#### **3. Discussion**

This study is focused on the uptake of mycotoxins in BSF and LM larvae, grown on naturally contaminated substrates, obtained from residues of cereal and vegetable processing.

In particular, BSF were reared on two different substrates based on corn distillation residues, which had been found to be contaminated by DON, FB1 and FB2. In agreement with other studies from the literature [21], no uptake of mycotoxins in BSF larvae was observed for both trials, while DON, FB1 and FB2 were found only in the residual fractions collected from the BSF reared on 100% of pure substrate. In this case, the concentration of DON, FB1 and FB2 found in the rests were higher as compared to the concentrations detected in feed. Interestingly, ZEN, lower than LOD in the initial feed, was detected in the residual fraction at 334 ± 44 μg/kg. Since the insects were exclusively grown on the selected substrate, the occurrence of ZEN after harvesting suggested its possible cleavage from the matrix, due to a hydrolytic activity carried out by the insect. An overall increase of ZEN in BSF larvae growing substrates was already observed by Camenzuli et al. [20], although the authors measured the parent compound together with its major phase I metabolites, α- and β-ZEL.

It is well-known that *Fusarium* mycotoxins can be biotransformed by plants into phase I and phase II metabolites, being the glycosylation the most common pathway [16]. Conjugates mycotoxins can be however cleaved by microbial enzymes, as reported by several authors [16,23,24]. Although a hydrolytic activity in insects towards modified mycotoxins has never been described so far, it cannot be ruled out and can be a possible explanation for the observed data.

In addition, it is known that *Fusarium* mycotoxins are often associated to the matrix, and this binding strongly affect the extractability [25,26]. Therefore, taking into consideration the increase of mycotoxins from starting materials to post-growing residues, it can be argued that BSF larvae may induce the substrate degradation upon growing, thus increasing the overall extractability of mycotoxins.

In contrast with BSF larvae, DON was found in LM larvae grown on contaminated substrates, mainly in those containing wheat middlings in combination with corn gluten. When 100% corn gluten was used as growing substrate, DON, FB1 but not ZEN were transferred to LM larvae.

The lack of ZEN uptake in Lesser Mealworm is in agreement with the literature [19], while the possible transferal of DON and FB1 in LM larvae grown on naturally incurred substrates, was observed in this study for the first time.

In case of LM residual fractions, the overall mass balance never exceeded 60%, clearly indicating that mycotoxins are partially metabolised by the larvae to unknown compounds, in agreement with the literature [19,20]. It should be noticed that the occurrence of DON in residual fractions was lower than the one reported in previous studies performed on Yellow mealworm larvae under similar conditions [18]. However, uptake, biotransformation and excretion of mycotoxins in insects could be affected by a range of factors, among them the substrate, the species, and the dose, as well as the use of naturally incurred or spiked growing material [20].

Different from other studies, in which larvae were grown on a substrate artificially contaminated by mycotoxins, at higher concentrations than those found in the substrate samples considered in this study [18–20], the present work clearly demonstrated that DON and FB1 can be found in LM (but not in BSF) larvae. The amount of mycotoxins anyway never exceeds the starting levels, indicating that there is no active uptake in insects, and there is rather a degradation or an excretion.

In addition, the mass balance calculation clearly indicated that biotransformation is rather the operating mechanism instead of simple excretion. Further experiments will be needed in order to investigate the mycotoxin biotransformation pattern in insects.

#### **4. Conclusions**

The present study reported on the possible uptake and/or excretion of mycotoxins in two insect species, LM and BSF, reared on naturally contaminated substrates. As feed, organic side streams recovered from cereal and vegetable processing were considered under a circular economy perspective.

Collected data clearly indicated that transfer from the waste to the insect of mycotoxins is possible, but without uptake into insects, and rather with an overall decrease of their amount. LM larvae were found able to transfer DON and FB in low amounts from naturally incurred growing substrates. Data were consistent with the possible biotransformation of mycotoxins in unknown metabolites in insects. ZEN was detected in BSF residual fractions but not in starting materials, suggesting a possible hydrolytic activity carried out by larvae upon growing.

Taken all together, our results proved the urgency of better deciphering the ability of insects to uptake, transform, and excrete mycotoxins, in view of a safer use of insects as an alternative protein source.

#### **5. Materials and Methods**

#### *5.1. Chemicals*

Mycotoxin standard solutions of aflatoxins B1, B2, G1 and G2, fumonisin B1 and B2, A and B trichothecenes (nivalenol, deoxynivalenol, 3-acetyl-deoxynivalenol, fusarenone X, diacetoxyscirpenol, T-2 toxin, HT-2 toxin), zearalenone, ochratoxin A, and patulin were obtained from Romer Labs (Tulln, Austria). All the solvents applied for both the extraction and analysis steps, methanol, acetonitrile formic acid and acetic acid, were HPLC-grade and were purchased from Sigma-Aldrich (Milan, Italy), while Bi-distilled water was produced in-house by using a Milli-Q System (Millipore, Bedford, MA, USA). Salts used for extraction as for the preparation of the eluents as sodium chloride and ammonium acetate were obtained from Sigma-Aldrich (Milan, Italy).

#### *5.2. Insect Treatments and Sampling*

For the experiments described herein, two insect species were selected: *Alphitobius diaperinus* (Lesser Mealworm, LM) and *Hermetia illucens* (Black Soldier Fly, BSF). The larvae were grown on different substrates prepared using different feed materials, prevalently coming from the processing of cereals (wheat, corn, rice and rapeseed) and vegetable (apple and olive), as indicated in Table 1. The choice of these substrates was driven by the seasonality, the availability and the cost of different by-products of agriculture sector. The substrates were analysed previously for mycotoxins presence, then also larvae were extracted and analysed using the same protocol applied for feed samples. Moreover, rests of insects grown on substrates resulted positive to the presence of mycotoxins were also collected and analysed.

Insects were grown on naturally contaminated feed, and in particular 2 substrates were used for BSF cultivation, while 13 different feeds were prepared for LM production (Table 2). The insects rearing was conducted as indicated in a previous work by Leni et al. [27]. Briefly, BSF eggs were initially placed in a specific incubator at 28 ◦C for 2 days, then the eggs were transferred in the rearing bins and the new-born larvae treated for 2 days with a started feed composed of chicken feed, the feed materials selected for the experiment and water, with a total dry matter of about 30%. During this time the temperature was set at 28–32 ◦C and humidity at 60% minimum. After that, the growth substrate was removed and substituted with that selected for the experiment, provided ad libitum. The larvae were reared under these conditions for 15 days. After this period, larvae were removed and quantified, and samples of remaining fractions were also collected as listed in Table 3. Similarly, LM larvae were reared utilizing the selected feed materials chosen for the experiments, under the same controlled conditions of temperature and humidity used for BSF growth, providing feed daily ad libitum. In this case, larvae were harvested for 28 days and then collected. As for BSF, also samples of remaining fractions were recovered and weighted.

Larvae of BSF and LM were killed at −18 ◦C and stored at the same temperature before each analysis. At the same time, samples of growth substrates and remaining fraction were stored at −18 ◦C until analyses.

#### *5.3. Mycotoxins Extraction and Purification*

Mycotoxins class is represented by several organic compounds with different chemical and physical properties. For this reason, we decided to apply different extraction protocols, selected for a specific class of toxins. In addition, all the samples of insect larvae and samples of remaining fractions were subjected to a lyophilisation process (Freeze dryer Lio-5P, 5Pascal, Milano, Italy) for 48 h and milled using a laboratory miller. The dried powders obtained from these steps were stored at −20 ◦C until extraction and analysis.

For the extraction of aflatoxins, 1g of sample added with 0.2 g of NaCl was extracted using 4 mL of a mixture of methanol/bi-distilled water, 80/20 *v*/*v* on a shaker at room temperature, at 200 strokes/min for 90 min. After that, the extract was centrifuged at 10,621× *g*, at 25 ◦C for 10 min. 1 mL of the supernatant was transferred in a tube, diluted with 4 mL of bi-distilled water and submitted to a purification step using immuno-affinity columns (VICAM, Afla Test®, mycotoxin testing system; VICAM, Milford, MA, USA). The cartridges were conditioned with 10 mL of bi-distilled water and subsequently with 10 mL of pure methanol. The diluted extract was then eluted through the column and, after the elution, a washing step with 10 mL of bi-distilled water was performed. The analytes were recovered with 1 mL of pure methanol. The purified sample was dried under a gently nitrogen flow, suspended in 1 mL of bi-distilled water/methanol, 80/20 *v*/*v*, and analysed by HPLC-FLD technique.

The contemporary extraction of fumonisins, ochratoxin A, zearalenone, patulin and A and B trichothecenes was performed on the basis of different protocols with slight modifications [28,29]. Briefly, 1 g of sample was extracted adding 4 mL of a solution composed of bi-distilled water/acetonitrile/methanol 50/25/25 *v*/*v*. The sample was positioned on a shaker at room temperature, at 200 strokes/min for 90 min. After that, the extracts were centrifuged at 10,621× *g*, for 10 min at 25 ◦C. 1 mL of the supernatant was collected and dried under a gently nitrogen flow. The residue was then dissolved in 1 mL of bi-distilled water/methanol 80/20 *v*/*v*. The samples were then subjected to UHPLC-MS/MS analyses.

#### *5.4. Mycotoxins Analysis*

Aflatoxins B1, B2, G1 and G2 were analysed on a HPLC Waters Alliance 2695 separation module, coupled with a FLD detector (Waters, Multi λ Fluorescence detector 2475) and an UV detector (Waters, Dual λ Absorbance Detector 2489) (Waters, Milford, MA, USA). The analytes separation was achieved on a C18-RP XTerra (Waters, Milford, MA, USA; 250 × 2.1 mm, i.d. 5 mm) column using as eluents bi-distilled water (A) and methanol (B), in isocratic conditions (65% A and 35% B). The flow was set at 0.25 mL/min and the column oven temperature was kept at 30 ◦C. A volume of 10 μL was injected. For the detection of aflatoxins, the UV detector was set at λ = 365 nm, while for the FLD λ = 365 nm and λ = 425 nm were chosen as the typical wavelength of absorbance and of emission, respectively.

For the quantitative determination of aflatoxins, a calibration curve was prepared starting from the commercial standard which contained AFB1 and AFG1 at the concentration of 2 mg/kg and AFB2 and AFG2 at the concentration of 0.5 mg/kg. Starting from this solution, 5 different dilutions in pure methanol were performed obtaining AFB1 and AFG1 at 0.5, 0.75, 1, 1.5 and 2 μg/kg, while for AFB2 and AFG2 concentrations of 0.125, 0.187, 0.25, 0.375 and 0.5 μg/kg, obtaining a good linearity (R2 > 0.99) for the both calibration ranges.

Fumonisins B1 and B2, ochratoxin A, zearalenone, patulin and A and B trichothecenes (nivalenol, deoxynivalenol, 3-acetyl-deoxinivalenol, fusarenone X, T2 toxin, HT2 toxin and deacetoxyscirpenol) were determined on an UHPLC–MS/MS apparatus consisted of an UHPLC Ultimate 3000 separation module (Dionex, Sunnyvale, CA, USA), coupled with a TSQ Vantage triple quadrupole (Thermo Fisher, Waltham, MA, USA) equipped with an ESI interface. The separation of the analytes was achieved on a RP-C18 EVO Kinetex column (2.6 μ, 100A; 100 × 2.10 mm) from Phenomenex (Torrance, CA, USA). Ammonium acetate 5 mM in bi-distilled water and methanol were used as eluent A and B respectively, both acidified with the 0.2% of acetic acid. A gradient was applied as follows: the elution started with 2% of B and these conditions were maintained for 1 min, then at 2 min the percentage of B was increased at 20% and kept for 6 min, at 17 min the column was flashed with the 90% of B for 3 min, then in 1 min the initial conditions were re-established and the column was re-equilibrated for 9 min, with a total run time of 30 min. During the analyses the column temperature was maintained at 40 ◦C while samples were maintained at 20 ◦C. The flow was 0.35 mL/min and for each sample 4 μL were injected into the system.

PAT, NIV, DON, 3ADON, FUSX, and ZEN were monitored in negative ion mode with a spray voltage of 3500 V, a capillary temperature of 270 ◦C, a vaporizer temperature of 200 ◦C, a sheath gas flow of 50 units and an auxiliary gas flow of 5 units. T2, HT2 toxins, DAS, FB1, FB2, and OTA were monitored applying a positive ionization mode, with the following parameters: spray voltage of 3000 V, a capillary temperature of 270 ◦C, a vaporizer temperature of 200 ◦C, a sheath gas flow of 50 units and an auxiliary gas flow of 5 units. All the other parameters as S-Lens RF amplitude values were obtained and set by tuning methanolic solutions of each considered molecule (1 mg/kg).

Detection of all the considered analytes was performed in SRM modality (Single Reaction Monitoring) monitoring the characteristic transitions for each considered mycotoxin (Table 4).

**Table 4.** Characteristic transitions monitored for the target mycotoxins: fumonisins B1 and B2 (FB1, FB2), ochratoxin A (OTA), zearalenone (ZEN), patulin (PAT) and A and B trichothecenes (nivalenol (NIV), deoxynivalenol (DON), 3-acetyl-deoxinivalenol (3ADON), fusarnone X (FUSX), T2 toxin, HT2 toxin and deacetoxyscirpenol (DAS)).

In order to quantify these mycotoxins, a calibration curve containing all the considered analytes was prepared starting from the commercial standard. For this purpose, 6 different dilutions were prepared considering the following concentrations: 50, 100, 200, 500, 750 and 1000 μg/kg, obtaining a good linearity (R2 > 0.99) for the calibration range.

#### *5.5. Mass Balance Calculation*

The mass balance was calculated as described by Camenzuli et al. [20] on the basis of the amount of substrates used for insects' growth, the amount of harvested larvae and of the residual fractions (frass). Furthermore, the accumulated, extracted and potential metabolized mycotoxins were calculated as follow:

$$\% \text{ accumulated money} = \frac{\text{amount of hrested insets x concentration mycotution detected in assets}}{\text{amount of substrate x concentration mycotution detected in substrate}} \times 100,$$

$$\% \text{ executed mycotution} = \frac{\text{amount of fraas x concentration my cotocation detected in grass}}{\text{amount of substrate x concentration my cotocation detected in substates}} \times 100,$$

% metabolyzed mycotoxin = 100 − % excreted mycotoxin − % accumulated mycotoxin.

Metabolized mycotoxins were referred to the undetected compounds which could be metabolized in different structures not yet identified. All the measurements of mycotoxin below the LOD were considered as equal to LOD values.

**Author Contributions:** Conceptualization, S.S.; Data curation, G.L. and M.C.; Formal analysis, G.L. and M.C.; Methodology, M.C., J.J., S.D. and N.G.; Supervision, C.D.; Writing–original draft, G.L., M.C., S.S. and C.D.

**Funding:** This project received funding from the Bio Based Industries Joint Undertaking under the European Union's Horizon 2020 research and innovation programme under grant agreement No. 720715 (InDIRECT project).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**6.1. Capacity analysis**

The capacity for the averaged synthesized patterns according to the number of sampling antennas across the ECC (Electronic Communications Committee) standard's UWB band was analyzed using (26). The term *pi* is taken from the power spectral density levels of the ECC UWB spectral mask, *λi* is the Eigenvalue of the subchannel from the matrix **S** and *σ*2 noise = *kTB*, where *k* is the Boltzmann constant, *T* = 297 K and *B* = 100 MHz. Fig. 6 shows the capacity of the synthesized radiation pattern using 2×2×2 till 5×5×5 sampling antenna elements within the defined 5×5×5 cm3 physical space. It can be seen that the higher the number of sampling antennas, the more the capacity increases, agreeing with the theory in [18]. Noting that the rise in the capacity is decreasing with the higher element configuration used, we conclude that the 5×5×5 configuration is nearing the capacity saturation limit.


**3. Recycling PTFE scrap material**

Use of plastics has the capability to cause danger to both environment and human life. PTFE scrap can be neither incinerated nor dumped! In the waste incineration processes which have been usual up to now, highly-corrosive vapors are released which also damage the incineration plant itself. Dumping of waste will in future be restricted due to nationwide regulations. In order to avoid this, Environmental Protection Agencies have taken up an action to recycle all the PTFE materials.

Recycling PTFE involves two different processes with two different end results. One involves irradiation of the PTFE scrap to heavy dose of ionizing irradiation which will reduce its molecular weight. On pulverizing, the irradiated PTFE scrap turns into *micro powder* which finds certain applications. The second method involves pulverizing the PTFE scrap without irradiation so that it becomes reusable like virgin PTFE itself.


A crucial role in the search for compromise and in finding a universal standard involves incrementalism, which would allow for identification of new elements protected within the framework guaranteed by the Convention. It consists in developing the scope of the right, even without finding its infringement in a specific case. Under this way of reasoning, the Court and the domestic courts engage in a complex and dynamic dialogue, seeking to define mutually approvable positions in all categories of cases discussed in this book. Development of the scope of a pregnant woman's right to privacy is clearly demonstrated – especially in abortion cases. Relevant formulations of this right are also found in cases where there is no substantive resolution. A good example is the case of *Boso v. Italy*, in which the Court deemed the provisions allowing abortion on the grounds of risk to the woman's health to be in compliance with the provisions of the Convention, although the case concerned the rights of the father (which the Court did not find violated). And, in *A., B. and C v. Ireland*, in relation to applicants A. and B., the Court found that the prohibition of the abortion for reasons of health or well-being leads to the interference with their right to respect for their private lives,8 although, in the opinion of the Court, the interference in these cases remained within the limits of admissibility. In the case of *R.R. v. Poland*, the Court – as if on a side note – defined the right to privacy anew besides the existing elements of the right to privacy, holding that the notion of private life applies to decision either to have or not to have a child or to become parents. This is a conclusion taken from a case dealing with issues other than access to abortion.9 Confirmation of the existence of such a right in this case appears to be a step towards extending the right to respect for private life in the context of the decision to terminate a pregnancy, although it did not form the basis of the ruling on the applicant's complaints.10 Again, the use of incrementalism and the gradual development

8 A., B. and C. v. Ireland, par. 216.

9 Evans v. the United Kingdom, par. 71.

10 R.R. v. Poland, par. 180.

of the scope of the right to privacy in this aspect is well illustrated in cases involving the right to die with dignity. The rulings in *Pretty v. the UK* and *Hass v. Switzerland* are good examples of this.

In the vast majority of cases examined in the book,11 the extension of the scope to include the new element did not lead to the conclusion that there had been a substantive infringement of the right. This made it possible to prevent conflict on the grounds of the established scope. However, these statements were made and constitute the body of jurisprudence – they therefore provide the foundation for further proceedings, where they are expected to be cited, also for the purpose of establishing an infringement of law, such as in a much more categorical context.

Therefore, this way of reasoning is particularly indicative of the Court's intention and aim, which is to shape the justifications dialogically and to gradually develop the meaning of the rights whose scope it decides upon. The Court is aware that its statements are intended not only to respect socially established attitudes and approaches, but also to shape them among a broader audience. Therefore, it does not make statements concerning world views; it does not value them, that is, it does not question or single out any of them. This is very evident when it comes to the issue of abortion, which clearly has divided European countries into two – albeit obviously not equally sized – camps. Instead, it attempts to address the reasons and procedures that led to the contested regulations, avoiding statements about their moral foundations and merely indicating their existence. Respect for the ethical order presented and protected by the domestic legislation and decisions of the authorities requires the initiation of a dialogue by finding a platform on which the dispute will be possible without criticism or questioning of the moral assumptions themselves. This approach makes it possible to equip the recipient with the attitude of commitment to look for a common consensual solution to the practical problem, and thus to the underlying dilemma and conflict of values.

The margin of appreciation, in turn, is meant to leave room for differences where agreement is not necessary. In fact, the margin of appreciation is used precisely as an instrument to show that the agreement as to the content of Article 8 and Article 2 of the Convention – two provisions most frequently referred to in the respective applications – is not absolutely needed, and that changes in the interpretation of these provisions are permissible. The margin of appreciation in respect of the cases in question is no longer just a way of interpreting the Convention norms and searching for their scope, but a strategy for convincing people of the position adopted in the adjudication. This aim is especially subordinate to the definition of the object of the margin. In cases concerning the admissibility of abortion, it is easy to see how the subject

11 Still, it must be added that in a small number of cases discussed in the book, the emergence of a new element of a right protected under the Convention led to establishing an infringement (Dickson v. UK, Mennesson v. France).

of the Court's application of this device is changing, along with the notion of consensus among European states, which constitutes its element. In our book we identify three subjects of margin of appreciation in abortion cases: the first one, concerning the will of the parties to consider the foetus as a subject of the right to life (cases decided by the Commission: *Brüggemann and Scheuten v. Germany, X v. the UK, H. v. Norway*); the second, pertaining to the assessment of when life actually begins (*A., B. and C. v. Ireland,* the meaning of the concept drawn from *Vo v. France)*; and the third, concerning the freedom of the State to balance the interests of the woman and foetus, implicit in the wording of public interest (*A., B. and C. v. Ireland*, further developed in *R.R v. Poland* and *P. and S. v. Poland*). This noticeable evolution indicates the subordination of the use of the margin of appreciation device to serve rhetorical and argumentative purposes. When properly applied, the margin of appreciation tool makes it possible to demonstrate, wherever needed, that there is no need for the Court's interference, and the Court's declared "moral sensitivity" of the decided cases as an exposure to worldview and moral issues is, as it were, of primary importance to the subsequent findings and provides an excellent context for establishing the object of the States' discretion. The Court stops short of delving into the structure of either the moral or the legal conflict that may arise.

Equally creative is the Court's treatment of the argument concerning the lack of consensus at the level of the States on the assumptions concerning both the legal solutions adopted and their axiological foundations, such as the fundamental element conditioning the granting of the margin of appreciation. The various ways of applying it, as shown here, are subordinated to the objective of deciding on an infringement of the right to privacy. Indeed, it is no longer an instrument to interpret the Convention norms, but to justify that they mean different things in different States, which in turn serves to argue the reason why the Court did not determine any violation.

#### **Worldview neutrality – pragmatism**

What draws attention in the cases we are discussing is the almost complete lack of appeal to moral considerations. The Court does not present its clear position in relation to the ethical aspects of the decided cases, which would be possible if the legitimacy of introducing limitations to the rights and freedoms of individuals in the analysed areas was examined against the premise of morality. Nevertheless, its position in this respect can be read indirectly from the arguments used. The consequence of resorting to some of them is that the ECtHR makes significant moral and ethical pronouncements. Others, on the other hand, aim at avoiding such a statement or presenting it as narrowly as possible.

It is true that in a number of cases the Court does acknowledge that the case concerns a particularly sensitive issue, but in none of the analysed rulings did it explain what moral sensitivity would mean. It thus avoids explicitly identifying the moral dimension of the case and gives itself the option of making partial pronouncements. In this context, it expresses its approval for example, for: a) the use of assisted procreation techniques abroad, which are not permitted in Austria for moral reasons (*S.H. and Others v. Austria*); b) surrogacy, which is *de facto* prohibited in France – also for moral reasons – by requiring that its legal effects be taken into account in the domestic legal system, provided that it has been carried out lawfully abroad (*Mennesson v. France*); and c) the protection of the potential for life of embryos – which clearly demonstrates a moral stance – by accepting domestic regulations prohibiting their transfer for the purposes of scientific research (*Parrillo v. Italy*). Similarly, in cases involving allegations of inadmissibility of abortions, the Court does not pronounce on the permissibility of abortion. Instead, it confines itself to acknowledging – as regards Irish cases – the entrenchment of the protection afforded under Irish law to the right to life of the unborn which was based on profound moral values concerning the nature of life which are reflected in the stance of the majority of the Irish people while at the same time accepts that domestic law allows for performing the abortion abroad and even formulates certain information obligations upon authorities in this regard.12 In these matters it avoids setting a moral minimum on a general level at all costs.

A similar outcome is reached after delving deeply into domestic legal arrangements, procedures and decision-making processes. The Court in some of these cases – such as *A., B. and C. v. Ireland* and *Lambert v. France –* reproduces and analyses with great insight and care not only the regulations involved, but the process that led to their adoption, the rationale and arguments of the drafters, the consultations, as well as the administrative, medical and judicial procedures applied with respect to the applicants. The Court thus focuses on the procedural side, examining decision-making at the State level and – although only to the extent of examining the decision-making procedure – with regard to the applicant's individual case. Such an analysis is intended to examine whether the law and decision-making process are designed to optimise protection of substantive rights. Ultimately, however, it usually brings about the possibility of adjudication and justification without the need for verification of the substantive content of the applicant's rights. By replacing such an evaluation with reconstruction of the legislative process in particular and by indicating its democratic legitimacy (*A., B. and C v. Ireland*) in the views of the competent authorities (*Lambert v. France*), as well as by the creation of a catalogue of positive obligations, that is to say, an indication of efforts seeking to create the conditions for the exercise of the rights guaranteed by the Convention within the framework of the State (such as with *Koch v. Germany* and *Haas v. Switzerland*), it is often possible to give the impression that the evaluation is objective, although in fact it is only a partial response to the claims raised in the applications.

A fundamentally pragmatic approach is also presented by the Court with respect to the distinctive tool of argumentation based on the margin of appreciation. In a certain special way, this mechanism is complemented by another construct, its reverse, such as the phenomenon of migration for the purpose of realisation of certain demanded rights in European countries other than the country of origin, as observed and, according to the Court, relevant for the protection of Convention rights. Abortion tourism is a good example of this because it leads to the creation of a coherent and repeatable concept of the "right to travel" – that is, accepting the practice of undergoing a particular medical procedure abroad (*A., B. and C. v. UK*). This reasoning is also present in other cases discussed in the work (*H.S. and Others v. Austria*). Arguing through the right to travel is an excellent means of withholding a finding as to the merits when the right can be exercised elsewhere. The effect of the dependence of the scope of the right on the place where it is exercised is discreetly omitted here, yet the effect of the actual possibility to benefit from a broader scope than in the country of origin is noticed. This is a purely pragmatic solution, referring to the actual effects of and opportunities to exercise the right, and not to its substantive content.

Pragmatism is most prominently used by the Court when defining the permissible limits of exercising the right, that is, when delving into the substantive assessment. In some cases concerning assisted suicide (*Pretty v. UK* and *Nicklinson and Lamb v. UK*) and medically assisted procreation (*S. H. and Others v. Austria*), under the influence of the State party's actions and arguments, it outlines the limits of the realisation of the right under Article 8 of the Convention, indicating the restriction of the protection of the rights and freedoms of others as the legitimate aim. This is perfectly understandable from the neutral worldview perspective and the liberal doctrine underlying the axiological system of conventional protection. According to classic liberal approach, only harm to another person can constitute a reason for restricting freedom.13 The choice of this legitimate aim is therefore the only one that can be taken into account in cases concerning such a fundamental right as the right to private life. It is important that in abortion cases the question of the balance between the rights of the woman and the foetus has been left out of the assessment due to the axiological entanglement of the dispute about the priority of their respective goods. The cases have been decided from the perspective of the fulfilment of positive obligations. Conversely, in the two remaining categories – the cases of termination of life and medically assisted procreation – although it is difficult to find those who, by exercising their right to suicide or by medically assisted parenthood, directly cause, or are even likely to cause, harm to other persons, the Court decided to adjudicate those cases with the use of the legitimate aim of "protection of others." In order to introduce such a legitimate aim, the Court is forced to define a group of individuals who are hypothetically threatened by the exercise of the right under Article 8. This is possible by combining the unquestionable and strongest premise of protecting the rights of others with resorting to an argumentative strategy of considering the possible negative social effects of the decision. By using such reasoning about the undesirable future implications of certain decisions, including the development of potentially unwelcome human attitudes and behaviour under the influence of authorising a particular outcome, the Court effectively persuades the need to limit the exercise of the right to privacy by creating an image of the threats faced by the community and especially its most vulnerable members. This persuasion evades the risks of ethical evaluation and the charge of moral majoritarianism in deciding on the limitations. It makes it possible to abandon ethical appeals altogether. It makes use of purely practical considerations, common sense and appeals to the emotional side. It is a question of concern for those who require it, and who, for particular reasons, are described by the Court as vulnerable to the possible abuse of rights that might arise if the applicants' claims were upheld. The ECtHR followed this line of reasoning, among others, in *Pretty v. UK*, sharing and reiterating the reasoning of the UK court and drawing attention to its precedent-setting nature and the necessary caution when making decisions that will inevitably result in certain legal, but also by extension social consequences. It thus refers to social practice, pointing out the fear of decisions and of harm to people which may potentially be enforced as a result of the creation of a certain social attitude of permitting behaviour which may expose the weak to take desperate and detrimental steps due to the impact and pressure of that attitude.

In fact, the Court thus makes the public interest and the good of the entire community tangible, although it almost never referred to this category explicitly in the cases in question, since it is a concept that falls outside the catalogue of legitimate aims contained in Article 8 of the Convention. Moreover, even an indirect reference to the public interest, such as the good of the community as a whole, would certainly give rise to accusations of moral majoritarianism – imposing the will of the majority on the individual – thus depriving him or her of the exercise of certain right. On the same grounds, there is no place for morals, public morals, and other goods and values, as they are not comparable to the human right to self-determination. But this one principle implies duty of a very wide scope which is sometimes difficult to be intuitively determined. Here, the Court ventures into the territory of pragmatic but also highly hypothetical assessments.

This is an invaluable argumentative tool, although, as indicated in the relevant literature14 and discussed in our study, it raises numerous questions.

14 Bossuyt, "Categorical Rights and Vulnerable Groups." 717–8, 726–34, M. O'Boyle, "The notion of 'vulnerable groups' in the case law of the European Court of Human Rights." Report delivered for *Conference on the Constitutional Protection of vulnerable groups: a judicial dialogue*, Santiago Chile 2015, Peroni, Timmer, "Vulnerable Groups." 1056–85.

#### 210 *Conclusion*

Hence, axiological and dogmatic doubts are raised by the hypothetical character of the identification of the group deserving protection special or juxtaposing it with the specific interests of the applicant, as well as by the heterogeneity of the group deserving protection. The only thing they have in common is the exposure to harm by the possible recognition of the applicants' claims and ad hoc creation of such a group for the purpose of persuasion, which is difficult to conceal. However, this device opens the way for appeals to common sense, indicating purely practical and plausible outcomes of possible decisions, as well as appeals to collective responsibility and a sense of concern, which is very convincing. On the other hand, it allows for an argument to be carried out on completely morally neutral ground, omitting ethical considerations through pragmatism and references to social practice.

This observation leads us to formulate a conclusion, or rather a supplement thereto, about the replacement of morality by the concept of human rights in the contemporary legal discourse. In current legal discussions on the subject, human rights have replaced morality and refer to moral theory, sometimes expressing the nostalgia of present-day society for lost moral values15 it pretends to substitute the moral and ethical discourse. Yet under conditions of pluralism of values and attitudes, as well as the firmly established liberal view of human beings and their rights as moral claims and moral entitlements possessed by persons,16 it is extremely difficult to adjudicate in situations of conflict of rights at the normative level. If the good means respect for and protection of individual rights – and these are universal, innate and unalienable, being derived from the human dignity and worth of every human being – the formulation of permissible limits of human behaviour encounters formidable obstacles.

The only logic that can be followed is that of the fear of harming another human being – this creates a normative system based on the personal responsibility of living in the community with others under the basic moral rule: to behave in a way that is not harmful to others. And this single principal system is evident in cases described in our book, even if sometimes it is not explicitly articulated. Thus, instead of the concept of moral limitation of the right to decide about life and death, we are given the solution that sets limits to the right to self-determination due to the necessity to protect the rights of others.

Other ways of reasoning singled out in our work (argument from authority or plasticity of concepts) have merely an ornamental or auxiliary character in the cases discussed herein. When assessed separately they do not produce a noticeable effect in the form of creating reasoning that independently persuades

15 Haule, "Some reflections." 367–95, 368; Frederic Megret, "International human rights law theory." in *Research Handbook on the Theory and History of International Law*, ed. Alexander Orakhelashvili, (Edward Elgar Publishing, 2020), 168.

16 Richard Wasserstrom, "Rights, human rights, and racial discrimination." *Journal of Philosophy* 61 (20) (1964): 628–41.

to the given decision. Undoubtedly, however, their use together with the appropriate selection of documents, positions or words can perfectly strengthen the intended effect. In the field of medically assisted procreation, this can be seen in the Court's use of reasoning based on the reference to binding and non-binding acts of international law, which allowed to strengthen, for instance, the thesis on the considerable liberty of states in shaping the adopted solutions. In the context of abortion, this is particularly evident in the use of arguments based on the plasticity of concepts, which allowed the Court to avoid emotional involvement through the use of words such as "mother" or "unborn child", in favour of more neutral ones: "woman" and "foetus".

#### **What do recurrent ways of reasoning mean for the human rights case law?**

Our book also provides insight into something more than just reasoning in the Court's oeuvre. Certain consequences of the judgments on the substance of rights and the direction of the development of jurisprudence are outlined, and we seek to define the functions of this kind of reasoning.

#### *Incrementalism – gradual build-up of significance, predictability of judgments*

The reasoning used by the Court and drawing on incrementalism has profound implications in terms of shaping the scope of rights and freedoms and influencing States to introduce specific arrangements favourable to individuals.

As an example, we can point to the inclusion of the right to recognition of the legal bond with both the biological and the legal parent as a consequence of birth of a child by surrogacy under a contract concluded abroad in the scope of Article 8 of the Convention, starting with *Mennesson v. France*. In this particular case, the Court's pronouncement translated positively into the fate of individual applicants. From the other side, the conclusion concerning the need for States to recognise the effects of foreign surrogacy, even in the absence of authorisation of surrogacy in their own legislation,17 has far-reaching consequences in that the Court expressed its acceptance of such procedures and obliged States to introduce practical regulations in that regard.18

In its reasoning in cases concerning euthanasia, the Court has incremented the right to privacy by applying the strategy of assumption as to the existence of a right to die with dignity without finding a violation of that right (*Pretty v. the UK*), or without finding substantive infringement of that right (*Haas v.*

17 Paul Beaumont, Katarina Trimmings, "The European Court of Human Rights in Paradiso and Campanelli v. Italy and the way forward for regulating cross-border surrogacy." University of Aberdeen, Working Paper no. 2017/3, p. 12, available at: www.abdn.ac.uk/law/documents/ cpil%20working%20paper%20no%202017\_3.pdf.

18 Ibidem, p. 11.

#### 212 *Conclusion*

*Switzerland*). However, determinations like these are of utmost importance when it comes to shaping subsequent decisions based on the presumed scope of the right to self-determination, as well as the legal framework and decisions left to the discretion of domestic authorities. Therefore, we see a gradual shaping of the right, already referred to, albeit in the form of "even assuming" until establishment the right to *facilitate the act of suicide with dignity.*19

Consequently, one observable outcome of the application of this technique is the predictability of subsequent judgments, built on previously carefully constructed foundations, though without finding a violation of the disputed right (or finding violations for other reasons). In employing the incrementalism, the Court, in a way, indicates and anticipates how and where the development of the right is heading, and thus what elements are required for recognising it as worthy of protection.

#### *Proceduralisation – meaning the right through the lenses of procedural requirements*

The intensity of the ECtHR's use of procedural reasoning supports the thesis expressed in the literature that the Court is more inclined to attach value to the quality of legislative process in hard cases with a high degree of sensitivity.20

Argumentation referring to the assessment of the State's fulfilment of its procedural obligations comes to the fore in cases arising out of legal disputes having strong ethical undertones. At the same time, the Court grants States a wide or even very wide margin of discretion in these cases. So, it can be hypothesised that there is a strong link between proceduralisation and margin of appreciation.

The application of procedural arguments by way of examination of the fulfilment of procedural obligations (requirements) obviously leads to different outcomes. Firstly, the Court may hold the States responsible for infringements against particular rights in cases where it would not have been able to hold them responsible for a substantive violation of the right (such a conclusion is manifested in the literature in relation to procedural obligations under Articles 2 and 3), or the finding of a substantive violation would involve taking a morally conditioned position. This was the situation in the abortion-related cases (*A., B. and C. v. Ireland, Tysiąc v. Poland, R. R. v. Poland* and *P. and S. v. Poland*) and in the cases relating to assisted suicide and euthanasia (*Lambert v. France*). To date, the Court has limited itself to finding a violation of the rights guaranteed by Article 8 only in those situations where it found noncompliance with positive obligations in the area of ensuring a proper decisionmaking process and the possibility to appeal to a court.

19 Haas v. Switzerland, par. 61.

20 Kleinlein, "The Procedural Approach." 95.

Secondly, the Court chose to employ proceduralisation in each of the three areas examined, in cases which apparently required it to make its own in-depth balancing of the applicants' interests. There, it recognised the State's drawing of the "bright line" (the terminology explicitly used in *Evans v. UK* and referred by the Court itself to *Pretty v. UK*21), namely the formulation of clear legal principles, even if they do not provide for the possibility of regulating exceptional situations differently, to "serve both to produce legal certainty and to maintain public confidence in the law in a sensitive field." The protection of those values – such as certainty and confidence – was placed above the legitimacy of the Court's independent balancing of conflicting interests in the individual circumstances of cases.22

Accordingly, it may be concluded that the Court's use of procedural reasoning in the cases in question led to conclusions that differed from those presented in relation to the use of procedural reasoning in the overall jurisprudence. Authors argue that procedural argumentation cannot replace substantive control, serving only as an auxiliary reasoning at most.23 By contrast, in the reviewed cases, this reasoning constituted an equally important element of the justifications. It can be pointed out, for example, that, based on the assumptions of proceduralisation, the Court has held that the fact that some legislation does not provide for the weighing of competing interests in each individual case is not per se contrary to the Convention requirements (so, for example, in *Evans v. UK* and *Pretty v. UK*).

Although there is a general perception that resorting to proceduralisationbased reasoning is of a more "neutral" character, the Court's use of procedural tactics does not always lead to smoothing the relationship between the ECtHR and the respondent State, as best exemplified by the years-long nonenforcement of abortion-related decisions against Poland. On the other hand, a mere finding of a procedural violation of the Convention may often be insufficient and disappointing for the applicant.24 This is where the Court's conclusions, formulated using incremental arguments, can meet the expectations of applicants and persons in a similar situation.

Undoubtedly, the analysis of the judgments shows that the procedural approach has served to augment the scope of the Court's review in situations where it would grant states a wide margin of appreciation or in situations

21 Evans v. UK, par. 65 and 68 and Joint Dissenting Opinion of Judges Türmen, Tsatsa-Nikolovska, Spielmann and Ziemele, par. 1 and 8.

#### 214 *Conclusion*

where it would be beyond the Court's competence to engage in the adjudication in the substantive aspect of the right in question.25

Clearly, the intensity of the use of the different types of arguments in a case – including those pertaining to procedures – will depend on the result that the Court wishes to achieve by appealing to the individual arguments. If the Court wants to redefine substantive standards, then less attention will be paid to procedural reasoning.

Meanwhile, the nature of positive obligations allows the ECtHR's judgments also to retain their subsidiary character vis-à-vis national legislative and judicial actions without substantive adjudication on end-of-life issues.26

#### *Margin of appreciation – shaping the right at home*

The wide margin of appreciation granted to States by the Court in cases involving moral considerations leads to the recognition and approval of a wide variety of national solutions. By means of this mode of argumentation, the Court is certainly not pursuing the aim advocated in the doctrine in respect of its jurisprudence, namely the harmonisation of law.27

In none of the cases reviewed, in which national legislation was called into question, did the Court find the existence of European consensus reflecting at least in principle a common moral standard. Establishing the existence of such a consensus would undoubtedly have an impact on limiting the margin of appreciation granted to those States whose legislation does not coincide with it. Granting States such a wide margin of discretion, as was done in *H.S. and Others v. Austria*, prompts the conclusion that almost any decision of the legislature, irrespective of its consistency and rationality, will fall within that margin.28

The reasoning used by the Court, built on references to the margin of appreciation, proves that it has accepted that States have the right to reflect certain views on moral and ethical issues in their legislation and to shape

25 Arnardóttir, "Organized Retreat." 6. An example of cases in which it would be impossible for the Court to pronounce on the merits of the case without engaging the procedural obligations approach are cases under Article 2 of the ECHR in which the circumstances leading to death occurred outside the temporal scope of the Court's competence in relation to the given country.

provisions in such a way as to be consistent with those views. The close association of the width of the margin of appreciation with the moral views of society, as was the case in *H.S. and Others v. Austria* and *A., B. and C v. Ireland*, deserves special mention. Clearly, the Court leaves it up to the national authorities, and in particular the parliaments of the Member States, to resolve ethical issues and conflicts in this area. Meanwhile, it pays close attention to the way the decision is reached: the consultation processes (*Lambert v. France*), the participation of the public and parliament in resolving the issues giving rise to the complaint (referendum – *A., B. and C. v. Ireland*), or the process of considering the rights and claims of the applicant in court proceedings (*Koch v. Germany*). In this respect, therefore, the State's demonstration of its thoroughness in examining the social background and the views held in society is a very strong argument in disputes – all the stronger the more difficult it is to resolve a worldview dilemma.

#### *The need to protect specific groups of persons as a limit of right to self-determination*

Throughout our book, we have assumed that when deciding on morally sensitive cases, the Court uses the premise of public morality as a legitimate aim for restricting the rights and freedoms of individuals very infrequently, hardly ever in fact. Be it in euthanasia or medically assisted procreation cases, the rationale it accepts is the protection of the rights and freedoms of others. In cases involving medically assisted procreation, the Court also makes auxiliary reference to the premise of protection of public health (*S.H. and Others v. Austria*) and occasionally the premise of protection of public order (*Paradiso and Campanelli v. Italy*). Defining the group whose interests – values belonging to whom – are protected by these premises is crucial for the Court's assessment of the application of these premises, instead of the seemingly appropriate premise of public morality.

It seems relatively easy to identify the interests that are protected under the premises of public health and public order. However, it may be difficult to identify the interests that are protected under the premise of "rights and freedoms of others." The key question that arises here is how concrete the identification of other persons must be for the balancing test to be reliable. In both categories of cases in which such a legitimate aim has been formulated, namely the end-of-life and medically assisted procreation cases, the rights and freedoms of persons concern a hypothetical group of wronged persons, a category that represents an extrapolation of the Court's fears about the possible and future harm that would be caused by extending the limits beyond the national decisions. The protection of the group so defined counterbalanced the individual interest of the applicants and effectively led to the limitation of their rights. As we pointed out previously, this strategy of determining the socially undesirable effects of a ruling is quite controversial, albeit convincing on pragmatic grounds. This tactic reveals that the courts seek justification in the realities of social life, referring to the motives which may guide such persons, and thus – indirectly – to develop a certain attitude of socially approved consent to certain behaviours. When it comes to marking the limit of the right to assisted suicide, it is the fear of pressure being exerted on weak and vulnerable persons to end their lives. Similarly, in the medically assisted procreation cases, the Court abstractly identifies a circle of "other persons," such as unspecified disadvantaged women or children potentially born as a result of the surrogacy technique. In either case, such a designation of a hypothetical group appears insufficiently precise to provide a background and reference for evaluating the interference into the applicants' rights by balancing their rights against those of this abstract group of persons. However, the Court accepts the governments' argument that it is precisely the protection of these potential victims that will justify the interference with the applicants' rights by the need to ensure the protection of the rights of other persons, identified as groups deserving special attention. Identification of these groups is without doubt an element contributing to the position of a judicial authority in the context of moral standpoint on the issue of admissibility of specific actions by referring to social practice.

This pragmatic instrument of reasoning through the indication of possible risks of a contradictory decision seems to be one the most interesting among those discussed in the study. It may mark a certain tendency to use different ways of reasoning and to look for consensus where it is particularly difficult to do so. However, from the point of view of the consequences for the system of protection, it should be noted that there is a risk of creating such a group ad hoc for the purposes of almost any adjudication, not only within the scope of the discussed cases. This may produce unexpected effects and limitations of rights not yet considered. From the point of view of the stability of jurisprudence and its predictability, this is in fact a sort of game-changer. The argument derived from the need to protect the rights of others is a powerful one; it has the most unquestionable justification in the human rights order, and the values represented by the legitimate aim thus formulated are capable of counterbalancing any right guaranteed under the Convention, among all those that may be restricted under the Convention norms. This is neither dogmatically nor ideologically questionable. Yet for this very reason, constructing a group of hypothetically vulnerable persons for the purpose of arguing about the limits of the exercise of a right – and in particular of the rights guaranteed by Article 8 – is a move with consequences that are difficult to foresee.

#### **Conclusion**

As the analysis we have carried out demonstrates, in all the cases it has decided, the Court employed several ways of reasoning at once. Some of them have been known for a long time and take the form of methods or tools of interpretation. Our study shows, however, that they are used primarily as means of convincing people of the decisions rendered, and thus they are derivative of the decision. They serve to substantiate it, and not only to give meaning to the norms of the Convention. In addition, their use is much broader and includes not only law, but also social practice and decision-making processes.

The use of particular ways of reasoning is demonstrated in great detail against the background of cases involving moral dilemmas.

By applying some of the argumentative devices, the Court clearly strives to avoid taking a position on issues of a morally sensitive nature. This will be the result of proceduralisation and appeals to authority, including the margin of appreciation.

In turn, other ways of reasoning favour the possibility of presenting the Court's view on certain moral issues, although such pronouncements are constructed with great prudence and usually indirectly. Undoubtedly, these ways include incrementalism, which consists in extracting new constitutive elements of the right vested in the applicants, arguments referring to potential secondary effects on groups deserving special attention, or the Court's use of flexible terms describing its attitude towards specific entities or phenomena (a foetus, rather than an unborn child; a woman, rather than a mother wishing to undergo an abortion). These last two lines of argument allow the Court to indirectly present its position in relation to ethical issues.

A prominent outcome of our study is the conclusion that although the Court generally avoids pronouncing directly on significant moral issues, it does not remain indifferent to the need to foster desirable social attitudes. Its single most important action in this regard is the development of the concept of "vulnerable groups" or "groups deserving special protection." One major advantage of these concepts is the creation of a moral and empathetic duty of care towards others, which, according to this case law, is enshrined in the Convention's order of human rights. If the limitation of the right to self-determination involves the protection of a group potentially exposed to the indirect effects of such a broad conception of the right, then such a limitation implies treating the duty to coexist within society more broadly than apparent *prima facie*. Hence, if in a neutral and liberal setting human rights supersede the ethical order, then the conclusion can be read from the case law in these particularly sensitive cases that, within the protection of human rights, only they alone can act as boundaries for the realisation of individual claims. However, designing limitations based on the rights and freedoms of others may go beyond action directly aimed at violating them. The relationship between the exercise of the right under Article 8 and the potential outcome of harming some hypothetical group of persons as a consequence of accepting the moral and social stance adopted is still rather vague and intriguing, but it seems to set clear limits as to individual action.

Thus, notwithstanding the fact that the Court has not been assigned the role to pronounce on moral issues with authority, it remains the "Conscience of Europe."29 As opposed to the traditional conscience, however, the Court – in its capacity as the most effective international tribunal – must rationalise its rulings and convince all those subject to its jurisdiction of its arguments.

29 *The Conscience of Europe. 50 Years of the European Court of Human Rights*, Council of Europe 2010, Kanstantsin Dzehtsiarou, "The Conscience of Europe that Landed in Strasbourg: A Circle of Life of the European Court of Human Rights." *The European Convention on Human Rights Law Review* 1 (2020): 1–6.

### 218 *Conclusion*

#### **Bibliography**

Van Hoof, Wannes and Guido Pennings. "The consequences of S.H. and Others v. Austria for legislation on gamete donation in Europe: An ethical analysis of the European Court of Human Rights judgments." *Reprod Biomed Online* 25(7) (2012): 665–669.

Wasserstrom, Richard. "Rights, human rights, and racial discrimination." *Journal of Philosophy* 61(20) (1964): 628–641.

### **Index**

abortion 1, 3–4, 7, 38, 40, 44–45, 58, 94, 100, 106–107, 110–112, 135–137, 139, 141, 200, 203, 211–213; access to 101, 103–104, 108–109, 114, 123, 125, 128, 130–131, 135–136, 139, 142, 201, 204; father's rights 102, 111, 130; legality 100–102, 109, 113, 120, 122; penalisation of 101, 103–105, 125–126, 132, 134, 140–141, 207; permitting 103–106, 109, 111–112, 114–116, 119–120, 124–129, 132–134, 136–137, 199, 201, 204–205; prohibition of health reasons 115, 118, 204; right to 49, 120, 122–124, 127–131, 134–135, 142n164, 204, 208, 217; tourism 117–119, 202, 208 access to unauthorised medicinal products 170 adoption 66, 74, 82–86, 92–93 American Convention on Human Rights (1969) 107 anchored in law concepts 31 argumentation: *ad auctoritatem* 66–78, 106–120, 151–166, 192; *ad verecundiam* 47, 113, 161; deontological 78–87, 120–143, 166–182; teleological 87–95, 182–191, 199–200 argumentative tools 2–4, 20, 38–42, 45–46, 58–59, 95, 97, 119, 155, 159, 192–193, 199–202, 209; authority (*ad auctoritatem*) 3, 21, 39–48, 52, 199–201, 210, 216–217; deontological 40, 48–56, 78–87, 115, 182, 192, 199; teleological 2, 40, 54–58, 120, 143, 193, 202 assisted dying/suicide: access to 146–148, 151, 154, 156–157, 159–160, 170,

democratic debate 173 discretion of the State 15, 79n63, 110, 113, 157, 159 disobedience 53

ECtHR rulings: *Ada Rossi and Others v. Italy* [2008] 149, 202; *A., B. and C*. *v. Ireland* [2010] 38, 94, 103–104, 109, 113–116, 118, 121–122, 125–127, 132–134, 139, 141–142, 158, 200, 202–204, 206–208, 212, 215; *Boso v. Italy* [2002] 102, 111, 121, 124, 126, 130, 138, 198, 204; *Brüggemann and Scheuten v. Germany* [1977] 101, 107, 110–111, 120, 123, 125, 130, 137, 206; *Burke v. UK* [2006] 149, 157, 168, 178; *Dickson v. UK* [2007] 64, 67, 70–71, 75–76, 78–79, 84–85, 95, 205; *D*. *v. France* [2020] 79; *D*. *v. Ireland* [2006] 39, 103, 115; *Evans v. UK* [2007] 6, 53, 64, 66–67, 70–72, 76, 78–79, 81–85, 87, 95, 128, 162, 200, 202–204, 213; *Foulon and Buvet v. France* [2017] 79; *Gard v. the UK* [2017] 150, 153, 157–158, 162–165, 169–170, 179, 181, 202; *Glass v. the UK* [2004] 149–150, 152–153, 163, 165, 167, 179; *Gross v. Switzerland* [2013] 148, 150, 159, 171, 177; *Haas v. Switzerland* [2011] 148, 155–157, 170–171, 176–177, 184–185, 188–189, 192, 202, 207, 212; *H. R*. *v. Norway* [1992] 10, 102, 108, 112; *Koch v. Germany* [2012] 148, 150, 156–157, 160, 171–172, 175–177, 202, 207, 215; *Laborie v. France* [2017] 79; *Lambert and Others v. France* [2015] 48, 150, 153–155, 157–158, 164–165, 168–169, 178, 180–181, 200, 202, 204, 207, 212, 215; *Marcx v. Belgium* [1979] 80n64; *Mennesson v. France* [2014] 65, 68–70, 73–77, 79, 81–82, 87, 89–91, 93–96, 205, 207, 211; *Nicklinson and Lamb v. UK* [2015] 147, 149, 173, 179–180, 187–189, 191, 193, 204, 208; *Paradiso and Campanelli v. Italy* [2017] 66, 68, 70–71, 73–74, 79–81, 86–87, 89–90, 92–96, 215; *Parrillo v. Italy* [2015] 65–66, 68, 70, 74, 80–82, 86, 90, 95–96, 207; *Pretty v. UK* [2002] 148, 155–156, 159–160, 168, 170, 172–177, 183–187, 189–190, 192–193, 198, 202, 204–205, 208–209, 211–214; *P. and S*. *v. Poland* [2012] 104, 115–116, 122, 128, 135, 140–142, 206, 212; *R. R*. *v. Poland* [2011] 103, 109, 115, 121–122, 127–129, 134–136, 138, 140, 142, 204, 212; *Sanles Sanles v. Spain* [2000] 148, 174–175, 202; *S.H*. *v. Austria* [2010, 2011] 38, 64–65, 67, 69–74, 77, 79, 81–82, 84–91, 94–95, 202–203, 207–208, 214–215; *Tysiąc v. Poland* [2007] 103, 108, 115–116, 121–127, 131–132, 134, 136, 138, 140–141,

185, 201, 212; *Vo v. France* [2004]

life prolongation 151, 161n69

medical evidence 161n69

	- instruments/means of 45, 59, 191

principles of states 72n35, 77

taboo 4, 7–8, 26, 156, 182

wrongful birth 103


*2.3.1 The effect of total solid (TS)*

Solid-state anaerobic digestion (SS-AD) systems usually operate at TS contents of higher than 15% [4]. A research has been conducted [20] about the effect of

**Table 3.** *Research material needs.*

**Figure 5.** *Cumulative biogas yield per g TS based on C/N ratio.*

## *Biogas Production from Water Hyacinth DOI: http://dx.doi.org/10.5772/intechopen.91396*

solid-state anaerobic digestion (SS-AD) on biogas production using water hyacinth leaves. In this study to make the total solid content increased, drying method was used. The water hyacinth leaves from Rawa Pening Lake have an initial total solid content of 13.52%. The first variable was dried for 2 days, and the second variable was dried for 1 day. After that, the water hyacinth leaves that have been dried in the sun were examined for their total solid content using Eq. (1). For the first variable, the total solid content was 48.26% and for the second variable was 36.36%. After that the water hyacinth leaves that have been dried in the sun were added to the cow rumen in a ratio of 1:1. In studies using the SS-AD method, no additional water was given [20]. The combination of the variables is shown in **Table 4**.

The variable with TS of 24.34% produced biogas with a total of 34.79 ml/g TS, and the variable with TS of 17.67% obtained 52.98 m/g TS. The result is shown in **Figure 6**.

Further research had been conducted [24] to know about the optimization of total solid (TS) and carbon to nitrogen (C/N) ratio of biogas production from water hyacinth leaves by adding microbial consortium as much as 3%, 6%, and 9%. Meanwhile the total solid contents from the research were 15%, 27.5%, and 40%. And the C/N ratios were 20, 35, and 50. To get the optimum conditions, calculation had been done by the central composite design method with the following variables in **Table 5**.

Variations of variable values in each reactor were obtained using Statistica software as shown in **Table 6**.

**Table 4.**

*Initial and final total solid of water hyacinth leaves.*

**Figure 6.** *Cumulative biogas yield per gram TS.*

### *Biogas - Recent Advances and Integrated Approaches*

### **Table 5.**

*Variable values in the central composite design.*

### **Table 6.**

*Variable values in experiments using central composite design.*

In this SS-AD method, variations in the total solid concentration used were 15%, 27.5%, and 40%. The total solid for each reactor was adjusted to the total solid of the water hyacinth. Water and rumen were added to regulate the total solid in each of the reactors. **Figures 7–9** show the graphs of biogas results produced at certain reactors which were compared between reactors with the same C/N and microbial consortium ratio values against different TS values [24].

The graph in **Figure 7** shows the production of biogas produced from Reactor 1 and Reactor 5 where the reactors had the same concentration variations for the same C/N ratio and microbial consortium variables, with the lowest value of each variation of 20 for the C/N ratio and 3% for the concentration of the microbial consortium. The difference was in the total solid concentration (**Table 6**). Based on the graph in **Figure 7**, the total cumulative biogas production for Reactor 1 was 27.367 ml/g TS while for Reactor 5 was 5.1 ml/g TS. Reactor 1 with a lower TS which was 15% produced biogas with a greater total than that of the Reactor 5 with TS of 40% [24].

Reactors 4 and 8 had varying concentrations for the same C/N and microbial consortium variables (**Table 6**), namely, a C/N ratio of 50 and a microbial consortium concentration of 9%. Both of these variation values are the highest values

**Figure 7.** *Effect of TS on biogas production (Reactors 1 and 5).*

**Figure 8.** *Effect of TS on biogas production (Reactors 4 and 8).*

among the range of values for these variables. The TS concentrations in Reactors 4 and 8 were 15% (lowest value) and 40% (highest value). Reactor 4 with a lower TS value of 15% produces more biogas production than Reactor 8 with a higher TS value (40%). **Figure 8** shows that the total biogas production for Reactor 4 was 43.87 ml/g TS and for Reactor 8 was 6.15 ml/g TS [24].

The biogas production graph in **Figure 9** came from a reactor with a C/N ratio of 35 and a microbial consortium concentration of 6% (**Table 6**). This value was the middle value of the variation of concentration for each of these variables. Biogas production varies in each of the reactors. It can be seen in **Figure 9** that the total biogas production for Reactors 9, 10, and 11 was 22.65 ml/g TS, 87.85 ml/g TS, and 10.09 ml/g TS. Reactor 10 with TS concentration of 5.45%, C/N ratio of 35, and

**Figure 9.** *Effect of TS on biogas production (Reactors 9, 10, and 11).*

**Figure 10.** *Effect of C/N ratio on biogas production (Reactors 1 and 3).*

microbial consortium concentration of 6% produces the largest biogas production when among Reactors 10 and 11 [24].

## *2.3.2 The effect of C/N ratio*

The variations in the C/N ratio used in this study were 20, 35, and 50. First the C/N ratios of the water hyacinth leaves were tested. To obtain variations in the concentration of the C/N ratio as determined, urea was used to adjust the N value of the water hyacinth leaves [24].

Different C/N ratios were tested with the same total solid and microbial consortium concentration in Reactor 1 and Reactor 3 (**Table 6**). Reactor 1 with a C/N ratio

### *Biogas Production from Water Hyacinth DOI: http://dx.doi.org/10.5772/intechopen.91396*

of 20, total solid of 15%, and microbial consortium concentration of 3% produced a total biogas of 27.37 ml/g TS. Reactor 3 with a C/N ratio of 50, total solid of 15%, and microbial consortium concentration of 3% produced biogas with a total of 51 ml/g TS. Reactor 3 with a higher C/N ratio of 50 produced more biogas volume than the Reactor 1 with a C/N ratio of 20. The graph is shown in **Figure 10** [24].

The graph in **Figure 11** was a biogas graph produced from Reactors 6 and 8. The concentrations of the total solid and microbial consortium variables in Reactor 6 were the same as those in Reactor 8 which were 40% and 9%, respectively (**Table 6**). The C/N ratio of Reactor 6 was 20, while Reactor 8 is 50. For the total biogas production produced, based on the graph in **Figure 11**, it can be seen that Reactor 6 has a higher biogas than that of the Reactor 8 which was 13.14 ml/g TS for Reactor 6 and 6.15 ml/g TS for Reactor 8. Thus, reactors with lower C/N ratios produce higher biogas under conditions of total solid concentration of 40% and microbial consortium of 9% [24].

The graph in **Figure 12** was taken from the calculation of biogas production produced in Reactors 9, 12, and 13. The reactors have the same total solid concentration and

**Figure 11.** *Effect of C/N ratio on biogas production (Reactors 6 and 8).*

**Figure 12.** *Effect of C/N ratio on biogas production (Reactors 9, 12, and 13).*

microbial consortium (**Table6**) of 27.5% and 6%, respectively, with a C/N ratio different from Reactor 9 with a C/N ratio of 35, Reactor 12 with a C/N ratio of 8.54, and Reactor 13 with a C/N ratio of 61.45. The total biogas production in Reactor 9 was 22.65 ml/g TS, whereas in Reactor 12, the total biogas production was 4.76 ml/g TS. For Reactor 13, the total biogas production was 31.24 ml/g TS. The volume of biogas production in Reactor 13 was greater than the volume of biogas production in Reactors 9 and 12 [24].


*2.3. Statistical Analysis*

To compare both baseline patient characteristics and postoperative outcomes between the pre- and post-COVID cohorts, Pearson chi-square tests were used for categorical variables and independent samples T-tests were used for continuous variables. Continuous variables were presented in terms of the mean and standard deviation (SD), and categorical variables were reported with frequencies and percentages. Statistical tests were 2-sided with *p* < 0.05 denoting statistical significance.


**1. Introduction**

The persistence and the resistance of *Escherichia coli* O157:H7 to disinfection are associated with its ability to form biofilms on food contact surfaces. Biofilms are communities of microorganisms embedded in an aqueous matrix of extracellular polymeric substances (EPS) produced by the attached cells; EPS are mainly composed by polysaccharides, proteins, lipids, and nucleic acids, which can vary in composition among strains and environmental conditions [1]. The adhesion and the biofilm formation of *E. coli* on food contact surfaces causes cross-contamination, and its consequences are observed on continuous outbreaks every year [2]. It has been reported that *E. coli* O157:H7 biofilms on stainless steel can lead to the release of embedded cells to contaminate other surfaces [3]. This information highlights the importance of studying the characteristics of *E. coli* biofilms to assure effective disinfection procedures.

Exopolysaccharides are secreted during *E. coli* O157:H7 biofilm development, and some of them include cellulose, colanic acid, and the adhesin poly-β-1,6-N-acetyl-glucosamine, and these polymers are involved in the maintenance of biofilm structure and cellular protection against disinfectants [4]. It has been reported that cellulose is the major EPS component of *E. coli* biofilms, and it is essential for its structure and strength, creating cell–cell and cell–surface interactions, retaining water, and avoiding the effect of disinfectants [5]. Previously it was demonstrated that degradation of the EPS matrix of *E. coli* O157:H7 biofilms (mainly composed by glucans) increased their susceptibility to disinfectants. The synthesis and the secretion of glucans are carried out by the enzyme glucosyltransferase, consisting of three transmembrane proteins (BcsA, BcsB, and BcsC) [6]. BcsA is the catalytically active subunit located within the cell, and it is responsible for the uridine diphosphate glucose (UDP-glucose) condensation, then the product is transferred to BcsB and BcsC subunits for processing and extracellular secretion [6]. Thus, blocking this enzymatic process could lead to the inhibition of biofilm production, leaving planktonic *E. coli* more susceptible to disinfectants.

The essential oil (EO) of lemongrass (*Cymbopogon citratus*) is rich in terpenes such as citral (85%) and geraniol (1.5%). *C. citratus* EO has been effective in inhibiting the planktonic growth of *E. coli* O157:H7 with a minimal inhibitory concentration (MIC) of 0.63 mg/mL [7], while Singh et al. [8] reported an MIC value of 0.008 mg/mL. On the other hand, citral and geraniol also showed antibacterial activity against *E. coli* as well as anti-quorum sensing activity at concentrations of 0.01 and 0.06 mg/mL, respectively [9]. On the other hand, *C. citratus* EO in combination with *Allium cepa* EO reduced the presence of *E. coli* in lettuce and spinach [10]. However, its antibacterial activity on planktonic cells could differ from the expected response against biofilms. In addition, *C. citratus* EO was able to inhibit *Staphylococcus aureus* and *Streptococcus mutans* biofilms [11,12]. Previous evidence described the ability of citral and geraniol-like terpenes to traverse the bacterial membrane and interact with vital metabolic enzymes [13]. Previous studies also evidenced the potential of citral to inactivate several enzymes [14,15]. Therefore, the objective of this study was to explore the effect of *C. citratus* EO, citral, and geraniol on the glucans production, glucosyltransferase activity, and biofilm formation of *E. coli* O157:H7.


*Article* **The Impact of Education and Residential Environment on Long-Term Waste Management Behavior in the Context of Sustainability**

**Andreea Simona Saseanu 1, Rodica-Manuela Gogonea 2,\*, Simona Ioana Ghita 2,3 and Radu ¸Serban Zaharia 4,\***

Received: 13 June 2019; Accepted: 8 July 2019; Published: 10 July 2019

**Abstract:** Currently, the problem of waste reduction is a permanent concern for all countries of the world, given the need to ensure the sustainability development. In this context, the research aims to highlight the impact of education and demographic factors by residence areas on the long-term behavior of the amount of waste generated in 29 European countries during 2013–2017. The study is based on statistical and econometric modeling aimed at identifying, testing and analyzing the existence of long-term correlation between the amount of waste per capita recorded in each country and four factors of influence considered significant for waste reduction: *Pupils and students by education level* and *Classroom teachers and academic staff by education level*, representing exogenous variables which quantify the educational outcomes, as well as *The population by degree of urbanization* (cities, rural areas), as demographic factors. As a result of an analysis based on correlation and regression method, a cointegration relationship between the analyzed variables was identified. Considering the amount of waste as an important component of the environmental pressure, the obtained results show the significant long-term effect that education and the demographic factor can have on its long-lasting behavior, as well as the ways through which these factors can act to strengthen sustainability.

**Keywords:** sustainability; waste; education; degree of urbanization; cointegration model; Vector Error Correction models; autoregressive vector patterns