Hygienic assessment of fish handling practices along production and supply chain and its public health implications in Central Oromia, Ethiopia

Fishborne diseases are among the major causes of morbidity and mortality worldwide. Contamination of the aquatic ecosystem and unhygienic handling practices along the fish supply chain can lead to a contaminated fish. Consumption of raw or under cooked fish and fish products is a major source of fishborne infections in humans. Despite reports of fish contamination with foodborne pathogens in Ethiopia, information regarding the hygienic status of fish handling practices is limited. We assessed fish hygienic handling practices at production sites and along the fish supply chain in three towns in east Shewa zone of Oromia. Data were collected using a semi-structured questionnaire interviews and personal observations. The study consisted of purposively selected respondents comprising of 50 fishermen, 10 retailers, 20 food establishments serving fish, and 120 consumers. Descriptive statistics and Chi-square test were used to present the proportion of various actors along the fish production and supply chain and to compare the proportions of observations among the different categories respectively. We observed that the lakes were accessible to animals and exposed to chemical and microbial contaminations through rainwater run-off. Fish were processed under unhygienic practices like washing of filleted fish with lake water, indiscriminate processing at unhygienic landing sites, use of a single knife for processing all fish with infrequent washing and with no disinfection in between. Majority (70%; n = 10) of the retailers and all the food establishments transported fish in vehicles with no cold chain facilities. Good hygienic practices we observed were the use of refrigerators for storage in all retailers and 70% (n = 20) of the food establishments; 30% of retailers used vehicles with a cold chain facility for the transportation of fish. Over three-fourths (77%; n = 120) of the consumers preferred consuming raw fish; 80% of them lacked the knowledge of fishborne diseases. The study revealed a wide range of unhygienic handling practices along fish production and supply chain; lack of infrastructure for post-harvest fish handling and processing, lack of appropriate transportation facilities and presence of knowledge gaps regarding fish borne diseases.

In Ethiopia, few studies have reported the occurrence of common fishborne pathogens. A review by 32 showed that Edwardsiella tarda, Salmonella, E. coli, S. aureus, Aeromonas and Vibrio species are among the bacterial pathogens reported in fish in the country. The prevalence of these pathogens in fish varies considerably in different lakes across the country. For instance, Salmonella was reported at prevalence of 5.2% from Hayike 33 , 7.5% from Abaya and Chamo 34 and 30% from Tinike lakes 35 . Similarly, 1.5% 36 and 2.3% 37 prevalence of Shiga toxin producing E. coli O157:H7 were reported from fish samples tested at lake Hayike and Tekeze dam, and lake Hawassa, respectively.
Several bacterial pathogens cause infections and mortalities in fish and humans. People can be infected while handling infected fish on fish farms, at retail shops, or through ingestion of raw or inadequately cocked contaminated fish and/or fish products 38 . The widespread occurrence of these pathogens in fish along the fish supply chain could be due to lack of basic hygienic practices. Unhygienic handling practices lead to contamination of fish ready for human consumption with potentially foodborne pathogens; specially if fish is consumed raw or undercooked, it poses a significant public health risk 32 . Despite the availability of information on contamination of fish with common foodborne pathogens, data regarding fish hygienic handling practices along the production and supply chain is scarce in Ethiopia. The aim of this study was to assess the hygienic status of fish handling practices along production and supply chain in three towns of east Shewa zone of Oromia, Ethiopia that have natural lakes and potential reservoirs known to support fish production. Supply chain analysis is essential to closely examine the hygienic practices at each step along the production and supply chain to ensure fish quality and safety to increase consumer confidence. Information regarding the handling practices of fish at each step along the supply chain is critically needed to identify the critical control points and driving factors to devise preventive measures to ensure fish quality and safety. Moreover, it can pinpoint the extent of the problem to all stakeholders to initiate collective plans and actions to reduce associated costs and health risks and, optimize revenues to improve the livelihood of all actors relying on fishing business.

Materials and methods
Study area and study design. A cross-sectional study was conducted from December 2020 to June 2021 in Bishoftu, Koka and Batu towns of east Shewa zone of Oromia, Ethiopia. East Shewa zone is found in Central Oromia, connecting the western and eastern parts of Oromia. The zone has a total population of 1,356,342 within an area of 8,370.90 square kilometers with a population density of 162.03. While the majority (74.9%) of the population are mixed crop-livestock farmers or other rural dwellers, the remaining part of the population were urban inhabitants (25.1%) or few pastoralists (0.05%) 39 . Most of the fish population in Ethiopia is found in the rift-valley lakes, including a natural lake Dambel in Batu town, the crater lakes Babogaya and Hora-Arsedi in Bishoftu and reservoirs: Barbara or Koftu few kilo meters eastern to Bishoftu, and Koka reservoir near the town of Koka. Bishoftu, Koka and Batu towns are the three major towns in the east Shewa zone where fish is routinely harvested and consumed 40 .
Lake Babogaya is among the crater lakes of Bishoftu. Three fish species, namely Oreochromis niloticus (Nile tilapia), Clarias gariepinus (African Catfish) and Tilapia zilli were introduced into the lake by the Ministry of Agriculture to enhance the fishery resource of the lake. The most dominant species is Oreochromis niloticus; followed by Clarias gariepinus 41 . Lake Hora-Arsedi consists of only O. niloticus which was introduced around 1943 42 . Currently it supports a piscifauna, which is exclusively composed of Nile tilapia and Tilapia zillii although not much fishing is done except a few tons of fish caught for consumption by the town residents 43 . Barbara also known as (Koftu) is a man-made reservoir located few kilometers away from the eastern part of Bishoftu town. Oreochromis niloticus (Nile tilapia) is the only fish species inhabiting the reservoir. It was artificially introduced to the reservoir by the Bishoftu town office of livestock and fisheries with the objective of boosting fish production in the area 40 . Koka reservoir is the most important source of fish for small scale fisheries in general and riparian societies in particular 16 . It provides about 542 tons of fish annually. The currently existing commercially important species and their contribution to the total annual catch are common carp (Cyprinus carpio = 36%), catfish (Clarias gariepinus = 35%), tilapia (Oreochromis niloticus = 18%) and barbs (Labeobarbus intermedius = 11%) 44 .
The six indigenous fish species at Lake Dambel include Barbus ethiopicus, Barbus paludinosus, Labeobarbus intermedius, Garra makiensis, Garra dembecha and oreochromis niloticus 45 . In addition, three more exotic fish species such as Tilapia zillii, Carassius carassius and Ccarassius auratu were introduced into the lake with the objective of enhancing fish production, while the 4th exotic species, namely, Clarias gariepinus, is believed to have been slipped into the lake accidentally. Among others, tilapia (Oreochromis niloticus and Tilapia zillii), carp (Carassius auratus and Carasiuscarrasius) and catfish (Clariasgariepinus) are the most demanded and highly exploited type of fish species in the area 46 .
Sample size and sampling. The sample size was estimated according to Shari 47 who suggested a minimum required sample size of 50 participants for qualitative survey study. We collected data from a total of 200 selected respondents comprising 50 fishermen, 20 food establishment workers, 10 fish retailers and 120 consumers. Except for the later, the participants with more than one year of experience handling fish were purposively included in the study. Consumers were selected as convenience sample in the study based on their availability at the time of data collection at the restaurants or lakes, and their willingness to participate in the study. Questionnaire survey and direct observation were used to collect the data (supplementary file). All methods were performed in accordance with accepted ethics and regulatory requirements.
Ethical clearance. The 48 . Descriptive statistics was used to calculate the frequency and proportion of various actors along the fish production supply chain involving handling, processing, transportation and storage practices, and consumption preferences. Chi-square test was used to compare the proportions of observations among the different categories. P < 0.05 was used for a statistical significance of an estimate.

Results
Demographic characteristics of the participants. Among a total of 200 respondents participated in the study, 90% of them were males. The mean age of the participants was 33.3 years (range 17-56 years) ( Table 1).

Fish production and supply chain.
In the study area, fish is primarily harvested from freshwater lakes and reservoirs. The main actors in the supply chain were fishermen, fish retailers, food establishments and consumers. The fishermen take the lion's share in harvesting and processing fish. All fishermen were male in this study (Table 1). Consumers can acquire fish through five different gateways each of which require their own standard of hygienic handling processes as shown in the fish production and supply chain conceptual framework depicted below (Fig. 1). www.nature.com/scientificreports/

Hygienic conditions at lakes and reservoirs. All the respondents reported that the lakes and reservoirs
are accessible to animals, and most of them are watering points. They also indicated that an occasional open defecation by the people living and working around the lakes, and exposures of the water bodies to run-off water during the rainy season. We also observed animal feces, and cattle and cart horses roaming and grazing around the study lakes.
Post-harvest handling and processing practices. It was observed that all the fishermen handled and processed fish under unhygienic conditions at the lake shore. All of them were performing the activity with no basic processing facilities such as clean and impervious processing room/area, clean/potable water, equipment washing and disinfection facility, hand washing facility, product handling facility. A single knife and the same cutting board were often used for the processing of all fish with infrequent washing and with no disinfection in between the processing. The fishermen visually inspect each fish for any external defects followed by manual incision to separate the head, tail, scale, and skin, gut removal and filleting. Filleted fish were washed using unclean water directly obtained from the lakes. Table 2 summarizes fish processing, transportation, and handling practices by the fishermen. Majority (88%, n = 50) of the fishermen reported that their customers prefer processed fish at the shore compared to whole fish. Also, 84% of the fishermen reported selling the processed fish at the lake shore; and 64% of their customers were direct consumers. The most frequently used container by the fishermen for fish handling and transportation was a sack, and in some instances a plastic bag. They also used the same container (either sack or plastic bag) to transport a filleted fish for home consumption using a public transport with no cold chain.
Only 30% of retailers transported their fish using vehicles equipped with cold chain facilities. Overwhelmingly 70% of them used either ordinary trucks or a public transport with no cold chain using sacks and/or crate containers. All retailers stored fish in a refrigerator for a maximum duration of 7 days, but they never checked the storage temperature.
Similarly, none of the food establishments used a cold chain facility for fish transportation; and 70% of them stored fish in a refrigerator for a maximum duration of 24 h. However, none of them checked the storage temperature. The remaining 30% of the food establishments have reported that no fish will remain from the daily supply, and they did not store fish.
Fish consumption. Among the 120 consumers who completed the questionnaires, 13.3% of them preferred raw fish only, while the majority (63.3%) of them preferred either raw or heated/fried fish. Thus, about 77% of the consumers had the preference of consuming raw fish. There was statistically significant difference in the fish consumption preference based on gender and study sites (P < 0.05) ( Table 3). The majority (81.8%, n = 11) of female consumers preferred consumption of heat-treated fish. However, there was no difference in the fish consumption preferences among the consumers based on age, religion, educational status, occupation, or spatial location (study site) (P > 0.05).
Majority (83.3%) of the consumers had no knowledge about the significance of avoiding cross contamination between heat treated fish and raw fish including other raw foods (Table 3). Furthermore, 80% of them had no knowledge about fishborne zoonotic diseases. Consumers believed that the use of spices like red pepper, commonly consumed with a raw fish, and consumption of local alcoholic beverage called katikala would avoid the risk of acquiring fishborne illnesses. Three times per week 24 (12) Where to sale At the lake shore 82 (41) At restaurants 12 (6) Used for personal consumption 6 (3) Where to process At the lake shore 88 (44) At home 12 (6) Who will process Fishermen 88 (44) Other processors 12 (6) Fish customers Consumers 64 (32) Retailers 14 (7) Hotels/restaurants 16 (8) Used for personal consumption 6 (3)

Discussion
In this study, we assessed the hygienic status of fish handling practices at the production sites and using a combination of questionnaire survey and direct observation approaches to identify the fish supply chain and to qualitatively measure the hygienic conditions along the supply chain. The approaches we used are essential in generating relevant information needed as inputs in food quality assessment model. However, we did not investigate the prevalence and concentration of pathogenic microorganisms and examine the effects of unhygienic handling practices on fish microbial quality and safety of fish along the supply. Further comprehensive food safety risk assessment that consists of hazard identification at each critical point, hazard characterization (dose response relationship), human exposure assessment to the hazards via consumption of contaminated fish and risk characterization (e.g., illness per exposed people) is required to quantify the probable risk and identify the risk mitigation measures to implement efficient and feasible fish safety management system along the supply chain 49,50 . Nonetheless, our study revealed significant basic hygienic gaps in the fish handling practices that can be targeted for interventions. Considering the inherent differences in the scope and outcomes of the different food safety assessment approaches, we discussed the convergences and divergences of the findings from the perspectives of national and global sets of standards and regulatory requirements. Specifically, the major findings at each critical points in the fish supply chain were discussed in view of Ethiopian national proclamations (Ethiopian Public Health Proclamation No. 200/2000 and Ethiopian Food and Medicine Administration Proclamation No. 1112/2019), and internationally recommended hygienic standards and regulatory requirements (FAO, WHO and Codex Alimentarius Commission, CAC) for hygienic handling of fish to prevent fish contamination to ensure fish safety and to reduce financial loss due to fish spoilage. The free access of cattle to the freshwater lakes and reservoirs, the exposure of the water bodies to contaminated water run-off and open defecation by people in the area are potential factors that can lead to the contamination of the natural habitats for fish production. We also observed the use of contaminated water for  (1) Daily worker (n = 5) 0.00 (0) 20 (1) 80 (4) Farmer (n = 10) 10 (1) 20 (2) 70 (7) Fish cooker (n = 8) 0.00 (0) 50 (4) 50 (4) Fish retailer (n = 4) 25 (1) 25 (1) 50 (2) Fish processor (n = 6) 33.33 (2) 16.67 (1)  www.nature.com/scientificreports/ fillet washing which is contrary to FAO and WHO code of practice for fish and fishery products 51 .The code affirms the availability of adequate supply of clean sea water or potable water for washing fish prior to filleting or cutting, fillets after filleting, skinning, or trimming and filleting equipment and utensils. The codex general principle of food hygiene also declares the control of water quality to minimize the risk of potential biological, chemical, and physical hazards 52 . The Ethiopian public health proclamation no. 200/2000 prohibits the supply of any food which is unhygienic, contaminated, and unwholesome, or does not meet the standards of food safety and quality 53 . Therefore, the indiscriminate contamination of freshwater lakes and reservoirs from which fish is massively produced and consumed coupled with the use of potentially contaminated water for fillet washing at the production sites are important areas of concern for public health where interventions can be targeted. Fecal contamination that may contain pathogens from animals and people particularly as the result of the observed open defecation can reach the lakes through run-off water. This contradicts the general principle of food hygiene 52 , which states that the control of fecal contamination minimizes the potential for contamination with many foodborne pathogens such as Salmonella, Campylobacter, Yersinia and pathogenic strains of E. coli. Most importantly, contaminated water run-off may carry antimicrobial residues and resistant pathogens that can play an important role in the dissemination of antimicrobial resistance and transmission of such resistant pathogens among fish, animals and humans 50 . According to FAO and WHO 51 , bilateral code of practice for fish and fishery products, contamination of fish depends on the environment and the bacteriological quality of the water from which it is harvested. Once the aquatic environment is contaminated with a zoonotic pathogen, it is highly probable for the aquatic fauna including fish to acquire the pathogen and become a potential threat for fish consumers 54 . Thus, limiting the access of animals to the water bodies, diverting the direction of water run-off against the natural habitat of fish, and circumventing the open defecation by providing access to public restrooms around the beaches and at the shores are the most likely intervention strategies. Our study found significant breaches in the basic hygienic practices where the environment is likely contaminated by animal and human feces.
It was observed that, fish was washed with lake water having a potential risk of contamination with pathogenic microbes. According to FAO 54 , cross-contamination and contact with contaminated water may lead to dissemination of microbial contaminants into other fresh or heat-treated fishery products. Thus, due to unhygienic landing sites and unrestricted cross contamination of the processed products, the situation worsens the safety of consumers.
Our study indicated that fish processing was practiced by all fishermen at unhygienic landing sites at the lake shores that can result in the contamination of the fish. This contrasts with the recommendations of FAO, which states unhygienic post-harvest handling and processing procedures deteriorate the safety of consumers 54 . Fish requiring gutting on arrival at the processing facility should be gutted efficiently, without undue delay and with care to avoid contamination 55 . A study by Huss et al. 56 also showed that contamination of fishery products is primarily due to unhygienic processing or poor water quality. Training of fishermen and infrastructural development at the landing sites are important interventions to promote hygienic fish processing in the area.
Our observation of the use of the same knife and one cutting board for processing all fish without disinfection of equipment shows risky practices that contribute to contamination and cross contamination of fish during production. The European commission health and consumer protection directorate requires that equipment and premises approved for slaughtering, carcass preparation and meat cutting to be carefully cleaned and disinfected several times during and at the end of the working day, and before being re-used, to prevent contamination by pathogenic microbes or other microbes associated with meat spoilage. In addition, the European union meat hygiene directives require that during the production process the temperature of water used to decontaminate hand-held tools (knives, hooks, saws) should be maintained at 82 °C or higher 57 . However, these basic facilities were not present in the present study area and intensive training of the fishermen on the concepts of contamination, cross contamination and an overall hygienic production and processing of fish are significant tools to reduce fishborne illnesses and to increase the shelf life of the fish.
Fish transportation and storage practices are not in line with the required standards. All food establishments did not have refrigerated vehicles for transportation; and only 30% of the retailers shipped fresh filleted fish using vehicles equipped with cold chain facilities. The use of unhygienic sacks or plastic bags as a packaging material is also a common practice. However, according to FAO 54 fish is one of an exceptionally perishable product and post-harvest handling, processing and transportation channels should be performed under hygienic and cool temperature conditions to prevent microbial contamination and fish loss due to spoilage. The transportation and storage methods of fish should also be environmentally friendly, and assure the safety of consumers 58 . The recent recommendations of FAO and WHO 59 also declares that fresh fish should be transported using clean and suitable vehicles at a temperature closer to 0 °C, preferably in containers with dry freezer bags instead of ice while frozen products should be maintained at -18 °C or below. It also obliges the use of clean, sound, and durable packaging materials and maintaining adequate protection against contamination from dust, exposure to higher temperatures and the drying effects of the sun or wind 59 . The Ethiopian Food and Medicine Administration and Control Proclamation No. 1112/2019 also enforces the use of clean and suitable packaging material, adhering to the national and international quality and safety standards 60 .
The Codex Code of practice for fish and fishery products requires that fish should be maintained at the chilling temperature of 4 °C for short time storage and freezing temperature of ≤ − 18 °C for long time storage with regular temperature monitoring 51 . However, all retailers and 70% of the food establishments in the study area stored fish in a refrigerator for a maximum duration of 24 h and 7 days respectively, but none of them had known the storage temperature at which the fish were kept. Interestingly, 30% of the food establishments did not store fish; they supply consumers with freshly produced fish though the way it has been harvested, processed, and transported is still under sub hygienic conditions. This entails that fish retailers and food establishments should make every effort to store fish at required temperature to avoid fish spoilage and to ensure consumer safety. www.nature.com/scientificreports/ The present study showed that consumption of raw fish is popular in the study area where about 77% of the respondents had the preference of consuming raw fish. In addition, 80% of them did not know the potential risk of fishborne pathogens associated with the consumption of contaminated fish. FAO 54 indicated that food poisoning including consumption of contaminated fish, occurs because of eating raw or undercooked products or cooked products that have been cross-contaminated. Several studies elsewhere showed the linkage of the raw fish consumption with fishborne diseases. For instance, a study by Kalimuddin et al. 61 showed that 283 infections due to Streptococcus agalactiae was linked to the consumption of raw fish in Singapore. Austin et al. 38 also reported the presence of several bacterial pathogens of public health concern, causing serious morbidity and mortality in humans, through consumption of raw or inadequately cocked contaminated fish and/or fishery products. As stated above Shiga toxin producing E. coli O157:H7 was reported from fish examined at various lakes in Ethiopia 36,37 implying the potential risk of consuming raw or inadequately coked fish or fishery products. However, the Ethiopian food safety regulations almost exclusively target commercial markets while most people consume what they produce or buy from the local informal markets 62 . Therefore, adoption, establishment and maintenance of operational safety standard protocols will ensure the safety of fish and fishery products to protect consumer health 22,58 .
Although the number of female participants included in the questionnaire survey was small, the study identified a significant gender difference with respect to raw fish consumption. In addition, in the study area, most of the fish handling practices (processing, transportation and storage) are managed by the male counterparts under unhygienic conditions and without adhering to the national or international safety standards. This is contrary to the FAO and WHO, code of conduct for responsible fisheries 59 , which declares a remarkable importance of naturally occurring food safety hazards in the environment from which fish are harvested. To that end, risks to consumer health associated with fish and fishery products obtained from unpolluted marine environments are low provided that the products are handled in line with the principles of good manufacturing practices. However, it would be increased when fish is mishandled along the value chain 59 . The report of Austin et al. 38 also showed the potential for public health risk of handling infected fish on fish farms or in retailer shops. It implies that in men the risk of occupational exposure to fishborne pathogens (hazards) is more likely. So, women are less likely to be exposed to fishborne pathogens as compared to men and also by avoiding raw fish consumption.
Taken all together, the study indicates that the identified unhygienic handling practices that could be due to less stringent food safety regulation practices in Ethiopia calls for an urgent timely intervention to establish and maintain effective national fish quality and safety plans to ensure public safety. More importantly, consumption of a heat-treated fish should be promoted as effective risk mitigation strategy through public education to prevent risk of acquiring fishborne zoonotic diseases.
Moreover, the informal fish marketing system that dominates in Ethiopia should be regulated for implementation of effective supply chain management system. To reduce the perceived risk associated with food consumption and to increase the consumers trust, many countries have introduced Food Traceability System such as HACCP (Hazard Analysis and Critical Control Points), blockchain technology and other electronic systems; which can provide an information platform for all the supply chain members 12,63-65 . Blockchain technology in particular is a digital ledger platform which is becoming an important tool in different industrial production and Agri business in managing food supply chain by providing secure and immutable information 12,14 . Blockchain is a digital, decentralized, and distributed ledger in which transactions are logged and added in chronological order with the goal of creating permanent and tamper-proof records 33,66,67 . One of the most critical aspects in the use of blockchain applications is related to monitoring social and environmental conditions to control and avoid the occurrence of health and safety problems 68 . It provides the ability to track food such as fish and fish products through all stages of production, processing, and distribution to signal quality in the food supply chain 12,21 .
Under the current Ethiopian context, it is more prudent to transform the prevailing informal fish production and marketing system by strengthening the existing fish producer and supplier cooperatives to a large-scale commercial enterprise, establishing well networked formal marketing system, and regulating the market dynamics and improving infrastructural development. These will be foundational to evaluate the application of the digital technologies such as blockchain and adopt the technology in future studies for sustainable management of fish production and supply chain by addressing perishability of fish, demand-supply mismatch, unfair prices, and quality related data to satisfy the demands of intervening actors across the supply chain to encourage transparency and accountability in the entire value chain in Ethiopia 5 .

Conclusion
Our study revealed a wide range of unhygienic handling practices along fish production and supply chain such as the use of contaminated lake water for fillet washing; the use of a single knife and cutting board to process multiple fish with infrequent cleaning and without any disinfection between processing; and the use of unhygienic fish containers and packaging materials. Furthermore, the study identified lack of infrastructure for post-harvest fish handling and processing, evidence of conspicuous cross-contamination during processing, lack of appropriate transportation facility and lack of adequate knowledge about fish borne diseases linked with the consumption of raw fish. The unhygienic handling practices and the raw fish consumption preference of the consumer imply the potential risk of fishborne diseases in the area necessitating timely interventions. Infrastructural development, establishing standard fish processing plant/house with basic facilities, provision of food safety training to all actors along the production supply chain and the use of easy to clean containers and cold chain facilities for transportation of fish are critically needed to ensure fish safety. Further studies should aim at investigating the role of the unhygienic handling practices, microbial quality of fish and other risk factors for the occurrence and contamination level of fish with foodborne pathogens to identify critical control points along the production and supply chain at which future interventions will be targeted. In addition to investigation into the intrinsic www.nature.com/scientificreports/