Question	Answer
Questions related to the superchilling process
1/ What is the target fish temperature set by the processor (e.g. in the HACCP) for the superchilling? How is the superchilling process managed/controlled, e.g. by time (depending on the load of the blast equipment) or by monitoring the fish temperature real‐time?	The target fish temperature set by the processor for the superchilling lies between –0.5°C and –2°C. The temperature is measured (as regular temperature measurement requires) in the core of the product. Different variables are important to manage/control the superchilling process. In general, these are the size of the fillets, chiller/blast temperature, fillet shape, quantity of fillets in the chiller and the starting temperature of the product. Temperature is measured before and after chilling. Please see Annex B – part I (‘Homogenisation of fish temperature after superchilling in closed packaging’) & Annex B – part II (‘Superchilling temperatures a different establishments origin’).
2/ Is there data available on the freezing point of Nile perch and the proportion of ice within the fish matrix just after the superchilling process (or after equalisation)?	There is no data available on the freezing point of Nile perch and the proportion of ice within the fish matrix just after the superchilling process. Modelled ice content for salmon gives an idea of the freezing point.
3/ In Figure 3.1.2 and Figure 3.1.3, it seems that there is an inversely proportional correlation between the treatment duration and the fish core temperature distribution. What are the factors impacting on the fish core temperature distribution (e.g. fish size, starting temperature before superchilling)?	Treatment duration depends on different variables like chiller temperature, load, type of chiller (blast, spiral, deep, etc.), size of the fish, starting temperature of the fish and method (e.g. using stands, plastic crates, metallic cans, etc). See also Annex B – part II (‘Superchilling temperatures a different establishments origin’).
4/ Which are the factors explaining the treatment duration in Figure 3.2.3	See above. The procedure (in addition to depending on physical factors) is not the same for the different companies but is adopted according to its own self‐control plan. In any case, the output is common, i.e. superchilling with a product at a temperature between –0.5°C to –2.0°C (Attached some process procedures). See also Annex B – part II (‘Superchilling temperatures a different establishments origin’).
Questions related to the transport of the fish
1/ What was the size of the EPS boxes used in the study?	Boxes used in the report were of conventional sizes used in daily supply chain. Depending on volume of the products 3 kg or 6 kg boxes were used. In general, the sizes of the boxes are: o 1 box of 3 kg → 388 × 290 × 95 mm (L × W × H) → Nile perch fillet o 1 box of 6 kg → 400 × 300 × 147 mm (L × W × H) → Nile perch fillet o 1 box by 15 kg → 595 × 395 × 220 mm (L × W × H) → Nile because headed and gutted (H & G) OR o 3 kg – 40 × 12 × 30 cm (L × W × H) → Nile perch fillet o 6 kg – 40 × 14 × 30 cm (L × W × H) → Nile perch fillet o 15 kg – 65 × 22 × 40 cm (L × W × H) → Nile because H&G
2/ During the (validated?) study, the boxes were filled with six fillets per box. It is indicated in the report that usually those boxes are full of fillets? What would have been the usual number of fillets? Were the boxes full of fish in the self‐monitoring program?	See above. The procedure (in addition to depending on physical factors) is not the same for the different companies but is adopted according to its own self‐control plan. In any case, the output is common, i.e. superchilling with a product at a temperature between –0.5°C and –2.0°C (Attached some process procedures). In self‐monitoring program boxes are full of fish (number of fillets depending on size of fillet and weight in the box). Size of the boxes is adjusted to the content. In the validated research in two situations, 6 kg size boxes were used instead of 3 kg size boxes with around 3 kg content. See Annex B – part III (‘Dataloggers’) for more information.
3/ Can it be confirmed that no ice pack has been placed inside the boxes with superchilled fish?	We can confirm there are no ice‐packs inside the boxes with superchilled fish. The boxes were directly sent to the EU reference lab to check.
Questions related to the data loggers
1/ What was the position of the data loggers inside the box? This is important to understand what is the temperature that the loggers represent (e.g. surface fish temperature of a fish in the upper part close to the lid or of a fish located in the inner/central part of the box).	The loggers were located in the middle of the box. See attachment ‘datalogger’ for more information about the position of the dataloggers in the validated research. Also see extra validation in Annex B – part IV (‘Temperatures cold store and transport’).
2/ Is there a difference in location of the data loggers for the self‐monitoring programme and the validated study?	The loggers are placed in the middle of the box. In the box, the temperature will be fully homogenised. The position on the pallet could influence the temperature slightly. Product temperature will be measured and documented at production plant before arrival and at destination. For the self‐monitoring programme loggers and temperature measurements were done in the same way as in the validated research. In both cases, boxes were filled like in a commercial supply chain (full boxes) except of two boxes in the validated research (see Annex B – part III (‘Dataloggers’)).
3/ Can an actual profile be explained? In some profiles, two lines are shown. What does this represent?	We do think it is the temperature loss through the box to the outside when the box is outside the cold chain. See Annex B ‐ part III (‘Dataloggers’). In this attachment you can read that the blue line represents the logger temperature (air temperature) and the green line represents the probe temperature (core temperature product).
4/ In two profiles, a temperature increase was noticed (FRE, page 65; EAS, page 67). What is the reason for this and how can the temperature drop after this increase?	The temperature increase was realised by exposure to higher outside temperatures. The temperature drops again due to the temperature of the product that homogenise again. Annex B ‐ part III (‘Dataloggers’). In the attachment you can read that in these examples the used boxes were too big for the amount of samples.
5/ Only the temperature increase during transport is reported. Can the temperature of the data logger at the African establishment (just before the transport) and at the arrival be obtained?	Yes. On both sides accredited cold stores are used, HACCP accredited by local authorities. See attachment ‘temperatures cold store and transport’.
6/ Can the raw data of these temperature profiles be obtained?	Information is available on request at cold stores. See Annex B ‐ part IV (‘Temperatures cold store and transport’).
7/ Are data available of the temperature profiles outside the fish boxes?	There is no data available about the temperature profiles outside the fish boxes. See Annex B ‐ part IV (‘Temperatures cold store and transport’). The holds of aircraft are pressurised at controlled temperatures, as other fresh products (e.g. flowers, vegetables) are stowed together with super chilled fish. The transportation from the EU airport to the consignee is at controlled temperature (0–4°C), normally with other fresh fish products. At receiving the air temperature of the truck is occasionally checked and the carrier provides recordings of the transportation temperatures on demand.
Questions related to the fish temperatures
1/ For measuring the fish temperatures at the EU establishment (end of transport): where was the fish located inside the box (close to the lid or more internally?). Is there variation in the temperature depending on the location of the fish inside the box?	Location does not matter, because product temperature is homogenised throughout the box. See Annex B – part I (‘Homogenisation of fish temperature after superchilling in closed packaging’)
2/ The report included the self‐monitoring data. Is the temperature verification at different points of the supply chain an implemented practice (e.g. within the HACCP plan). If so, what is being verified?	Yes, it is an implemented practice. At the processing plant the product temperature and chilling time are verified. As well in the African establishment as in the EU establishment temperatures are measured and registered. For African establishment the temperatures are measured for every batch. At receiving at the establishment in EU the temperatures are measured based on the EU establishments HACCP plan (HACCP plan of the establishment).
3/ In the Nile perch study report data (although very few) included temperature from 1 to 2.5°C. Which is the maximum temperature tolerated by the EU establishment at arrival? Is this a critical limit set in the HACCP plan, thus measured and recorded in every batch	Temperature to be accepted at the establishment are legally required temperatures for fresh fish products.
Questions related to the supply chain
1/ How are the fish kept before the superchilling process?	The whole fish are continuously stored on ice before getting into the factory and between processing steps (during filleting).
2/ How is the gutting/filleting done (manual/equipment)? How long does it take?	Gutting/filleting is done manually and takes around 2–3 min per whole fish. The entire process before chilling (washing, filleting, skinning, trimming, fillet washing, grading, arranging on trays and stands) takes around 30 min. Between the different processing steps the fish is iced.
3/ Are the superchilled fish wrapped together in plastic before placing them in the fish box?	The products are not individually wrapped packed (IWP). There is a food grade cover sheet at top of box to protect the fish from potential contamination after opening the box.
4/ In case of conventional transport (in boxes with ice), what would have been the practice and how much ice would be added?	For 25 years, the fresh Nile perch supply chain products are superchilled and no ice is used during transport by air. So, this needs to be redesigned. In the current boxes probably 30–50% of the weight of the fish ice is needed to keep the products chilled. However, you will have to change box‐type (more isolation value) and size etc. if you export with ice. NOTE: boxes need to be free of leaking (insulated and sealed boxes). Melted ice will stay in the box if transporting in a conventional way. In the flow of the conventional product to share some data of the imported fish refrigerated with ice pack, we observed: By way of example, other chilled product delivered by air freight refrigerated with ice pack or gel pack: expanded polystyrene (EPS) 4 kg box With 1,200 g gel pack (fillets from Senegal) EPS 15 kg box with 2.7 kg icepack (whole fish from Australia) EPS 7 kg box with 900 g gel pack (chunks from Sri Lanka)
5/ Can it be confirmed that the type of boxes used for that chain is expanded polystyrene (EPS)? Are these boxes also used for other chains transporting superchilled fish?	We can confirm that the type of boxes used for that chain is EPS. We have no experience with other chains.
6/ What are the usual sizes of fish boxes used for transporting superchilled fish?	For fillets in general 3–6 kg. For other (whole and H&G), bigger sizes are used. See above.
7/ Once the superchilled fish arrives at the EU establishment, how is it stored – transported to subsequent establishment (retail) and displayed on the market?	Fish is stored and transported in a refrigerated chilled environment. Majority of the fish are sold via fresh fish counters on ice or are provided directly to the end user (restaurant/foodservice).
8/ Are you aware of the commercialisation of any other superchilled fish and/or other supply chain of the superchilled fish? In this case, which would be the similarities and the differences from the Nile perch supply chain?	Iceland cod supply to the EU
Questions related to the HADH enzymatic test
1/ HADH is supposed to be released extracellularly upon cell damage due to crystal formation. Is this dependent on the size of ice crystals, that is affected by the freezing method and thus, the freezing speed?	The HADH enzymatic test is based on https://link.springer.com/article/10.1007/s002170050481 operational working with the mitochondrial enzyme HADH that is released in cell damage from ice crystal formation. A faster freezing process will form smaller ice crystals. The smaller the ice crystals will be the smaller the resulting cell damage will be and thereby the amount of HADH. In conclusion: Yes, it depends on the conditions of the freezing process.
2/ Is there a sensitivity threshold, i.e. a value of slope below/above, which a fish may be considered positive (i.e. previously frozen)?	The threshold is determined experimentally per animal species in the study. A sample with a known history (i.e. fresh and not frozen) is used for this research.
3/ It is mentioned in the report that ‘an EU reference laboratory’ carried out the HADH analysis’. Can it be confirmed that this laboratory is not a reference laboratory for HADH?	RIKILT Wageningen University is not an EU reference laboratory for HADH.
4/ In the report, it is said that ‘the calculations of the ratios tell us that the sample was not previously frozen’. Which ratio was calculated? Is this the absorbance between time 0 and end of absorbance measurement? Or between the one frozen sample and the superchilled samples?	A received sample is split into two parts with one part measured directly for 3 min (Xo) and 1 part stored at –18°C for 2 days and then measured for 3 min (X1). The ratio (R1) is calculated using Xo/X1.
5/ What were these ratios (the values) and was there a threshold value?	For the positive reference sample the value 0.1113, for the superchilled samples this ratio was on average a factor of 10 smaller. The rejection or approval limit value has not been determined.