**3. Biohazard**

Biohazard in the production of biogas may be related to feedstock and digestate. Wastes of animal and human origin contain various pathogenic bacteria (e.g. *Salmonella*, *Enterobacter*, *Clostridia*, *Listeria*), parasites (e.g. *Ascaris*, *Trichostrongylidae*, *Coccidae*), fungi, viruses [11, 12] and could represent an occupational biohazard. In the biogas production from co-digestion of animal manure and biogenic wastes, the microbiological quality of raw materials of animal origin is guaranteed only through the application of specific veterinary and sanitary measures (e.g. control of livestock health, hygiene control of raw materials entering the digester). High-risk biomasses such as those from sick animals must be excluded from use; for biomass categories such as slaughterhouse residues, pre-sanitation measures are required through pasteurization or sterilization as stipulated by European Regulation EC 1069/2009 [13]. In case of feedstock categories, which do not require separate pre-sanitation, the combination of AD process temperature and a minimum guaranteed retention time provides an effective pathogen reduction/inactivation in the digestate [14]. In Italy, the digestate quality standard is monitored by several checkpoints [15]. In a biogas plant, exposure levels to biological agents are highly dependent on site activities and tasks undertaken by workers. It is the site operator's responsibility to identify potential hazards, carry out suitable risk assessments and provide adequate protection to their workforce to control such risks. During AD, the microbial reactions take place inside the digester under containment conditions and, therefore, there is no workers' exposure. However, activities such as inoculation, sampling and harvesting the microbial flora during the monitoring of the fermentation process, could involve worker exposure and, therefore, the workers' activities should be checked to define the exposure characteristics. According to European classification, the microorganisms with infection potential, which take part in the anaerobic fermentation process, are mainly assigned to the risk group 1 and to a small extent to the risk group 2 [16]. Some of these microorganisms should be considered opportunistic agents, which do not cause any infections in healthy employees, but they can lead to diseases when body defences are defective. In general, good work practices and simple but effective personal hygiene measures are sufficient to prevent workers from infection risk, including provision of adequate hand-washing facilities. Biological risk assessment should take into account that specific activities, such as biomass reception, temporary storage, biomass handling, digestion drainage and maintenance work, may pose exposure risks to organic dust, bioaerosol and biological components conveyed such as particulates (i.e. bacterial endotoxins, fungal spores). Evidence from epidemiological data shows that these biological agents can cause allergic reactions such as hypersensitivity pneumonitis, allergic rhinitis, some types of asthma and organic dust toxic syndrome (ODTS) [17]. In Italy, the biogas industry expansion is quite recent, and there are not many data available on biological risk in these plants. Recent findings on airborne workers' exposure in two full-scale plants of anaerobic digestion in North Italy showed different biological contamination levels in relation to the involved biomasses (silage, vegetable waste, animal slurry and biomass from dedicated crops) and to the technological and building characteristics [18]. This evidence suggests that every biogas plant requires a specific approach. Contamination and occupational risk must be evaluated individually for each plant, because numerous variables influence risk magnitude, with particular regard to digested sludge treatments, such as input biomass nature, storage, movement conditions, building configuration and technological processes [18]. The results of the air microbiological monitoring, performed during the biomass movement in some biogas plants investigated in Italy, showed that organic dust (PM10) and its endotoxin content are limited [18] and widely below the occupational safe guidelines [19, 20]. The particulate is not a relevant risk for workers in the plants monitored, because it reached rural environmental

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The assessment of biological risks is seriously hampered, since neither universally approved criteria for assessing exposure to biological agents nor agreed dose-response estimates and occupational exposure limits (OELs) are yet available. Lack of a standardized sampling methodology has made it difficult to compare data derived from different studies and relate exposure levels to effects on health. Potential seasonal variation of microbial exposures also adds difficulties in comparing data. Establishing the prevalence and incidence rates of diseases related to exposure to biological agents is not easy: data on occupational diseases from biological agents are difficult to collect, because the infections could often be in subclinical form, with atypical incubation periods and/or transmission routes [21]. Moreover, the exact role which, is played by biological agents in the development or aggravation of symptoms and diseases, is only poorly understood. Human response to exposure to biological agents depends on the organic material involved and individual's susceptibility to infections and allergies. In addition, microorganisms constantly interact with the environment and are able to modify their pattern of gene expression rapidly in response to the environmental signals [21]. A variable human response has also been described, following the exposure to organic dust in different workplace settings, and it was shown that the composition of the dust may play an important role in determining the potency [22]. The assessment of biological risk in the biogas sector is a complex task, even considering that the biogas industry is still in its infancy in some countries such as Italy. Limited public domain information is also available from ongoing health and injury surveillance of biogas workers, particularly for health outcomes of highest concern (e.g. respiratory, irritation, sensitization). There is a need for improving the collection of work-related diseases in the biogas sector, and an ad-hoc accident

The proposed approach for biological risk assessment is that certain areas or activities, resulting from the biogas industry, could be categorized using fairly simple descriptive expressions of risk and a corresponding set of control measures, which depend on the perceived risk associated with the area or the activity. The qualitative checklist approach can represent a reasonable tool in order to overcome the current knowledge gaps in establishing agreed monitoring protocols and developing reliable dose-response data. In absence of such information, the potential risk should be managed in a precautionary manner. Exposure levels to biological agents are highly dependent on site activities and tasks undertaken by workers, and an adequate workers' protection requires a detailed site and task risk assessment. Potential exposure can be controlled by changing the work process to minimize the generation of bioaerosols or dustiness. In order to achieve compliance, employers should demonstrate that adequate control measures have been developed in accordance with the hierarchy of controls, detailed

levels recorded in North Italy [18].

reporting system should be created.

**3.1. Biological risk assessment**

movement conditions, building configuration and technological processes [18]. The results of the air microbiological monitoring, performed during the biomass movement in some biogas plants investigated in Italy, showed that organic dust (PM10) and its endotoxin content are limited [18] and widely below the occupational safe guidelines [19, 20]. The particulate is not a relevant risk for workers in the plants monitored, because it reached rural environmental levels recorded in North Italy [18].

#### **3.1. Biological risk assessment**

**3. Biohazard**

186 Advances in Biofuels and Bioenergy

Biohazard in the production of biogas may be related to feedstock and digestate. Wastes of animal and human origin contain various pathogenic bacteria (e.g. *Salmonella*, *Enterobacter*, *Clostridia*, *Listeria*), parasites (e.g. *Ascaris*, *Trichostrongylidae*, *Coccidae*), fungi, viruses [11, 12] and could represent an occupational biohazard. In the biogas production from co-digestion of animal manure and biogenic wastes, the microbiological quality of raw materials of animal origin is guaranteed only through the application of specific veterinary and sanitary measures (e.g. control of livestock health, hygiene control of raw materials entering the digester). High-risk biomasses such as those from sick animals must be excluded from use; for biomass categories such as slaughterhouse residues, pre-sanitation measures are required through pasteurization or sterilization as stipulated by European Regulation EC 1069/2009 [13]. In case of feedstock categories, which do not require separate pre-sanitation, the combination of AD process temperature and a minimum guaranteed retention time provides an effective pathogen reduction/inactivation in the digestate [14]. In Italy, the digestate quality standard is monitored by several checkpoints [15]. In a biogas plant, exposure levels to biological agents are highly dependent on site activities and tasks undertaken by workers. It is the site operator's responsibility to identify potential hazards, carry out suitable risk assessments and provide adequate protection to their workforce to control such risks. During AD, the microbial reactions take place inside the digester under containment conditions and, therefore, there is no workers' exposure. However, activities such as inoculation, sampling and harvesting the microbial flora during the monitoring of the fermentation process, could involve worker exposure and, therefore, the workers' activities should be checked to define the exposure characteristics. According to European classification, the microorganisms with infection potential, which take part in the anaerobic fermentation process, are mainly assigned to the risk group 1 and to a small extent to the risk group 2 [16]. Some of these microorganisms should be considered opportunistic agents, which do not cause any infections in healthy employees, but they can lead to diseases when body defences are defective. In general, good work practices and simple but effective personal hygiene measures are sufficient to prevent workers from infection risk, including provision of adequate hand-washing facilities. Biological risk assessment should take into account that specific activities, such as biomass reception, temporary storage, biomass handling, digestion drainage and maintenance work, may pose exposure risks to organic dust, bioaerosol and biological components conveyed such as particulates (i.e. bacterial endotoxins, fungal spores). Evidence from epidemiological data shows that these biological agents can cause allergic reactions such as hypersensitivity pneumonitis, allergic rhinitis, some types of asthma and organic dust toxic syndrome (ODTS) [17]. In Italy, the biogas industry expansion is quite recent, and there are not many data available on biological risk in these plants. Recent findings on airborne workers' exposure in two full-scale plants of anaerobic digestion in North Italy showed different biological contamination levels in relation to the involved biomasses (silage, vegetable waste, animal slurry and biomass from dedicated crops) and to the technological and building characteristics [18]. This evidence suggests that every biogas plant requires a specific approach. Contamination and occupational risk must be evaluated individually for each plant, because numerous variables influence risk magnitude, with particular regard to digested sludge treatments, such as input biomass nature, storage,

The assessment of biological risks is seriously hampered, since neither universally approved criteria for assessing exposure to biological agents nor agreed dose-response estimates and occupational exposure limits (OELs) are yet available. Lack of a standardized sampling methodology has made it difficult to compare data derived from different studies and relate exposure levels to effects on health. Potential seasonal variation of microbial exposures also adds difficulties in comparing data. Establishing the prevalence and incidence rates of diseases related to exposure to biological agents is not easy: data on occupational diseases from biological agents are difficult to collect, because the infections could often be in subclinical form, with atypical incubation periods and/or transmission routes [21]. Moreover, the exact role which, is played by biological agents in the development or aggravation of symptoms and diseases, is only poorly understood. Human response to exposure to biological agents depends on the organic material involved and individual's susceptibility to infections and allergies. In addition, microorganisms constantly interact with the environment and are able to modify their pattern of gene expression rapidly in response to the environmental signals [21]. A variable human response has also been described, following the exposure to organic dust in different workplace settings, and it was shown that the composition of the dust may play an important role in determining the potency [22]. The assessment of biological risk in the biogas sector is a complex task, even considering that the biogas industry is still in its infancy in some countries such as Italy. Limited public domain information is also available from ongoing health and injury surveillance of biogas workers, particularly for health outcomes of highest concern (e.g. respiratory, irritation, sensitization). There is a need for improving the collection of work-related diseases in the biogas sector, and an ad-hoc accident reporting system should be created.

The proposed approach for biological risk assessment is that certain areas or activities, resulting from the biogas industry, could be categorized using fairly simple descriptive expressions of risk and a corresponding set of control measures, which depend on the perceived risk associated with the area or the activity. The qualitative checklist approach can represent a reasonable tool in order to overcome the current knowledge gaps in establishing agreed monitoring protocols and developing reliable dose-response data. In absence of such information, the potential risk should be managed in a precautionary manner. Exposure levels to biological agents are highly dependent on site activities and tasks undertaken by workers, and an adequate workers' protection requires a detailed site and task risk assessment. Potential exposure can be controlled by changing the work process to minimize the generation of bioaerosols or dustiness. In order to achieve compliance, employers should demonstrate that adequate control measures have been developed in accordance with the hierarchy of controls, detailed in the Directive 2000/54/EC [16]. Examples of control measures are exhaust ventilation to prevent exposure, adequate filters on the air intakes of vehicles (such as tractors used to move biomass) and personal protective equipment, such as suitably fitted respiratory devices, when working in areas close to where bioaerosols are generated.

**4. Explosion risk: formation of potentially explosive atmospheres**

1) **Zone 0**: an area in which an explosive gas atmosphere is present for long periods;

operation, but if it should occur, it would exist for a short period.

• black letters on a yellow background with black edging

explosive atmosphere in the shortest times.

reported in Technical Standards.

**Table 1.** Properties of gases.

normal operation and

• triangular shape and

features:

2) **Zone 1**: an area in which an explosive gas atmosphere can periodically occur during the

3) **Zone 2**: an area in which an explosive gas atmosphere is not expected during the normal

Directive 99/92/EC states that, places where potentially explosive atmospheres can occur are marked with specific signs (**Figure 1**), which are characterized by the following distinctive

In **Figures 2** and **3**, the classification procedure of hazardous areas (outdoor and indoor place) is shown. It may be used as a basis to support the proper selection and installation of work equipments in hazardous zones. Classification of indoor places is particularly important because ventilation system design plays a fundamental role in order to dilute the potentially

The first step of classification procedure consists of locating the potential sources of biogas release. On this subject, it has to be remembered that catastrophic elements failures are not considered as potential sources because they are beyond the concept of abnormality [27],

A plant component, such as valves, flanges, pumps, compressors, and so on, is considered as a potential source when its failures are expected during the operation. Zone classification depends on source release grade, ventilation degree and availability. Release grade

**, 40% CO2**

**) Methane Natural gas**

**Unit Biogas (60% CH4**

Heat value kWh/m<sup>3</sup> 6 10 10 Ignition temperature °C 700 600 650 Explosion range Vol (%) 7.3–28.3 4.4–16.5 4.4–15

Because of the presence of methane in its composition, biogas in combination with air can form potentially explosive atmospheres (**Table 1**). In Europe, safety measures against explosion risk are stipulated in Atex Directives 99/92/EC [24] and 2014/34/EU, which have inspired the preparation of checklist section, referred to the explosion risk. A crucial topic, reported in safety checklist, is the classification of plant areas [25], where explosive mixtures could be generated by biogas releases. This classification has to be carried out in terms of zones (Zone 0, Zone 1 and Zone 2), geometrical characterization (extent and volume) of hazardous areas [26] and persistence time:

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#### **3.2. Prevention and protection measures for occupational biohazard**

Design of workplaces and work processes, the choice of adequate equipment and working methods allow the control of occupational biohazard in the biogas plants. Any activities involving the movement of biomass and/or waste should be controlled, and site design and activities should be managed to avoid organic dust and/or bioaerosol release in the workplace. In particular, the biomass, such as silage, should be stored in closed silos or in platforms provided with containment walls and covered by a plastic material wrap. Livestock slurry storage tank should be equipped with immersion agitators to avoid air contamination, and moreover, the automatic transfer of slurry into the digester should be guaranteed by a pumping system. Working areas, where biomass is moved, should be considered as potential high exposure zones. An efficient system of forced ventilation is required if high-exposure activities are conducted within a confined space and, where practicable, employees should only work in these areas within a suitably controlled environment, such as a vehicle cab, or wear appropriate respiratory protective equipment (RPE). It is recommended that for exposure to bioaerosols, RPE is provided with the highest efficiency filters (P3). The replacement of the filters in the vehicle cabs' air handling system, cleaning of vehicle cabs and the instructions given to operators not to open cab doors and windows and remain in the vehicle have a significant effect on workers' exposure levels. These rules should be applied within a radius of 50 m from the operational areas, considering that bioaerosol levels typically return to background concentrations within this distance [23]. Such requirements clearly have an impact on site design and layout. In order to achieve these targets, the employers should amend working practices and operations and relocate office accommodation and welfare facilities to an area outside the potentially high-exposure zones. Dust control from the movement of vehicles is also recommended, and roadways should be properly constructed so that they can be cleaned and a vehicle wheels washing system should be planned. The workplace should be provided with adequate hand-washing and shower facilities and 'clean areas' in order to ensure that no contamination can affect external places. Employers should undertake an appropriate health surveillance of their workforce to ensure that early signs and symptoms of diseases, related to exposure to biological agents, are managed and reported. This may involve simple health screening or more detailed assessments, involving health questionnaires, lung function and blood serum test. All employees, who have undertaken health surveillance, should have a personal health record and the information must be kept for a period of 40 years and the findings of any health surveillance should be communicated to employees and any adverse findings should be deeply investigated and appropriate controls should be adopted. The training of site managers and personnel is a fundamental topic in order to verify the design and implementation of these prevention measures. It must be stressed that appropriate instructions, information and training, referred to the potential risks to their health and how they should be controlled, must be given to employees. Employers should also develop procedures for people who do not comply with the procedures and site rules.
