PLOS ONE RESEARCH ARTICLE Composition of nutrients, heavy metals, polycyclic aromatic hydrocarbons and microbiological quality in processed small indigenous fish species from Ghana: Implications for food security Astrid Elise Hasselberg1‡, Laura Wessels2‡, Inger Aakre1, Felix Reich2, Amy Atter3, a1111111111 Matilda Steiner-Asiedu4, Samuel Amponsah 3,5ID , Johannes Pucher 2‡ ID *, a1111111111 Marian Kjellevold1‡ a1111111111 1 Institute of Marine Research, Bergen, Norway, 2 German Federal Institute for Risk Assessment, Berlin, a1111111111 Germany, 3 Council for Scientific and Industrial Research, Food Research Institute, Accra, Ghana, a1111111111 4 Department of Nutrition and Food Science, School of Biological Sciences, University of Ghana, Legon, Accra, Ghana, 5 Department of Fisheries and Water Resources, University of Energy and Natural Resources, Sunyani, Ghana ‡ AEH and LW share first authorship on this work. JP and MK share last authorship on this work. OPEN ACCESS * Johannes.Pucher@bfr.bund.de Citation: Hasselberg AE, Wessels L, Aakre I, Reich F, Atter A, Steiner-Asiedu M, et al. (2020) Composition of nutrients, heavy metals, polycyclic Abstract aromatic hydrocarbons and microbiological quality in processed small indigenous fish species from The triple burden of malnutrition is an incessant issue in low- and middle-income countries, Ghana: Implications for food security. PLoS ONE and fish has the potential to mitigate this burden. In Ghana fish is a central part of the diet, 15(11): e0242086. https://doi.org/10.1371/journal. but data on nutrients and contaminants in processed indigenous fish species, that are often pone.0242086 eaten whole, are missing. Samples of smoked, dried or salted Engraulis encrasicolus (Euro- Editor: Marly A. Cardoso, Universidade de Sao pean anchovy), Brachydeuterus auritus (bigeye grunt), Sardinella aurita (round sardinella), Paulo, BRAZIL Selene dorsalis (African moonfish), Sierrathrissa leonensis (West African (WA) pygmy her- Received: March 31, 2020 ring) and Tilapia spp. (tilapia) were collected from five different regions in Ghana. Samples Accepted: October 26, 2020 were analyzed for nutrients (crude protein, fat, fatty acids, several vitamins, minerals, and Published: November 12, 2020 trace elements), microbiological quality (microbial loads of total colony counts, E. coli, coli- forms, and Salmonella), and contaminants (PAH4 and heavy metals). Except for tilapia, Copyright: © 2020 Hasselberg et al. This is an open access article distributed under the terms of the the processed small fish species had the potential to significantly contribute to the nutrient Creative Commons Attribution License, which intakes of vitamins, minerals, and essential fatty acids. High levels of iron, mercury and lead permits unrestricted use, distribution, and were detected in certain fish samples, which calls for further research and identification of reproduction in any medium, provided the original anthropogenic sources along the value chains. The total cell counts in all samples were author and source are credited. acceptable; Salmonella was not detected in any sample and E. coli only in one sample. Data Availability Statement: All relevant data are However, high numbers of coliform bacteria were found. PAH4 in smoked samples reached within the manuscript and its Supporting information files. high concentrations up to 1,300 μg/kg, but in contrast salted tilapia samples had a range of PAH4 concentration of 1 μg/kg to 24 μg/kg. This endpoint oriented study provides data for Funding: The here presented research activities were supported by funds of the Research Council the nutritional value of small processed fish as food in Ghana and also provides information of Norway and the Federal Ministry of Food and about potential food safety hazards. Future research is needed to determine potential Agriculture (BMEL) based on a decision of the sources of contamination along the value chains in different regions, identify critical points, Parliament of the Federal Republic of Germany via the Federal Office for Agriculture and Food (BLE). PLOS ONE | https://doi.org/10.1371/journal.pone.0242086 November 12, 2020 1 / 25 PLOS ONE Nutritional quality and food safety of processed small fish species in Ghana The conducted work is part of the project “Small and develop applicable mitigation strategies to improve the quality and safety of processed Fish and Food Security: Towards innovative small fish in Ghana. integration of fish in African food systems to improve nutrition” (SmallFishFood) funded within the ERA-NET LEAP-Agri which was co-financed by the European Union´s EU Framework Programme for Research and Innovation Horizon 2020 under the ERA-NET-Cofund under Grant Agreement No. Introduction 727715. The funders had no role in study design, With a growing global population and an increasing number of undernourished people, hav- data collection and analysis, decision to publish, or ing access to sufficient amounts of safe and nutritious food is crucial [1]. The significance of preparation of the manuscript. food and nutrition security (FNS) is anchored in the United Nations Sustainable Development Competing interests: The authors have declared Goals (SDGs), specifically SDG 2 “Zero Hunger” and SDG 3 “Good Health and Well-Being” that no competing interests exist. [2]. Fish is a source of many essential nutrients [3–7], and can substantially improve FNS espe- cially in a diet that largely consists of starchy staple foods such as cassava, yam, rice, maize and millet, which is the case in Ghana [8–11]. Compared with animal source foods, starchy foods contain low amounts of micronutrients, have poorer protein quality and may be a source of phytate which further aggravates the severity of undernutrition by inhibiting the absorption of essential minerals and limiting protein and lipid utilization [12, 13]. Fish is an essential part of the Ghanaian diet and is accessible at a low cost, with an apparent per capita consumption of 28 kg/year and constituting 50–80% of consumed animal protein [14–16]. In Ghana small fish is usually eaten whole [3], which represents the most beneficial form of consuming fish in terms of nutrient density, providing vitamins A, D and B12, minerals such as calcium and trace elements iodine, selenium, iron and zinc [17, 18]. In contrast, when consuming larger fish, the micronutrient-rich organs, viscera and bones are usually removed, which limits the potential nutrient content [19]. Different processing techniques, such as smoking, drying and salting enables the availability of marine and freshwater fish throughout the country [20]. To prevent spoilage and prolong shelf life, smoking is the leading technique of fish processing in Ghana constituting 70–80% of locally consumed fish, while the remaining percentage is consumed fresh or subjected to dry- ing, salting, frying or fermentation [21]. Commonly, a metal drum kiln or “Chorkor smoker” is used [22–24] in which fat and other fluids from fish may drip into the open flames and form harmful substances, such as polycyclic aromatic hydrocarbons (PAHs), which have reported carcinogenic and mutagenic properties [23, 25, 26]. Another potential hazard for consumers is microbial contamination, especially in countries where cold storage is not widely available to inhibit microbial growth. Here, the management and way of processing, like smoking or drying of the fish, plays an essential role [24, 27]. Further, improper handling and hygiene practices, storage, and insufficient smoking or drying can increase the likelihood of recontami- nation and growth of microorganisms including pathogenic bacteria from fecal sources [28– 31]. Additionally, fish may be a source of heavy metals e.g. mercury, arsenic, lead and cad- mium [32, 33]. The majority of fish consumed in Ghana is of marine origin and low levels of mercury have been registered in fillet of fresh marine fish from the Gulf of Guinea [34, 35]. Nonetheless, one of the largest e-waste recycling sites in Africa is located in Accra, resulting in large-scale environmental contamination with many toxicants, particularly lead, at high con- centrations through smoke emission [36, 37]. The elevated ambient burden of heavy metals in surrounding areas contributes to the toxic exposure of the general population, and quite possi- bly, to the fish markets situated nearby [36]. In the freshwater lakes and rivers, discharge of effluents from gold mines and other industries increases the presence of mercury and other contaminants [38, 39]. The current study is part of the SmallFishFood project (https://smallfishfood.org/), an international consortium collaborating to understand how socio-cultural, economic and PLOS ONE | https://doi.org/10.1371/journal.pone.0242086 November 12, 2020 2 / 25 PLOS ONE Nutritional quality and food safety of processed small fish species in Ghana institutional transformations of the fish food and value chain—from ecosystem to consumer— can contribute to improved, sustainable utilization of small fish resources for Africa’s low- income population. Access to local, reliable and up-to-date food composition data (FCD), par- ticularly on foods that are essential to vulnerable population groups, is scares but imperative to improve FNS. Although the main body of FCD comprises nutrients, data on potentially harm- ful food components such as heavy metals, polycyclic aromatic hydrocarbons (PAHs) and pathogenic microorganisms are also important for nutritional recommendations, as it pro- vides the basis for the estimation of exposure, risk assessments and risk-benefit analysis [40]. Combined, this knowledge may also be used to develop programs and policies to improve FNS [41, 42]. In most published data on nutritional quality and safety parameters in fish only the flesh of raw fish has been analyzed, and the analytical data on nutritional quality and safety of processed whole small fish are scarce. The aim of the present study was to generate analytical data on selected nutrients, as well as microbial, heavy metal and PAH contamination in six commercially important processed small fish species sampled from five regions in Ghana. Materials and method Sampling Selection of fish species sampled from Ghana were based on a market survey conducted in February 2018, where open markets in Accra, Techiman and Kumasi were visited to determine the most frequent fish species, fish sizes and the most common processing method used for each species. During November 2018, samples of processed small marine fish species Engraulis encrasicolus (European anchovy), Brachydeuterus auritus (bigeye grunt), Sardinella aurita (round sardinella), Selene dorsalis (African moonfish) and small freshwater species Sierra- thrissa leonensis (West African (WA) pygmy herring), and Tilapia spp. (tilapia) were sampled at selected fish markets throughout Ghana. The identification details of the sampled species, including scientific and Ghanaian names, are presented in Table 1. The species were sampled from six markets in five cities, representing five different regions in Ghana; Accra Agbog- bloshie and Adabraka Market (Accra, Greater Accra region, near coast), Kumasi Central Mar- ket (Kumasi, Ashanti region, about 215 km from coastline), Techiman Market (Techiman, Brong-Ahafo region, about 335 km from coastline), Tamale Market (Tamale, Northern region, about 600 km from coastline) and Bolgatanga Market (Bolgatanga, Upper East region, about 760 km from coastline). Sample size. The sampling was conducted according to Greenfield and Southgate’s requirements for retail sampling [43]. Sample size (number of specimens) was adapted from Öhrvik, von Malmborg [44], who estimated that each composite sample of fish should consist of minimum 12 specimens. The calculation was based on a formula for sample size by Green- field and Southgate [43] using the variability of key nutrients in fish including docosahexae- noic acid (DHA) [45], vitamin D and selenium [46]. This would require that for each fish species, more than 60 individuals in total and 12 individuals from each selected location should be collected. However, approximately 500 g of organic material was estimated to be needed to perform the targeted analyses, and more than 12 individuals of each fish species were thus col- lected from each location. Sampling procedure. Upon arrival at the fish markets, the sampling was started in one section of the market and the selected fish species were sampled from every third market stall. The selected species were identified by a taxonomist, and two separate batches of approxi- mately 100 g of fish was weighed and collected in zip lock plastic bags. Depending on availabil- ity of the selected fish species at each fish market, which was influenced by the unpredictable PLOS ONE | https://doi.org/10.1371/journal.pone.0242086 November 12, 2020 3 / 25 PLOS ONE Nutritional quality and food safety of processed small fish species in Ghana Table 1. Species, habitat, weight and length of fish sampled from five different locations in Ghana. Common Scientific name Local name Habitat Tissue Fish Processing Batches Composite Specimens in Batch/ name analyzed length method samples (n) each composite (cm)a composite weight (g) sample European Engraulis Amoni, Bonu, Marine, Whole 6.3 (5– Smoked 25 5 >250 100/500 anchovy encrasicolus Abobi, Saskawesi, pelagic fishb,c 7.6) Ablobid Bigeye Brachydeuterus Boeboe, Moe, Marine, Whole fish 12.4 Smoked 15 3 45 (42–50) 100/500 grunt auritus Hawui, Eboe, Ano benthopelagic without (11.8– kpeteid headb/ 12.9) whole fishc African Selene dorsalis Antele-wawaa, Marine, Whole 9 (6.6– Smoked 12 3 75 (35–100) 100/500 moonfish Ngogba lolotor, Epo demersal fishb,c 10.8) edwire, Tantemire ansoradze, Epo edwile, Ndademired Round Sardinella Kankama, Man, Marine, Whole 13.3 Smoked 24 5 49 (33–73) 100/500 Sardinella aurita Vetsimu, Eband pelagic fishb,c (12.7– 15.4) Tilapia Tilapia spp. Apatire Tidie, Freshwater, Whole fish 11.3 Salted 23 5 43 (22–73) 100/500 Akpaa, Apataa, benthopelagic without (10.2– Koobie gutsb/ 12.8) whole fishc West Sierrathrissa One man thousand, Freshwater, Whole 4.6 Dried or 7 3 230 (>100– 100/500 African leonensis Woevie pelagic fishb,c (3.4–6) smoked 500) pygmy herring a Numbers presented as mean and range (min-max). b Tissue analyzed for nutrients, heavy metals and PAHs. c Tissue analyzed for microbiological contamination. d Local names from [59]. e Local names. https://doi.org/10.1371/journal.pone.0242086.t001 nature of fish supply from small-scale fishermen and fishmongers, between 1–5 batches of each processed fish species were collected from different market stalls at each location. After sampling, the samples were stored in Styrofoam boxes and kept at room temperature along the sampling route from Bolgatanga to Accra. The sampling was conducted between the 2nd and 24th of November 2018. In Accra, the batches of each sampled fish species were pooled into composite samples for analysis of nutrients, heavy metals and PAHs, with each composite sample representing one species from one location. Subsequently, the pooled samples were shipped in boxes containing cooling elements via express airmail to Norway on November 29th, 2018. Un-pooled batch samples for microbial analysis were shipped via express airmail to Germany on December 17th, 2018. Chemical analyses of nutrients and contaminants Depending on the fish species, the tissue for analysis was prepared according to Ghanaian food customs, and may or may not include head, viscera, bones, scales or other parts (Table 1). The composite samples were homogenized as per edible parts prior to analysis in a food pro- cessor (Braun 3210, Neu-Isenburg, Germany), and subsamples of the homogenate were dis- tributed into tubes (Thermo Scientific Nunc A/S, Roskilde, Denmark) and stored at -20˚C or -80˚C pending analysis. Samples for analyses of metals, minerals, trace elements, and PAHs PLOS ONE | https://doi.org/10.1371/journal.pone.0242086 November 12, 2020 4 / 25 PLOS ONE Nutritional quality and food safety of processed small fish species in Ghana were freeze dried in accordance with the AOAC 930.15 method as previously described [47]. All composite samples were analyzed for total protein, total fat and fatty acid (FA) content including saturated fatty acids (SFA), sum monounsaturated fatty acids (MUFA), sum polyun- saturated fatty acids (PUFA), n-3/n-6 ratio, eicosapentaenoic acid (EPA), and DHA. Further- more, the concentration of vitamins A (A1 and A2) and D3, calcium and trace elements iodine, selenium, zinc, and iron were analyzed. The concentration of heavy metals arsenic (total arse- nic), mercury (total mercury), lead, and cadmium was analyzed in all composite samples in addition to levels of PAHs. All analyses were performed in singular parallels at the IMR labora- tories using accredited methods with NS-EN ISO/IEC 17025 standards, except for iron. The analytical methods including corresponding limits of quantification (LOQ) and measurement uncertainties are described in detail elsewhere [48]. Determination of crude protein, crude fat and fatty acids. The crude protein content was determined by burning the material in a combustion tube containing pure oxygen at 830˚C. Nitrogen (N) was detected with a thermal conductivity detector (TCD), and the con- tent of N was calculated from an estimated average of 16% N per 100 g protein using the fol- lowing formula N g=100 g � 6:25 ¼ protein g=100 g: The method is accredited according to AOAC Official Methods of Analysis [49]. For determination of crude fat, the fat content was extracted with ethyl acetate and filtered before the solvent was evaporated and the residual fat was weighed. The method is accredited in accordance with ISO-EN 17025 and standardized as a Norwegian Standard, NS 9402 [50]. The fatty acid content was determined using gas liquid chromatography (GLC). Lipids in the samples were extracted according to Folch, Lees [51], and the fatty acid composition of total lipids was analyzed as previously described in Fauske, Bernhard [52]. Methyl esters were separated on a capillary gas column (CP-sil-88, 50 m WCOT, ID:0.32) and peaks were identi- fied by retention time using standard mixtures of methyl esters (Nu-Check, Eylian, USA). Content of fatty acids per gram sample was subsequently calculated using 19:0 methyl ester as an internal standard. Determination of vitamins. To determine vitamin A1 (sum all trans-retinol and 13-, 11-, 9 cis retinol) and A2 (4,4 didehydro-all-trans retinol) content, the samples were saponified and the unsaponifiable material was extracted. Vitamin A1 and A2 was determined by high-perfor- mance liquid chromatography (HPLC) (normal phase) using a PDA detector (Photo Diode Array) and the retinol content was calculated by external calibration (standard curve) [53]. For determination of vitamin D3, the samples were saponified, and the unsaponifiable material was extracted and subsequently purified in a preparative HPLC column. The fractions containing D2 (ergocalciferol) and D3 (cholecalciferol) were pooled (normal phase) and injected on an analytic HPLC column (reverse phase). Vitamin D3 and D2 was determined by an ultraviolet (UV) detector and the content of vitamin D3 was calculated using vitamin D2 as an internal standard [54]. Vitamin B12 (Cobalamin) was released from the sample by extraction (autoclaving in ace- tate buffer) and mixed with growth medium before the microorganism (Lactobacillus del- bruecki -ATCC 4797) was added and subsequently incubated at 37˚C for 22 hours. The concentration of vitamin B12 was calculated by comparing the growth of the organism in the unknown samples with the growth of the organism in known standard concentrations by tur- bidimetric reading (Optical Density, OD, v / 575 nm) [55]. Determination of metals, minerals and trace elements. The concentration of calcium, heavy metals cadmium, arsenic (total), mercury (total), and lead as well as trace elements PLOS ONE | https://doi.org/10.1371/journal.pone.0242086 November 12, 2020 5 / 25 PLOS ONE Nutritional quality and food safety of processed small fish species in Ghana selenium, zinc, and iron was determined by Inductively Coupled Plasma-Mass Spectrometry (iCapQ ICP-MS, ThermoFisher Scientific, Waltham, MA, USA) equipped with an autosam- pler (FAST SC-4Q DX, Elemental Scientific, Omaha, NE, USA) after wet digestion in a micro- wave oven as previously described by Julshamn, Maage [56]. For determination of iodine, the sample was extracted with tetramethylammonium hydroxide (TMAH) before ICP-MS analy- sis. The elements were quantified using an external standard curve in addition to different internal standards for specific elements; scandium (Sc) was used as internal standard for cal- cium, rhodium (Rh) was used as internal standard for zinc and selenium, tellurium (Te) was used for iodine and either Rh, germanium (Ge), indium (In) or thulium (Tm) was used for the heavy metals. Determination of PAHs. The concentration of the PAHs benz(a)anthracene (BaA), benzo (a)pyrene (BaP), benzo(b)fluoranthene (BbF) and Chrysene (Chr), collectively referred to as PAH4, were determined by Gas Chromatography Mass Spectrometry (GC-MS/MS) after extrac- tion with dichloridemethane (DCM): cyclohexane (1:3) using an US EPA 16 PAH cocktail (CIL ES-4087) internal standard. The extract was evaporated and rinsed on an SPA column (Envi- chrom) before adding recovery standard and analyzing the samples by GC-MS/MS [57, 58]. Microbial analysis Sample preparation. After arrival at the German Federal Institute for Risk Assessment, the samples were stored at 4˚C until further processing. Samples were homogenized batchwise using a GRINDOMIX GM200 (Retsch GmbH, Haan, Germany) for 20 seconds at 4.000 rpm and stored in plastic cups with a screw-on lid (WMC Medical Consulting, Pulheim, Germany) at 4˚C until microbiological analysis. Rehydration of fish and preparation of pooled initial suspension. The batchwise sam- ples were processed as described in ISO 6887–3:2003. A batch sample aliquot of 11 g was mixed with 22 g buffered peptone water (Mast Group, Bootle, United Kingdom) in a peristaltic lab blender (Bag Mixer CC, Interscience, Saint Nom, France) for 10 seconds and rehydrated at room temperature for 1 h. The batchwise initial suspensions were prepared as a tenfold dilu- tion of the rehydrated fish by addition of 297 g of buffered peptone water and mixing it for 90 seconds. For microbial analysis, initial suspensions of batch samples were pooled per species and market, as described for the analysis of nutrients and minerals. Analysis of total colony count, E. coli, coliform bacteria and Salmonella spp.. The total colony count (TCC) analysis was based on ISO 4833:2015, however, instead of plate count agar, Columbia blood agar plates (Oxoid Deutschland GmbH, Wesel, Germany) were used. The pooled initial suspension was further diluted 1:10 in maximum recovery dilution (Oxoid Deutschland GmbH, Wesel, Germany) and 100 μL per dilution step were spread on agar plates. One set of plates were incubated at 30˚C for 72 h aerobically, and an additional set of plates were incubated at 30˚C for 72 h under anaerobic conditions in anaerobic jars with AnaeroGen™ sachets (Oxoid Deutschland GmbH, Wesel, Germany). The limit of detection (LOD) based on the detection of one colony per plate was calculated as 2.5 log CFU/g of pro- cessed fish. For quantification of E. coli and coliform bacteria, Brilliance™ E. coli/coliform agar plates (CM1046, Oxoid Deutschland GmbH, Wesel, Germany) were inoculated with 100 μL of the pooled initial suspension and their further dilutions. In addition, 1 mL of the pooled initial suspension was spread on three plates to increase the level of detection 10-fold (LOD 1.5 log CFU/g). The plates were incubated at 37˚C for 24 h, aerobically. The detection of Salmonella spp. was performed according to ISO 6579–1:2017. The 300 mL initial suspensions per batch were incubated at 37˚C for 18 h, aerobically. After incubation, PLOS ONE | https://doi.org/10.1371/journal.pone.0242086 November 12, 2020 6 / 25 PLOS ONE Nutritional quality and food safety of processed small fish species in Ghana enriched batch samples were pooled as described above. An aliquot of 1 mL of the pooled enrichment culture was added to 10 mL Mueller Kauffman Tetrathionate Novobiocin broth (MKTTn; Oxoid Deutschland GmbH, Wesel, Germany) and incubated at 37˚C for 24 h, aero- bically. Further, 100 μL was dripped as three droplets on modified semi-solid Rappaport Vassi- liadis agar (MSRV; Oxoid Deutschland GmbH, Wesel, Germany) and incubated at 41.5˚C for 24 h, aerobically. After incubation, 10 μL of MKTTn selective culture or a loop full of spread- ing growth from MSRV were spread to Xylose Lysine Deoxycholate agar plates (XLD, Merck, Darmstadt, Germany) and Gassner agar plates (GAS, sifin, Berlin, Germany) both incubated at 37˚C for 24 h aerobically to achieve single colonies. After incubation, plates were checked for presumptive colonies. Data management The analytical data was exported from Laboratory Information Management Systems (LIMS) and processed using Microsoft Excel 2013. When calculating mean and standard deviation, analyses below LOQ were included in the calculations as the respective LOQ value divided by two. Statistical analyses was performed using IBM SPSS version 26 (IBM Corp., Armonk, NY, USA) and for all tests a significance level of α = 0.05 was used. Normal distribution was tested using Shapiro-Wilk test. For normally distributed data, the homogeneity of variances was tested using Levene-test. If homogeneity of variances was given, the differences between the fish species was analyzed using one-way analysis of variance (ANOVA). As a post hoc test to discriminate different pairs, a Tukey-test was performed. If the homogeneity of variances was not given, a Welch-ANOVA was used to determine the differences between the fish species, followed by a Dunnet-T3-test. If the values were not normally distributed, a Kruskal-Wallis- test was performed to determine differences between the species followed by pairwise compar- ison of the fish species with a Bonferroni correction. Results and discussion In the present study, analytical data on 24 composite samples of processed, small fish (total length 4.6–13.3 cm) from several fish markets covering a large geographic range in Ghana are reported (Table 1). Based on the nutritional analyses all the processed fish samples, except tila- pia, had the potential to significantly contribute with essential nutrient to the intakes of vita- mins, minerals, and essential fatty acids. In all samples, the microbiological total cell counts were acceptable (Table 2). High levels of PAHs, mercury and lead were detected in some sam- ples (Table 2), which calls for further research and identification of anthropogenic sources in different regions and along the value chains. Nutrient content Proximate and fatty acid composition. The content of protein and fat as well as fatty acid composition of the processed fish species are presented in Table 2 and in S3 Table. Protein content ranged from 31.80 g/100 g in salted tilapia to 71.80 g/100 g in smoked European anchovy, thus constituting a significant source of complete proteins in the otherwise starchy- staple-dominant Ghanaian diet. The stability of proteins in fish have previously been exam- ined, and analyses of sardine (Sardinella spp.) from Ghana reported minimal variation in protein content between fresh and smoked specimens [60]. Furthermore, Usydus, Szlinder- Richert [61] found that in terms of both quality and quantity, the amino acids in smoked and salted fish remain stable and highly digestible according to WHOs protein standard [61]. Despite being subjected to a variety of processing techniques, dried, salted and smoked fish from Ghana can thus be regarded as a high-quality protein source [62]. PLOS ONE | https://doi.org/10.1371/journal.pone.0242086 November 12, 2020 7 / 25 PLOS ONE Nutritional quality and food safety of processed small fish species in Ghana Table 2. Analytical values (means ± standard deviation, wet product weight) for selected nutrients, microbial quality and contaminants of processed whole fish from Ghana. Unit Anchovy Bigeye grunt Round African WA pygmy Tilapia p-value sardinella moonfish herring Moisture content % 8.45±1.31AB 8.22±1.77AB 7.92±1.75A 10.36±1.66AB 7.87±2.04AB 28.50±7.96B 0.019 Nutrients Protein g/100g 71.80±2.05A 64.33±1.53B 64.80±3.19B 67.67±1.53AB 67.67±3.06AB 31.80±3.90C <0.001 Total Fat g/100g 6.44±0.77A 15.03±3.80AB 13.84±1.29B 6.37±0.97A 12.23±3.38AB 7.02±2.11A 0.001 Sum SFA g/100g 2.00±0.47A (37) 5.16±1.44B 4.94±0.77B (33) 1.99±0.21A (38) 4.02±0.78BC (39) 2.93±0.38AC <0.001 (%) (38) (41) Sum MUFA g/100g 0.75±0.13A (14) 3.20±1.01B 2.48±0.52AB (26) 0.92±0.10AB (18) 2.86±0.97B (27) 2.06±0.36AB 0.003 (%) (24) (28) Sum PUFA g/100g 2.33±0.58AB 4.25±1.04C 3.98±0.58C (35) 1.92±0.20AB (37) 3.12±0.63AC (30) 1.68±0.17B (23) <0.001 (%) (43) (32) EPA g/100g 0.41±0.13AB (7) 0.77±0.26B (6) 0.82±0.17B (7) 0.34±0.07AB (6) 0.29±0.06AB (3) 0.04±0.01A (1) 0.001 (%) DHA g/100g 1.41±0.32A (26) 2.21±0.47B 2.01±0.30B (16) 1.04±0.12AC (20) 0.77±0.22CD (8) 0.18±0.06D (3) <0.001 (%) (16) Vitamin B μg/100g 14±3AB 9±1A 23±1B12 14±1A 16±6AB 11±3A 0.014 Vitamin D μg/100g 12±3AB 15±6AB3 34±9A 9±3B 17±10AB 13±4AB 0.024 Vitamin A1 μg/100g 14±15AB 323±153B 10±3AB 290±46B 39±28AB 4±8A 0.004 Calcium mg/100g 2940±207AB 3533±577ABC 3040±207B 5467±153C 2633±379AB 2900±436AB 0.026 Iron mg/100g 25±6A 22±9AB 19±3A 50±12AB 78±89AB 10±3B 0.010 Zinc mg/100g 6.3±0.9ABC 3.4±0.4BC 5.1±0.2ABC 4.9±1.0ABC 14.7±2.9A 4.3±0.6BC 0.003 Selenium μg/100g 192±28A 113±12B 242±37A 173±15A 94±15BC 33±7C <0.001 Iodine μg/100g 170±48A 219±122A 142±31AB 233±25A 129±74AB 49±11B 0.002 Microbial TCC aerob log CFU/g 5.06±0.74A 4.58±0.23A 4.62±0.52A 4.93±0.30A 6.15±0.74A 4.23±0.64A 0.077 Quality TCC log CFU/g 4.36±0.92A 3.67±0.42A 4.05±0.92A 3.98±0.27A NAA 4.43±1.01A 0.505 anaerob Coliform log CFU/g 2.73±0.85A 2.86±1.05A 2.93±1.19A 2.69±0.07A 3.12±1.28A 30%) in round sardinella, thus indicating that some processed marine fish species are also a potential source of vitamin A2. Collectively, our results suggest that including smoked bigeye grunt or smoked African moonfish in the diet may contribute towards alleviating the persistent burden of vita- min A deficiency in Ghana [77]. All fish species contained considerable amounts of vitamin B12, ranging from 8.9 μg/100 g in processed bigeye grunt to 23.0 μg/100 g in round sardinella. Animal source foods are PLOS ONE | https://doi.org/10.1371/journal.pone.0242086 November 12, 2020 9 / 25 PLOS ONE Nutritional quality and food safety of processed small fish species in Ghana known as the major dietary source of vitamin B12 [78] and previous studies have documented that the concentration of vitamin B12 is up to three times higher in the viscera of fish compared to fillet [79]. This indicates that consuming small fish whole may be suitable, particularly for population groups with limited access to animal source foods. A study on round herring (Etru- meus teres) demonstrated that heating processes may decrease vitamin B12 levels by up to 62% [79], but the final concentration of vitamin B12 in smoked, dried or salted fish could neverthe- less be higher than in fresh fish. Still, the cumulative effects of high temperatures during both processing and cooking on vitamin levels are yet to be thoroughly investigated. Mineral composition. Of the analyzed processed fish species, tilapia was the least mineral dense, containing the lowest concentrations of selenium (33 μg/100 g), iodine (49 μg/100 g) and iron (10 mg/100 g) compared with all other analyzed species, and significantly lower levels of selenium and iodine than the marine species. Round sardinella had the highest levels of sele- nium (242 μg/100 g) while African moonfish contained significantly higher concentrations of calcium (5467 mg/100 g) and iodine (233 μg/100 g). The smallest fish analyzed, WA pygmy herring, had the highest concentrations of both iron (78 mg/100 g) and zinc (15 mg/100 g). Even though mean iron values of WA pygmy herring were not significantly higher than for other species (Table 2), high levels of iron were detected in the composite sample of dried WA pygmy herring from Accra (180 mg/100 g; S2E Table), which exceeds previous findings of iron in smoked-dried tilapia from Ghana twentyfold [64]. The high values of iron measured in the specific composite sample of WA pygmy herring from Accra may be linked to the Agbog- bloshie area in Accra where the ambient metal contamination is high due to burning of e- waste. But further studies are needed to verify this potential regional contamination pathway. Still, with a high prevalence of iron deficiency anemia [80], the inclusion of processed small fish could be a valuable addition to the staple-dominant Ghanaian diet by providing bioavail- able iron [3, 9, 81, 82]. Zinc is essential for optimal growth, and is closely interlinked with aggravated symptoms of iron deficiency anemia with its role as a catalyst in iron metabolism [83]. Although the preva- lence of zinc deficiency has not yet been evaluated in Ghana, it can be assumed that a diet comprising limited animal-source foods is inherently low in terms of both zinc content and bioavailability [13]. Zinc levels in the current study were similar to previously determined lev- els in smoked sardine (Sardinella spp) from Ghana [64] and notably higher than the concen- tration in small raw freshwater fish analyzed whole [84]. These findings also correspond with the reported stability and high retention of zinc documented in cooked foods [85, 86]. Incor- porating all processed fish species, but primarily WA pygmy herring, in the diet could thus be a valuable contribution towards increasing dietary zinc and simultaneously alleviating iron deficiency anemia in Ghana. Adequate iodine intake is essential for synthesis of thyroid hormones and neurodevelop- ment, however, the magnitude of iodine deficiency among children and other vulnerable pop- ulation groups in Ghana is not thoroughly mapped. Marine fish are regarded as the superior dietary source to iodine [87]. However, iodine content largely depends on environmental con- ditions, and large variations are expected both between and within different fish species [88, 89]. This great variation was confirmed in the present study, with iodine content in bigeye grunt ranging from 96–340 μg/100 g. As a freshwater fish, processed tilapia showed signifi- cantly lower iodine levels than most of the marine species. Interestingly, the freshwater species WA pygmy herring showed relatively high iodine levels which were comparable to the pro- cessed marine species. In line with our results, former analyses of iodine in whole smoked European sprat (Sprattus sprattus) have yielded high average levels of iodine (148 μg/100 g) and iodine retention ranged from 48–93% in a study on different cooking methods [90], thus underlining that processed and cooked fish may be considered a good source of dietary iodine. PLOS ONE | https://doi.org/10.1371/journal.pone.0242086 November 12, 2020 10 / 25 PLOS ONE Nutritional quality and food safety of processed small fish species in Ghana Selenium plays a key role in ameliorating the toxic effects of mercury [91] which is present in variable concentrations in fish [92, 93]. Previous analyses of smoked European sprat (Sprat- tus sprattus) reported a selenium content of 20 μg/100 g [94], which is markedly lower than for all fish species analyzed in the current study. With the high levels of selenium determined in our study we conclude that along with fresh seafood [95], processed fish may be considered an excellent dietary source of selenium. Food safety Microbiological quality. A total of 24 pooled samples were analyzed for bacteria related to microbiological quality. Total colony count (TCC) was used to describe the general bacterial load which can be used as an indicator for poor hygiene conditions and control along the value chain. Coliform bacteria and E. coli were used as indicator bacteria for fecal contamina- tion along the value chain and Salmonella was analyzed as their presence is associated with food-borne disease. The microbial quality parameters of the processed fish species are pre- sented in Fig 1 and Table 2. As shown in Fig 1, the highest aerobic TCC were found in WA pygmy herring from Bolga- tanga (6.67 log CFU/g) while the lowest counts were detected in tilapia from Tamale (3.85 log CFU/g). The two sampled freshwater fish species differed in two orders of magnitude in mean aerobic TCC with 6.15±0.74 log CFU/g in WA pygmy herring and 4.23±0.64 log CFU/g in tila- pia. While in marine species, mean aerobic TCC ranged between 5.06±0.74 log CFU/g (Euro- pean anchovy) and 4.58±0.23 log CFU/g (bigeye grunt). The Ghana Standards Authority provides limits for aerobic TCC in hot smoked fish with an acceptable limit (m) for TCC at 5.7 log CFU/g and a rejection limit (M) for TCC at 7 log CFU/g (GS 95:2013). None of the here Fig 1. Total Colony Counts (TCC), counts of coliform bacteria (coliforms) and the acceptable limit (m, 5.7 log CFU/g) for TCC, the rejection limit for TCC (M, 7 log CFU/g) and the rejection limit for coliforms (M, 1.6 log CFU/g) by the Ghana Standards Authority (GS 95:2013). I: Accra; II: Techiman; III: Tamale; IV: Kumasi; V: Bolgatanga. https://doi.org/10.1371/journal.pone.0242086.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0242086 November 12, 2020 11 / 25 PLOS ONE Nutritional quality and food safety of processed small fish species in Ghana Fig 2. https://doi.org/10.1371/journal.pone.0242086.g002 analyzed samples had an aerobic TCC above the rejection limit (Fig 2), but European anchovy samples from Kumasi (6.32 log CFU/g) and WA pygmy herring from Bolgatanga (6.67 log CFU/g) had TCCs above the acceptable limit. This means that more than 90% of all samples were within the Ghanaian acceptable limit for aerobic TCC. There are no scientific reports available on aerobic TCC in processed fish from Ghanaian markets and only few reports are available from other African countries. In Nigeria, Adegunwa, Adebowale [96] analyzed smoked herring (Sardinella eba) samples from three different markets and found aerobic TCC ranging from 6.36 log CFU/g to 7.47 log CFU/g, thus being more than one log-cycle higher than in the here presented samples in general. Another study from Nigeria by Akinwumi and Adegbehingbe [97] reported mean aerobic TCC ranging between 4.11 log CFU/g and 5.46 log CFU/g in freshwater fish species (tilapia and African mud catfish) and 4.70 log CFU/g and 5.46 log CFU/g in herring from three different markets. In smoked catfish from Malawi, high aerobic TCC (6.75 log CFU/g) were found by Likongwe, Kasapila [24], who compared two dif- ferent hot smoking techniques. In view of the few published market studies from other African countries, the here analyzed samples of Ghanaian processed fish showed lower aerobic TCC mainly within the acceptable limit. However, aerobic TCC can only be used as an indicator for general hygiene conditions along the value chain and should be combined with more specific microbial identification. PLOS ONE | https://doi.org/10.1371/journal.pone.0242086 November 12, 2020 12 / 25 PLOS ONE Nutritional quality and food safety of processed small fish species in Ghana In the sampled fish products, anaerobic TCC were showing low levels being one order of magnitude below the levels of aerobic TCC (Table 2). Consequently, the microbiota on these processed fish products were dominated by aerobic and facultative anaerobic organisms. For anaerobic TCC, no Ghanaian reference values are available. The presence of anaerobic grow- ing bacteria was low in the analyzed samples, but to exclude the presence of hygienically rele- vant bacteria, further identification is needed. The analysis of E. coli in sampled processed fish from Ghana revealed that only one com- posite sample (WA pygmy herring from Techiman) was positive for E. coli with a concentra- tion of 2.51 log CFU/g). Higher levels were found for coliforms in 70.8% of the fish samples. There was a strong variance in contamination of single fish species with coliforms between market samples, where the highest coliform counts were found in tilapia from Bolgatanga (6.23 log CFU/g, Fig 1) while tilapia samples from other markets were negative for coliform bacteria, which is calling for identification of sources within the affected value chains. The mean concentrations of coliform bacteria in European anchovy, bigeye grunt, and African moonfish were 2.73±0.85 log CFU/g, 2.86±1.05 log CFU/g, and 2.69±0.07 log CFU/g, respec- tively. European anchovy, bigeye grunt, WA pygmy herring tested positive for coliforms in all samples whereas round sardinella from two markets (Tamale and Kumasi) and African moon- fish from Accra tested negative for coliforms. The guidelines by the Ghana Standards Author- ity set the limit for coliform bacteria in hot smoked fish to 1.6 log CFU/g (GS 95:2013). Based on the LOD in the present study, all samples that tested positive for coliform bacteria thus exceeded the legal limit in Ghana. The high concentrations of coliform bacteria in most of the samples might indicate fecal origin, therefore contamination from a dusty environment, hygienic conditions, contact surfaces, or handling practices are likely sources. The large varia- tions in coliform counts, both between different products and between markets, indicate the potential to mitigate the bacterial load by improving hygienic conditions along the value chains. Critical points along the value chains might differ locally, which necessitates deeper investigation in future studies. Salmonella was analyzed in this study as a food-borne pathogen, but was not detected in any of the sampled processed fish. Similar to our results, Plahar, Nerquaye-Tetteh [29] did not detect the presence of Salmonella in two different processed marine species in Ghana and Ade- gunwa, Adebowale [96] did not detect Salmonella in smoked herring samples from three dif- ferent markets in Nigeria. In contrast, Likongwe, Kasapila [24] analyzed smoked catfish from Malawi and detected Salmonella with counts up to 4 log CFU/g in fresh and smoked fish. Fur- ther, Salmonella/Shigella was found in catfish, tilapia, and herrings collected from different markets in Nigeria [97]. Based on our results in combination with published data, Salmonella was generally not present in processed fish samples, but might be a possible food-borne hazard in certain fish value chains. The sampling approach in this study was endpoint oriented and showed that aerobic TCCs were in the acceptable range, while only one sample tested positive for E. coli and no Salmonella were detected. But the counts of coliform bacteria were exceeding the limit given by the Ghana Standards Authority (Fig 1) in the majority of the samples and might indicate fecal contamina- tion. As coliform bacteria and E. coli are foremost fecal bacteria and are classified as indicator bacteria for fecal contamination of food that harbors an increased risk to contain pathogenic bacteria [97, 98], several points along the value chains are possible critical points where contami- nation could have taken place. Either, the fish was primarily populated from its habitat and the smoking and drying of the fish was not sufficient to reduce coliform counts, or the smoked prod- ucts were re-contaminated along the value chain due to improper handling or storage. Distinct conclusions on the effect of different preservation techniques (e.g. smoking, salting, and drying) or the different habitats cannot be drawn from this study design and need further research. PLOS ONE | https://doi.org/10.1371/journal.pone.0242086 November 12, 2020 13 / 25 PLOS ONE Nutritional quality and food safety of processed small fish species in Ghana To develop suitable, locally applicable strategies to minimize the contamination by coli- forms, critical points along the specific value chains need to be identified in further studies. Such strategies might include training in best practice procedures and process control to improve hygienic conditions, processing techniques, storage conditions and transportation. At consumer level, it is recommended to thoroughly cook fish before consumption to minimize the risk of food-borne illnesses. Polycyclic aromatic hydrocarbons (PAHs). Different PAHs are known to have carcino- genic and mutagenic properties [23, 25, 26]. To determine the amount of the most important PAHs in food, the European Commission recommends an analysis for benzo[a]anthracene, chrysene, benzo[b]fluoranthene, and benzo[a]pyrene, summarized as PAH4 [25, 99, 100]. All processed marine fish products analyzed in this study were smoked. Processed European anchovy, bigeye grunt, round sardinella, and African moonfish showed high mean PAH4 con- centrations of 478±164 μg/kg, 553±155 μg/kg, 418±103 μg/kg, and 443±91 μg/kg, respectively (Table 2). The freshwater fish in this study were either smoked (WA pygmy herring from Accra and Bolgatanga), dried (WA pygmy herring from Techiman), or salted (all tilapia sam- ples) (Table 1). The PAH4 concentration of WA pygmy herring differed between the pooled market samples, probably because the pooled samples were differently composed of smoked and not-smoked batches. In salted tilapia, the mean PAH4 level (7.4±9.6 μg/kg) was lower than all composition samples of smoked fish species (Table 1). There are no legal limits avail- able from the Ghana Standards Authority for PAHs in fish products and the European limit value of 12.0 μg PAH4/kg of the product was therefore used as reference [101]. PAH4 levels exceeded the EU limit considerably in all market samples of processed European anchovy, big- eye grunt, round sardinella, and African moonfish as well as in WA pygmy herring from the markets in Techiman and Bolgatanga (Table 2). Only one composite sample of smoked fish did not exceed the EU limit for PAH4 (smoked WA pygmy herring from Accra, S2E Table). Generally, salted tilapia samples were below the EU limit value (except the samples from Bol- gatanga containing 24 μg PAH4/kg, S2F Table). PAHs are known to be produced during the smoking process, and therefore levels in non-smoked fish species are expected to be low. Based on the literature, levels of PAH4 are highly influenced by the type of smoking kiln, type of burning material, duration of smoking, re-smoking of fish products and fat content of the fish [23, 26, 102, 103]. Consequently, the PAH4 levels in fish products can be minimized by applying best practices within the smoking process. Given the high concentration of PAH4 in the majority of smoked fish samples in this study, applied smoking techniques and smoking practices in Ghana should generally be improved. The United Nations (UN) in cooperation with the Food and Agriculture Organization (FAO) developed an improved kiln called the FAO-Thiaroye Processing Technique (FTT) [23, 26]. In this improved kiln, fat and other fluids from the fish are not able to drip into the fire. Further, the fish are not in direct contact with flames and the smoking process can be performed in a more controlled way, achieving up to a 100-fold reduction in PAH4 concentration in smoked fish when using FTT compared to tradi- tional smoking methods [23, 26]. However, the FTT is more expensive, and the Ahotor oven [104–106] was therefore developed in cooperation with the United States Agency for Interna- tional Development (USAID) as a more affordable option. This oven is equipped with a fat col- lector, requires less burning material compared to the chorkor and is assumed to produce less PAHs. The awareness of the negative effect of PAHs on human health should be widely com- municated to the public and the use of safer ovens such as the FTT and the Ahotor oven and best smoking practices should be advocated to limit the health hazard for the consumer to a minimum. Heavy metals. Heavy metals can have neurodegenerative as well as nephro-, and immu- notoxic effects on humans and are important contaminants regarding food safety. Fish are PLOS ONE | https://doi.org/10.1371/journal.pone.0242086 November 12, 2020 14 / 25 PLOS ONE Nutritional quality and food safety of processed small fish species in Ghana known to be a source of heavy metals, given their ability to bioaccumulate heavy metals from the surrounding water and feed. Maximum levels for fish as food are available from Ghanaian regulations, the Codex Ali- mentarius Commission and the European Commission. As all these regulatory limits are given only for fresh fish or fresh fillet with a high moisture content, an application of these limits to processed/preserved fish products with lower moisture content requires a normalization of the analytical levels to a moisture content comparable with fresh fish [107]. EU legislation has established maximum permissible levels for cadmium (0.05 mg/kg wet weight (w.w.)), mer- cury (0.5 mg/kg w.w.), and lead (0.3 mg/kg w.w.), however, no value is available for arsenic [107]. The maximum level for lead in raw fish is in line with the guidelines provided by the Codex Alimentarius [108] but for arsenic compounds in fish, no maximum concentration has yet been established. The concentration of cadmium varied from unquantifiable levels (