Report on how stress and environmental conditions related to transportation affect food quality...

Project no: COLL-CT-2006-030421

Project acronym: CrustaSea

Project title: Development of Best Practice, grading & transportation technology in the crustacean fishery sector

Instrument: Collective Research Project

Deliverable Report 6.1 : Report on how stress and environmental conditions related to transportation affect food quality parameters.

Start date of project: 1st of September 2006 Duration: 36 Months

Organisation name of lead contractor for this deliverable: VRC, MR, IPIMAR

Project co-funded by the European Commission within the Sixth Framework Programme (2002-2006)

Dissemination Level

PU Public X

PP Restricted to other programme participants (including the Commission Services)

RE Restricted to a group specified by the consortium (including the Commission Services)

CO Confidential, only for members of the consortium (including the Commission Services)

Deliverable 6.1

Best practice for handling and storage at point of sale

Preface

Project acronym: “CrustaSea” Project full title: Development of Best Practice: grading & live

transportation technology in the crustacean fishery sector

The crustacean fishery sector faces challenges as high mortality and quality reduction of the live animals (lobster and crab) often occurs during transportation. To increase the competitiveness of the European Crustacean industry, the EU CrustaSea project was devised. It was funded from autumn 2006 and it lasted for three and a half years, till the 31

st of December of 2009.

The main goals of the project were to reduce or eliminate mortality and loss of quality of the crustaceans after capture, and to expand market opportunities for live crustaceans. The aim was also to improve the infrastructure and to establish best practice along the crustacean trade chain. Finally, the project expected to initiate innovation and exploitation of live transportation technology of crustaceans. To address these challenges, it was necessary to innovate and develop cost efficient production methods throughout the crustacean chain. Scientific knowledge and operational experience were exchanged between the EU partners, and the technological solutions of the grading, holding and transportation systems of live crustaceans were improved. The following knowledge & technological capabilities were investigated:

- The requirements of the Edible crab, Norwegian lobster and European lobster regarding their biological and physical factors to improve their health, growth and survival.

- Handling, storage and transportation systems of the live crustaceans were established

to increase survival and quality, hence adding value to the crustacean fisheries in the future.

- Cost effective crustacean grading units were evaluated regarding meat content of the crab. Both systems for onboard live grading and grading of dead crustaceans at a processing plant, were investigated.

- Transportation technology consisting of”intelligent” boxes, comprising a floodable system for flushing of the live crustacean animals, was developed. The system is able to discharge waste components from the metabolism of the crustaceans through their gills.

- The European crustacean market regards quality requirements at point of sale, was also mapped.

The results obtained by the CrustaSea project, which the partners have decided shall be public, will be archived at the web page www.crustasea.com from the 1

st of January 2009. Here, the

public reports and power-point presentations (ppts) delivered, will be open and access able according to the overview listed below.

The deliverables of the CrustaSea project decided public by the Consortium

Del. no.

Deliverable name WP no.

Leading participant

Nature

D2.1 Protocol on Best Practice for handling, grading & storage of crabs on board fishing vessel.

WP2 MR R

D3.1 Protocol on Best Practice for handling & storage of crustacean commodity at landing/recovery station.

WP3 Pera R

D6.1 Report on how stress and environmental conditions related to transportation affect food quality parameters.

D6.2 Establishment of a European guideline for quality of live crustaceans in retail and HORECA segments.

D7.2 Two papers presented at 4 conferences or major exhibitions.

D7.3 Two publications in the form of editorials, technical papers or trade press.

D9.1 Project Web page

WP9 NSS

D9.2 Project Presentation published on the project Web page

D = Deliverable, WP = Work Package, no. = number, R = Restricted, O = Open

On behalf of the Consortium of the CrustaSea Project R & D Coordinator Kristin Lauritzsen Norske Sjømatbedrifters Servicekontor (NSS) Project Coordinator

Contents

1. Introduction ........................................................................................................................... 6

2. Brown crab (Cancer pagurus) value chain and critical points/stress ................................... 6

2.1 Transportation - dry vs wet vs semi-dry: transportation plan.......................................... 6

2.2 Water quality ................................................................................................................... 7

2.3 Special critical points: nicking vs banding vs nothing ..................................................... 9

3. Langoustine (Nephrops norvegicus) special critical points & on-board handling ............... 12

3.1 Capture and post-capture handling .............................................................................. 12

3.2 Handling on-board ........................................................................................................ 15

3.3 Sorting/grading on board .............................................................................................. 15

3.4 Storage on board .......................................................................................................... 16

4. Brown crab - physiological changes related to stress ........................................................ 19

4.1 Knowledge from literature ............................................................................................ 19

4.2 Dry transport and physiological changes ..................................................................... 19

4.3 Effect of wet transportation and recovery at landing station ........................................ 21

4.4 Use of anaesthetic and effect of temperature during transport ................................... 26

4.5 Effect of acclimation and recovery at at different temperatures in stocking tanks ....... 30

5. Norway Lobster (Nephrops norvegicus) – physiological changes related to stress ........... 34

5.1 Knowledge from literature ............................................................................................. 34

5.2 Experiments on storage and transport by air ............................................................... 38

6. Brown crab - sensory, microbiological and nutritional quality............................................. 51

6.1 Sensory quality ............................................................................................................. 51

6.2 Microbiological quality................................................................................................... 54

6.3 Nutritional quality .......................................................................................................... 56

7. The effect of stress on final product quality ........................................................................ 68

7.1 Dry transport brown crab - sensory quality, stressed vs unstressed) ........................ 68

7.2 Norway lobster: transport-induced stress and product quality ..................................... 72

8. The effect of stress on product shelf-life ............................................................................. 73

8.1 Shelf life related to stress parameters - Brown crab .................................................... 73

8.2 Shelf life related to stress parameters – Norway lobster .............................................. 73

1. Introduction

The trade of live crustaceans faces serious challenges related not only to the increase in

economic costs and loss of purchasing power by consumers, but also with the huge trade chain

complexity and duration.

Work Package objectives

The objectives of work package six are to establish a European guideline for quality

requirements of live crustaceans and other typical food quality parameters such as appearance,

shape, colour taste, texture, odour, microbial load and toxic compounds. The work also looks

into how important stress indicators are in terms of the impact on end-user quality, and

establishes a European guideline for handling crustaceans.

The aim of this report is to collate existing knowledge on the effects of capture, storage and

transport of live crustaceans. We describe results of experiments and draw conclusions about

the best practice for delivering high quality crustaceans from source to market. Research

included investigation of storage regimes, simulated transport trials and food shelf live in

relation to various stress parameters. The document describing these is intended to benefit the

whole industry.

2. Brown crab (Cancer pagurus) value chain and critical points/stress

2.1 Transportation - dry vs wet vs semi-dry: transportation plan

The edible crab is harvested by pots of different types depending on environmental conditions

and local tradition. The crab is fished in two principal different ways; inshore in coastal areas

and fjords and on offshore grounds, i.e. outside 12 n. miles. In the offshore fishery the crab is

harvested with specialised vivier boats (super-crabbers) that fish the whole year round. Most of

the crabs harvested by these boats go to the live market in Europe. Inshore, the crabs are

fished by smaller boats, most of them without wells. This means that they have to transport the

crab dry in boxes or containers, only wetted by the hose-pipe or by a wet stacking-flow system.

The inshore fishery is mainly conducted from June to November. Most of the crab goes to the

processing industry.

In the vivier chain the crab is stored and transported in water; from harvesting to the market this

only interrupted by unloading. In UK and Ireland this is a common way of harvesting and

transporting live shellfish. The system is based on vivier boats that unload the crab to vivier

trucks. These transport the crab to the European market where the crab is unloaded to vivier

holding tanks before being distributed to different markets.

The vivier boats, also called supercrabbers, are specialised for holding and transporting the live

crabs. Utilising a flow-through system, animals can be kept in water for up to5 days The water

intake is in the bottom of the tanks and it exits at the top, i.e. an upstream system. The water is

directed beneath a false bottom, leading to an even distribution of the water flow and preventing

the dirt from the crab to be stored at the bottom of the tank. The water in a tank is renewed 6-7

times per hour. This is sufficient to keep the oxygen and ammonia content within safe limits.

When the boat passes through low salinity or dirty water, such as in the harbours, the intake is

closed and air-bubbling is connected in order to give sufficient oxygen for the crabs.

A vivier boat normally carries 6-7 tonnes of crabs and delivers once or twice a week (usually on

Tuesday and Saturday). A crew of at least 6 persons is necessary. One person is responsible

for inactivating the crab‟s claws by nicking. During hauling he is sitting in the nicking chair and

every crab has to go through his hands. Buckets are placed nearby and work like a buffer,

holding the animals until he nicks the claws. In UK there are about 20 vivier boats and in Ireland

5. In the Norwegian crab fishery there is only one vivier boat which has been fishing crabs for 4-

5 years, however only on inshore grounds.

Figure 1. Value chains in the European fishery for Edible crab (Cancer pagurus).

2.2 Water quality

2.2.1 Flow-through systems

Flow-through systems are mainly used for storage of Edible Crab in Europe. These systems

impose few problems as long as the salinity and the temperature of the water is monitored and

controlled and the flow gives sufficient oxygen to the animals. Crabs generally survive well

(t°C)

Offshore On board handling Viviere system Flow-through system Ambient t

Viviere boats

On board Inshore boats On board handling Boxes/containers Dry Ambient

Wet (flow-through Ambient

stacking system) Ambient

Wet (cages in sea) Ambient

Holding Handling on land Live storage wet Flow-through system Ambient

facility Re-circulating Cool (4-5)

Holding E-C water treatment system

facilities

& / or Live storage dry Boxes dry Cool (4-5)

processing

Processing Production Several -

Transport Shipping method Live Viviere trucks Cool (6-12)

Dry by road Cool (3-5)

Dry by air Cool (0.5-2)

Processed Several products -

Transport

revitalising Revitalising Storage for crabs Live storage Flow-through system Ambient

Re-circulating Cool (4-5)

E-C water treatment system

Market

during short-term storage in flow-through systems. However problems do arise concerning

mortality rates during medium and long term storage, most likely as a result of starvation, limb

loss or chronic stress.

2.2.3 Closed systems

If sufficient oxygen is added, ammonia will be one of the main limiting water quality parameters

in a closed system. Ammonia (NH4+

/NH3) is mainly excreted through the gills and is toxic above

certain levels depending on the species. Toxicity seems to be related to the un-ionized

ammonia (NH3). It is uncertain if the ammonium-ion (NH4+) will affect the edible crab.

Regulations or changes in the water pH affects the balance between NH3 and NH4+, where

increase will give more NH3 and thereby a more toxic environment. In waters with high load, the

pH normally drops because of CO2 expired from the animals. In this instance ammonia gets

detoxified.

In a recirculating system, high levels of ammonia are prevented by nitrifying bacteria in a

biological filter. These bacteria convert ammonia to nitrite (NO2-) which is less toxic and then

convert nitrite to nitrate (NO3-). The system is considered to be in balance when the bacterial

colony has grown large enough to keep the ammonia and nitrite levels under control. Nitrite may

accumulate in aquatic systems as a result of an imbalance of nitrifying bacterial activity (Wickins

& Lee, 2002). A high level of nitrite in the water is potentially a factor triggering stress and can

cause high mortalities in aquatic organisms. Acute toxicity of nitrite has been investigated for

several fish and crustacean (Wickins & Lee, 2002).

2.2.4 Water quality in vivier trucks

In vivier trucks the normal practice for edible crab is to use a 1:1 ratio between weight of crabs

and weight of water. These values have been learned by trial and error and seem to be

acceptable for journeys between 24 - 36 hours duration. Longer journeys usually result in an

increase in mortality in the holding tanks at the market („in-pond mortality‟).

During vivier transport of edible crab dissolved ammonia levels have been shown to increase

greatly within the tanks. Ammonia levels close to 3,000 – 4,000 µM1 have been recorded in the

transport water for edible crab in vivier trucks by the end of the travel from Ireland to France.

Correspondingly high blood ammonia levels were found in the crabs. By the end of a journey

the crabs may have extremely high and toxic ammonia levels (Hosie, 1993; Schmitt, 1995;

Uglow & Hosie, 1995). The more direct stress the crab has been subjected to during their

marketing, the more acute the problem will be.

Hosie (1993) investigated the ammonia efflux rates in media with high dissolved ammonia

levels and found that they were related to concentration gradients between the internal and

external media. When animals were transferred to media with higher ammonia levels than those

in blood, a cessation of efflux, or even a net influx of ammonia (NH4+) occurred (Hosie (1993).

However, Schmitt (1995) observed that the concentration of total ammonia in the blood of C.

pagurus was always lower than that in the vivier tank water thus showing that animals were able

to use active mechanisms to excrete ammonia at such times.

1 Corresponding to 51 – 68 mg L

-1 TAN.

2.2.4 Water quality in recirculating tanks

Recirculating systems for storage of edible crabs are only used on a smaller scale, mainly for

short-term storage as a pre-treatment before export when cooling of the animals is essential.

Recirculating tanks are more common used for the more expensive commodities such as the

European and the American lobster. These species are held both in short-term storage, long-

term storage and in long-term storage with feeding.

The water quality for edible crab in a recirculating system is not well examined. The optimal and

lethal conditions for lobsters however, have been examined by several authors. Van Olst et al.

(1980) reviewed the water quality parameters for Homarus sp. in recirculating storage. The

optimal conditions, except for temperature, lies within the natural water conditions for lobsters.

As shown in Table 1, the lobster can tolerate relatively extreme environmental fluctuations.

AquaMedic AS produces recirculating system much used for shellfish and lobsters. In these

systems lobsters seems to be quite hardy in storage, while edible crab is more sensitive.

AquaMedic recommend 75% biomass of crabs compared to 100% lobster in their tanks. (pers.

com. Dr. Manfred Schlüter, AquaMedic). This may to some extent, be used as a guideline for

the tolerance of edible crab.

Table 1. Key water quality parameters for Homarus sp. in recirculating water (van Olst et al. 1980; Wickins & Lee 2002).

Parameter Optimal condition Natural condition Lethal condition

Temperature (°C) 18-22 1-25 <0, >31 Salinity (‰) 28-35 28-35 <8, >45 Oxygen (mg L

-1) 6.4 4.0-7.3 <1, >saturation

pH 8 7.8 - 8.2 <5, >9 NH3-N (mg L

-1) <0.14 0 – 0.3 >1.4

As previously mentioned the toxicity of ammonia depends mainly on the acidity (pH) of the

water, but temperature and salinity will also cause minor changes. For lobster levels of total

ammonia above 6 mg L-1

should be considered to be dangerous, between 4 and 6 mg L-1

should give cause for concern while lower than 2 mg L-1

should be safe for short periods (at a

temperature of 10°C, a pH of 8.0 and salinity of 30‰) (Beard & McGregor, 1991).

A guide for live storage and shipping of American lobster (Estrella, 2002) gives a brief overview

of the ammonia in a storage system. In such a system high levels of ammonia is controlled by

nitrifying bacteria in a biological filter. The system is considered to be in balance when the

bacterial colony has grown large enough to keep the ammonia and nitrite levels under control.

When this happens, a test for ammonia should be read < 10 ppm while nitrite drops to < 5 ppm.

Under these circumstances nitrate levels will continue to build up and should be kept below 100

ppm by a regular schedule of water exchange.

2.3 Special critical points: nicking vs banding vs nothing

Nicking

Holding edible crabs in tanks, containers or boxes without some kind of claw inactivation may

lead to self-amputation and mortality. This is due to damage caused by use of the claws in an

aggressive manner or stress in general (Anon, 1987; Uglow and Hosie, 1995). Inactivation may

be accomplished by nicking, banding or just stacking tightly in boxes.

Nicking is the traditional way of inactivating crab claws in the commercial live trade. It can be

conducted by cutting the tendon on the upper side of the dactylus (above the claw) which

makes the crab unable to open the claw but still able to close it („English nicking‟). Nicking can

also be conducted by the „French method‟ where the ligament underneath the dactylus

(between each claw) is cut instead of above (Figure 2). The French method leaves the claw

totally unusable and is the most common way to handle live crabs in Ireland and in the live trade

in general (Lawler, 2001).The process is estimated to take between 15-20 seconds per animals

(Jacklin & Lart 1995).

Correct nicking must be done with a fully opened claw so that the tendon splits under tension,

i.e. nicking relies upon the fisherman applying the lightest cut to a ligament that then breaks

under the strain of the powerful muscle in the claw. Done badly, nicking will cause excessive

bleeding; even when carefully nicked the crab will bleed slightly. Crabs should be returned to

seawater directly after the nicking as this will allow a quicker clotting of the blood.

Figure 2. Nicking on-board Scottish speed boat (a) and on-board offshore vivier boat using a nicking bar (b). Photo: LHH Nicking is generally claimed to weaken the crab and discolor the meat around the injury site

(Figure 3) (Anon, 1987). According to Jacklin & Lart (1995), the requirements to nick also

impacts on the market choice as nicked animals have to be marketed as a live product. The

potential damage to the valuable claw meat renders such crabs unacceptable to processors

who rely on the profit element generated by crab claws.

Figure 3. The result of English nicking after 3 weeks storage; a) the arrow shows the nicking site; b) discolouring of the claw meat. Source: Woll & Berge, 1987. Photo: AW

From a legal perspective there is an increasing body of legislation, in the form of EEC Directive

91/628 and UK orders, which seek to maintain the welfare of animals undergoing transportation

at the highest reasonable level. All this suggests that there is a need for an alternative to nicking

that would be suitable for all sectors in the edible crab industry. Several attempts have therefore

been conducted in order to find a method to inactivate the crab‟s claw with a non-invasive

technique which should reduce stress and mortality due to the injuries caused by nicking.

Tube and band restraints

A study of non-invasive restraint was conducted by Jacklin & Lart (1995). The criteria for the

restraint were set by the crab industry: improved condition and less mortality compared to

nicking techniques, the time to apply the restraint should not exceed that required for nicking,

acceptable cost, and maximum required endurance 21 days.

The results of laboratory and commercial experiments showed that more development work was

required before commercial use. Under experimental conditions tube restraints showed

potential to satisfy the design criteria with endurance times up to and exceeding 20 days.

However, a suitable applicator must be designed as the equipment proved to be too

complicated and could not accommodate the variation in claw size. The endurance time for the

band restraints were less than for the tube, ranging from 8–21 days. Applications of bands

proved difficult because of the requirement to orientate the pincers correctly and the slippery

nature of the material when wet. However, the work demonstrated that the non-invasive

restraints during storage and transport could reduce mortality and improve quality compared to

the current practice of nicking.

Rubber band

A procedure for banding the claws (Figure 4) is used in Canada for storage and transport of live

Dungeness crab to over-seas markets. The procedure does not hurt the claws, but prevents the

movements of the chelipeds. The banding procedure has been adapted to edible crab in

different storage and transport experiment as described by Woll & Berge (2007). In the

experiments the banding was applied manually and therefore time consuming (approximately

100 crabs banded per hour). The rubber band had a tendency to break during storage or the

crab manipulated the band so it only fitted around the merus.

Figure 4. “Cold water” – rubber band for crabs of medium sized claws application with the ”regular banding tool”. For bigger claws ”Cold water jumbo” rubber bands are used and applied with ”Jumbo banding tool”. Photo: AW Rubber band and tools from Vernon d‟Eon Lobster plugs Ltd. http://www.vernondeon.com)

During long storage in tanks (3 weeks) banded crabs also suffered some damage such as lost

legs and broken tips (dactylus) (Woll & Berge, 2007). However, when compared to banded and

nicked crabs (English nicking), no difference was found in the amount of damage during the 3

week experiment suggesting that the methods are equally efficient in preventing damage

caused by aggressive interactions between free-going crabs in tanks. The banding process

gave no discoloration or mortality due to the process.

3. Langoustine (Nephrops norvegicus) special critical points & on-board handling

3.1 Capture and post-capture handling

This is a fundamentally important stage since actions here drastically affect not only the

following stages but also the type of market that the product will be sold in. For example, if the

objective is immediate sale upon arrival at the destination country, then it is only necessary to

keep the animal alive for the duration of the storage and transport. If however, the animal is

destined for further storage upon arrival at the destination country, then it is essential that care

is taken from the earliest stages to ensure the highest long-term survival rate. The options

include trawling or use of creels (traps) to capture the Nephrops, and on-board storage in sea-

water or emersion for the duration of the trip.

When fishing for live marked the use of creels is a traditional method of capture for Nephrops,

particularly across the west coast of Scotland. These creels are tied together in fleets of 40-60

units, baited with salt herring and deployed for periods of 24-48 hours. Number of lobsters will

not increase after two days, however the stress factor increases with time and number of

lobsters. Typically, the taking on-board, emptying, re-baiting and deployment of a fleet takes

about 30 minutes. This speed ensures that animals experience the minimum amount of stress

during capture, with creel caught animals releasing significantly less of the stress hormone,

CHH, than trawled animals (Ridgway et al, 2006).

Additionally, both mechanical trauma and other physiological stresses are known to occur

during trawling. It has been found that more than 50% of a trawled catch will exhibit structural

damage, and the compression of animals within a net prevents individuals from getting sufficient

oxygen forcing them to utilise anaerobic pathways of metabolism. Figure 5. (from Ridgway et al,

2006) indicates the difference in levels L-lactate, the end-product of anaerobic respiration and

shows that the levels found in trawled animals are many times higher than those from creeled

animals.

Figure 5. Haemolymph lactate concentration in creeled and trawled animals.

SAM = Short dawn tow - SPM = Short dusk tow - LAM = Long dawn tow - LPM = Long dusk tow (Ridgeway et al. 2006).

An increased level of lactate associated with anaerobic respiration has been posited as a

causative agent of muscle necrosis, a condition originally found in Scottish Nephrops destined

for live export. Clinical diagnosis is the appearance of white, opaque lesions through to

complete opacity in the abdominal musculature and the inability to flex the abdomen. The

disease is terminal with death occurring within several days of the appearance of the first

lesions. The implications for production of a live export product are that if a high proportion of

animals in the catch experience greatly raised haemolymph concentrations of lactate, there is a

risk that many animals may die.

Nevertheless, trawling is successfully used as a capture method in Scotland, particularly when

the animals are to be sold immediately after arrival at the destination. A series of studies carried

out during 2006 by the Seafish Industry Authority generated the advice that if the length of time

for tows was kept to about 1 hour, damage levels would be low and survival rates would be high

enough to make this an economically viable means of Nephrops capture. In addition to this,

although lactate is known to be lethal in sustained high concentrations, Nephrops show a

surprising capacity for recovery when revitalised in seawater (either a transport or storage tank)

(Albalat et al., 2009).

It has also been shown recently that although trawling impacts on the immune function of

animals compared with creel-caught Nephrops, the effects are more important in trawls lasting a

longer duration. Ridgway et al. (2006) used to common measure of susceptibility to infection,

total haemocyte count (THC), to show that animals caught from five hour tows had significantly

lower THC than those from one hour tows and creel-caught animals. However, the THC from

creeled Nephrops and those from one hour tows was not significantly different, providing more

support to the practice of using short tows to capture Nephrops for live processing.

In addition to the method used to catch the lobsters, the immediate post-capture handling

greatly affects the quality of the landings. This species is entirely sub-littoral and although does

not have the same capacity to tolerate emersion as some other crustaceans, its physiological

features allow it to survive for a period of time in air under the correct conditions. The pathology

of aerial exposure is varied and complex but key aspects include a build-up of ammonia,

hypoxia and acidosis due to an increase in lactate concentration. The negative effects are

strongly associated with ambient temperature with Spicer et al. (1990) finding that the

progression towards mortality was greatly enhanced in animals held at 10°C compared to those

stored on ice. More recently, Ridgway et al. (2006) showed that as ambient temperature

increased, the rate of build up of L-lactate also increased whilst pH decreased (fig.6 & 7).

The significant point of this study is that at 25°C, none of the animals survived for longer than

four hours. In commercial practice, it is very feasible that ambient temperatures would reach,

and even exceed 25°C, either on summer days or especially when using a shelter deck. This

exemplifies the need to protect the catch in order to not only maintain quality but also to

facilitate survival between the boat and storage point.

Currently, a variety of procedures are used although these can be broadly classified into two

categories: aerial storage and seawater tank storage. Animals stored in air are protected from

desiccation by covering with an absorbent material (frequently old pieces of carpet or hessian

sacks) which is regularly drenched with seawater and heavily iced to keep their temperature as

low as possible.

More commonly, catches are stored in seawater tanks on board the boat until the end of the

trip. The tanks may be static, aerated or flow-through with static tanks being the most frequently

used on board smaller Nephrops boats, especially those using creels to fish. In larger boats,

particularly trawlers, aerated or flow-through vivier tanks are generally used. This is because, as

described previously, the build-up of metabolites is much greater in trawled animals due to the

trauma of this fishing method. Consequently, it is vital to provide the animals with a means of

releasing these metabolites and recovering normal homeostasis as quickly as possible.

In summary, there are several practices that are currently employed with success to catch

Nephrops for processing as a live product. The highest quality landings derive from creeled

animals that are stored on board in a flow-through system. However, a creel boat operating with

two crew over good fishing grounds can expect to land between 100 -150 kg per day.

Meanwhile, a small trawler operating with the same number of crew and over the same grounds

can catch 200-500 kg and can supply both the frozen and live market. This makes it an

attractive method although it is still applied on a small scale basis.

Pre-exp 4 8 12

Figure 6. Haemolymph lactate concentration (mmol /1) at 10°C, 15°C and 25°C. At 25°C, all animals were dead within 8 hours.

Figure 7. Haemolymph pH at 10°C, 15°C and 25°C. At 25°C, all animals were dead within eight hours.

3.2 Handling on-board

“Handle with care” could be the motto in the whole process of fishing, handling and storage on-

board when fishing Nephrops for live market. Good handling during fishing and on board

processing means a better product for the storage facility and transport. A higher price is

achieved for a better quality product.

It is important to avoid stressful conditions because they will lead to reduced quality and higher

mortalities. Stressful conditions are, for example, if the lobsters are emersed for long time,

especially if ambient temperature is high. Also if temperature and salinity in holdings tanks are

not close to their natural conditions. It is also important to pick the lobsters up carefully from the

creels or from the trawl. Limbs can easily be lost and lobsters missing arms are not suitable for

live market. Wound damages will also facilitate bacterial infections.

3.3 Sorting/grading on board

Size-grading should be carried out on-board; that will prevent double handling later on in the

process. All handling increases stress levels and should be kept to a minimum. Other

advantages to on-board grading are that it makes production quantities immediately apparent

and also prevents animals fighting and thus damaging each other.

Nephrops have chemo receptors that detect the proximity of other individuals by their

pheromones. They are very aggressive and territorial so will often fight. Their claws are very

powerful and they can damage each other considerably. Bleeding from big wounds can lead to

death and open wounds are a way for bacterial infection to enter. Nephrops are able to lose

their limbs by autonomy which means that they can come off easily under stressful conditions.

Nephrops missing arms or with big wounds or that have shell damage are not suitable for live

market so it is important to keep individuals apart.

The grading system used for Nephrops is like a wine-rack box with different sizes of

compartments for the lobsters. It consist of cortex cartons with plastic top and bottom (ca

35x55cm). It is important to size grade appropriately; approximate grades are predetermined by

weight categories although individual customers may request more specific grading procedures

(Figure 8).

Figure 8. Weight range of individuals in size-classes.

The same size grading protocol is followed on the

boat, in the holding facility and when transporting to

the customer. If you size-grade properly in the

beginning it saves both work and lobster stress later

It‟s best for the lobsters to go into a compartment of

the correct size. A small lobster in a big compartment

is not supported in the vertical position and there is a

greater risk of accidentally placing two or more

lobsters together putting a small individual to a

compartment that is too large – this causes

aggressive interactions. A big lobster placed into a

compartment that is too small is at risk of claw

damage and is difficult to remove without causing

injury.

The grading crates should be held in seawater whilst

being filled. The Nephrops needs to remain in

seawater to maintain respiratory function and

continue to excrete metabolites. It is best to have a

flow-through system or some kind of system where

the seawater is renewed. That will prevent the build

up of excreted products and replenish oxygen.

Nephrops eyes are adapted to light levels on the

seabed and surface light is many times stronger.

Exposure to intense light during handling can cause

eye-damage or retina breakdown in Nephrops. Short

periods of moderate daylight seem to be enough to

cause damage to a large portion of the retina and the

dioptric apparatus (Chapman et al. 2000). Although

this increases the stress level, it is not necessarily

life-threatening (Chapman et al. 2000). To keep the

stress level to a minimum use mats to cover the

boxes protects the lobster from direct sunlight.

3.4 Storage on board

Stress

It is important to keep in mind that increased stress level in the Nephrops will lead to reduced

quality and higher mortalities and therefore lower price for the catch. In order to prevent this, the

animals should be handled with care and emersed for as short time as possible. Nephrops

needs to be immersed in fresh seawater to be able to respire properly and to excrete

metabolites. Long emersion time results in lack of vigour and the lobster becomes unsuitable for

the live market. A period of emersion is unavoidable but this should be kept to a minimum.

The grading crates should be held in seawater whilst being filled and when full they should be

put into a holding tank (Figure 9). In both cases the best practice is to have a flow-through

system of seawater. Then it is certain that the animals will get enough oxygen and also it is

important to keep flow rate high enough to flush out toxic wastes. Waste metabolites produced

by the lobster under stressful conditions are mostly ammonia and lactate.

Figure 9. When the grading crates have been filled they are put into a holding tank with a seawater flow-through system and covered with mats.

The Nephrops gills are located under the carapace and they play a vital role in consumption of

oxygen and excretion of ammonia and CO2. The lobster is unable to respire correctly if not in

seawater. When it is emersed, the gill-lamellae can collapse and limit correct gill function. Also

the lobster will transfer its metabolism to anaerobic respiration and the lactate level will

increase. Both ammonia and lactate can quickly reach a toxic level for cells.

Ammonia

When protein is metabolized, the end product is ammonia (NH3) and it is released from the cells

into the blood. The vast majority of the ammonia is removed from the blood as it flows through

the gills.

When held out of water lobsters are unable to use the gills effectively. In humans ammonia is

removed in the kidneys and the nitrogenous waste (in this case urea) is excreted in the urine.

Under normal conditions lobsters are constantly excreting ammonia into their surrounding

seawater. Then there is a natural downhill gradient of ammonia between the animal and the

seawater. When large numbers of lobsters are kept in the same mass of water, levels of

ammonia continue to increase. When external ammonia level rise the animal continue to

maintain their ammonia gradient and the ammonia builds up in the blood and can reach a toxic

level (Uglow).

Lactate

The lobster needs to be immersed to be able to respire and excrete metabolites. If the oxygen

level drops a lot, the lobster will transfer its metabolism to anaerobic respiration and the lactate

level will increase. Lactate is the end-product of anaerobic respiration. If the levels of lactate

become too high, it will become toxic for cells. Lactate can be lethal in sustained high

concentrations. However, Nephrops show surprising capacity for recovery when revitalised in

seawater (Albalat et al. 2009).

The lactate levels are found to increase both with temperature and emersion time (fig.26)

(Ridgeway et al. 2006). If the lactate levels are very high, the lobsters become lethargic and if

not allowed to recover, can die.

Respiration

The lobster is unable to respire correctly if not in seawater. When it is emersed, the gill-lamellae

collapse and limit respiration. It is important to keep the time of emersion to a minimum and

have fresh seawater in the tub where the grading crates are filled and in the holding tank. A

flow-through system is ideal or some kind of system with renewal of seawater.

The concentration of dissolved oxygen in the system seawater is very important and should not

fall below 70%. If the levels are low the animals will suffocate or suffer high levels of stress

(Gerhardt and Baden 1998).

If the Nephrops do not get sufficient oxygen it will force them to utilise anaerobic pathways of

metabolism and the level of lactate will rise.

According to Gerhardt and Baden (1998) normoxia is defined as 70% oxygen saturation and

hypoxia as 30% oxygen saturation. Increased pleopod activity can be used as an indicator of

hypoxia as the Nephrops uses the pleopods to create ventilation current over the gills.

The oxygen concentration should always be kept as close to saturation as possible. The

saturation level for oxygen is affected by temperature and salinity. The capacity to hold oxygen

increases as temperature drops. Oxygen holding capacity also increases with drop in salinity

(Estrella, 2002). Oxygen consumption is an important factor and increases with rise in

temperature and deviation from optimum salinity values.

A flow-through system

The best practice in storage on-board is to have a flow-through system in the tub were the

grading crates are filled and in the holding tank. Using a flow-through system will prevent the

build up of excreted products and replenish oxygen.

By doing so it is possible to keep the lobsters in good condition for days (fig 37). If lobsters stay

in stressful conditions for to long the result will be a lack of vigour. They can not lift their claws

and have slow abdomen movements (fig 38).

It is stressful for the lobster if the temperature in the holding tank is a lot higher than the sea

temperature in which he was caught and if the

salinity level is not close to what he is used to. If the weather is warm, the temperature in the

tank can rise quickly if there is no system for renewal. You will also have evaporation that will

lead to an increase in salinity levels. It is better to cover the top of the tank.

Seawater taken from the surface layer of the sea can be lower in salinity than deeper waters,

especially in summer when ambient temperature is high. Therefore it is better to take seawater

for the holding tank from a few meters below the surface.

Hygiene

It is important to keep everything clean. Dirt can lodge on the gill surface and if the lobster is

unable to remove it, there will eventually be problems breathing. The lobster will be less likely to

survive during storage if dirty. When the lobsters are free, they can groom themselves and

clean the gills etc. However, when they are placed in the holding crates they are unable to do

so. If there is a film of oil on the sea surface, it will contaminate the lobster when he passes

through it and that can lead to breathing problems.

Dirty creels do not fish as effectively. When creels are placed on the seabed with the bait in the

middle, the scent of the bait should attract the lobster towards it. But if the creels are very dirty

with old bait, the smell from that will overwhelm that of the new bait and the lobster will not be

drawn into the trap, just on the outside.

4. Brown crab - physiological changes related to stress

4.1 Knowledge from literature

The process of harvesting, landing, storage and transport means that the crab will be out of

water for varying periods of time. The gills are the principal site for exchange of the respiratory

gases and ions. When in air, the gills tend to collapse and clump thus reducing the ability and

effective area for these fluxes.

The reduction in oxygen uptake usually causes several changes in the blood composition and

acid-base balance. A switch to anaerobic pathways of energy production generally occur

leading to an increased utilisation of carbohydrate and production of lactate. Accumulation of

lactate and increased levels of carbon dioxide due to the impairment of gas exchange leads to

decreases in blood pH.

An accumulation of ammonia in the Haemolymph occurs in Cancer pagurus following aerial

exposure (Danford, 2001; Hosie, 1993; Regnault, 1992). As water is required by crustaceans to

excrete ammonia from blood, accumulation during emersion may reach toxic levels causing

irreparable tissue damage which ultimately kills the animals. However, total ammonia production

during emersion is generally reduced in crustaceans (Regnault, 1992). In Cancer pagurus a

reduction in aerobic metabolism has been shown to be 13.6% of immersed levels when

exposed to air (Uglow et al. 1986).

Accumulation of haemolymph lactate and ammonia increases with increasing temperature.

Preliminary data for laboratory-held crabs showed increasing concentration of ammonia and

lactate with increasing temperature and time (Woll et al, unpublished data). According to these

preliminary data recommended maximum holding time in air at 21°C, 15°C, 5°C and 2°C should

be less than 6, 9, 50 and 72 hours, respectively. This would give mortalities less than 5% and

on average Haemolymph ammonia concentration of 550 µM and 20 mM lactate. The increase in

blood ammonia levels during emersion is usually accompanied by a large efflux rates followed

re-immersion (Danford, 2001).

During emersion water loss occur especially if the animals are exposed to moving air. During

the first minutes of emersion the crab empties the gill chambers. Thereafter the dehydration

continues gradually, however, quicker when the air temperature is high and humidity low. Woll &

Tuene (2001, unpublished data) found that crabs of medium to good quality kept cool and

humid lost 6% weight during the first 5 minutes of emersion due to emptying the gill chamber.

Thereafter weight was lost gradually. Over the subsequent 17 hours an additional 3% was lost.

For more information refer to D1.1 and D2.1 in the CrustaSea project.

4.2 Dry transport and physiological changes

Authors Møreforsking Marin: A. K. Woll, W. E. Larssen & I. Fossen.

Exposing the crabs to air (dry storage) is one of the key points to get right in order to keep the

crabs in good condition from catching, storage and transport to their final destination.

Laboratory studies were conducted aiming to give more knowledge about the crabs‟ capacity to

tolerate air storage (Woll et al, 2006 in Norwegian). Revitalised crabs were kept in air at

different time and temperatures (5, 10, 15 and 20 °C). Vitality indexes (Table 2) (strong, healthy,

weak, moribund and dead) and blood parameters (pH, lactate, total ammonia, and glucose)

were compared at the termination of dry storage. The relationship between the vitality indexes

and the hemolymph parameters were significant for pH, total ammonia and lactate, but not for

glucose (Pearson correlation test).

On average the strong crabs (index = 5, reference groups) had a lower pH and a higher TA and

lactate concentration than the healthy crabs (index 4). Moribund crabs (index 2) had lower pH

and higher TA than the weak (index = 3), while no differences were detected for lactate between

moribund and weak specimens. No differences between condition indexes were found for

haemolymph glucose.

Table 2. Criteria for vitality indexes for brown crab.

Index Description

5 (Strong) • Prompt, strong, aggressive response • Defensive position unable access to abdominal area. • Cling to hand when held upside down. • Eyestalk response. • Mouthpart strong.

4 ( Healthy) • Like index 5, however the aggressive / defensive response is quick, but not prompt.

3 (Weak) • Weak legs and claws, no aggressive response. • Slow reaction when touched in abdominal area. • Loosen the grip when held upside down. • Eyestalk response. • Mouthpart strong.

2 (Moribund) • No claw response. • No reaction when touched in abdominal area. • No grip when held upside down. • Slow eyestalk response. • Mouthpart slack.

1 (Dead) • Like index 2, but no movement in antennae and mouthparts when touched.

A conservative guidance for dry storage at various temperatures was given based on the

laboratory studies. This aims to help fishermen and industry to plan the best logistics to keep

the crabs healthy in different environmental conditions. Table 3 shows the expected time

allowed to keep the crab in good condition at different storage temperatures assuming that crab

were stored without draught and in damp conditions. The green line shows the time when no

mortality occurs and red line when approximately all crabs were moribund or dead. At 20 °C

these two temperatures were 6 and 9 hours, at 15 °C 10 and 18 hours, at 10 °C 24 and 36

hours, and at 5°C 48 and 72 hours.

It must be underlined that in a commercial fishery other factors are likely to be added, such as

stress factors during catching, air humidity and not at least the sorting of the crabs where weak,

damaged and low quality crabs such as soft and pale ones should be rejected.

Table 3. A guideline for available holding time during emersion at different temperatures. A)maximum hours expected to keep all crabs in good vitality. B)exceeding this holding time is likely to result in a marked reduction of overall vitality and rapidly increasing mortality.

Air temperature (°C) A) B)

5 60 72

10 30 44

15 11 18

20 5 9

Reference A. K. Woll, W. E. Larssen and I. Fossen. Physiological responses of brown crab (Cancer pagurus LINNAEUS 1758) to dry storage under conditions simulating vitality stressors. J.Shellfish Res. Vol. 29, No. 2. 2010.

4.3 Effect of wet transportation and recovery at landing station

Authors INRB I.P./L-IPIMAR: António Marques, Sara Barrento, Maria Leonor Nunes

The trade of live crustaceans in Mediterranean countries like Portugal is part of a long chain,

which is highly dependent on importation and centralized in holding/deposit facilities (Barrento

et al., 2008). Live crustaceans like edible crab are mostly captured in Britain (31,079 tones),

Ireland (11,525 tones) and France (5,724 tones) (data from Eurostat, 2009) to Southern

European countries. Transport is a critical step in which crustaceans are subjected to several

stressors, such as periods of air exposure, hypoxia, handling, interaction with other individuals,

seawater physicochemical variation (e.g. temperature, ammonia, pH, salinity, nitrite, nitrate) and

starvation.

The edible crab is mainly transported to Mediterranean countries in refrigerated trucks with

tanks filled with aerated seawater (around 12 ºC), which is not renewed or filtered during the

transportation period (from Scotland to Portugal, around 58 h; from France to Portugal, around

24 h), and each tank contains one crustacean species at a density of 1 kg/L (Barrento et al.,

2008). Consequently, seawater accumulates significant quantities of excreted products. Finally,

the unloading process is usually careless and promotes temperature changes, air and sun

exposure just to name some stressors associated with harvest and handling (Barrento et al.,

2008).

These stressors have been associated with losses due to high mortalities during transport (up to

50 %), but mainly during recovery at the importer‟s premises (15 to 70 %) (Uglow et al., 1986;

Barrento et al., 2008). Additionally, the transported water is usually spilled in coastal areas at

the importer‟s country, a procedure that may introduce non-native or exotic species and create

ecological problems to endemic coastal species in a similar way as ballast water. These events

culminate in significant economical losses in the short term and ultimately in the waste of an

important, but finite, resource and deterioration of coastal habitats.

The present study aimed to identify the critical points in the distribution chain from the fishing

vessel in England to a typical importer facility in Portugal and to link critical points with

physiological changes in haemolymph chemistry (pH, glucose, lactate, and haemocynine),

vigour (or vitality index; using the scale described in Table 2), physical injuries and mortality of

C. pagurus. The general procedure is summarized in Figure 10.

Crabs were harvested by a commercial fishing vessel in the English Channel with traps at 70

meters depth in late April, 2009. The fishing trip took eight days and harvested crabs were

maintained in recirculation water tanks in the boat. Upon arrival to Weymouth harbour, England,

seawater was removed from the tanks and 7 tons of edible crabs were unloaded from the boat

to the vivier truck tanks filled with seawater. This procedure was done with plastic buckets

taking approximately 40 kg crabs. Two hours were required to unload all crabs. Crabs waiting to

be loaded to the truck were kept exposed to air in plastic buckets.

Table 4. Vitality index employed to evaluate the vigour of edible crab.

Vitality index Claws Legs & abdominal area Eyestalk response Mouthparts & antennae

Strong/Reactive Strong / aggressive

response

Defensive position

prohibiting access

Yes Strong

Weak Weak / no aggressive

response

Slow reaction when

touching

Yes Strong

Moribund No movement No movement Eyes into holes,

slow reaction

Slack or open, slowly

moved when touched

Dead No movement No movement No movement No movement

Figure 10. Diagram summarizing the experimental procedure. Abbreviations: CW, carapace width; CL, carapace length; MY, muscle yield; GI, gonadosomatic index; HI, hepatosomatic index.

Transport (1 kg crab/L seawater at 13 ºC)

Vessel (6 tons) Truck (700 kg/tank)

60 crabs tagged

and sampled:

- haemolymph

- Vigour

Unloading 2 h

30 crabs top

30 crabs bottomEngla

Truck (6 tons)Unloading 2 h

Recovery

tanks (16 ºC) after:

56 crabs sampled:

- Haemolymph

- Vigour

Haemolymph analyses:

- Glucose - pH

- Lactate - haemocyanin

Biological data:

Weight, CW, CL

MY, GI, HI

Transport (1 kg crab/L seawater at 13 ºC)

Vessel (6 tons) Truck (700 kg/tank)

60 crabs tagged

and sampled:

- haemolymph

- Vigour

Unloading 2 h

30 crabs top

30 crabs bottomEngla

Truck (6 tons)Unloading 2 h

Recovery

tanks (16 ºC) after:

56 crabs sampled:

- Haemolymph

- Vigour

Haemolymph analyses:

- Glucose - pH

- Lactate - haemocyanin

Biological data:

Weight, CW, CL

MY, GI, HI

Meanwhile, 60 crabs were tagged and haemolymph samples were taken during the unloading

process (T0). Thirty tagged crabs were put in the bottom of a tank at the vivier truck, while the

remaining animals were put in the top of the same tank. Each tank carried about 700 Kg of crab

at a density of 1kg/1L water. Transport from England to Portugal lasted 58 h and animals were

kept in tanks with aerated seawater at 13 ºC. Upon arrival to the importer‟s facilities the 56

tagged crabs were sampled for haemolymph, and damages were also recorded (missing limbs

and claws). Subsequently, animals were maintained immersed in seawater for 96 h at 16 ºC

and 34-35 ‰ of salinity in tanks at the importer‟s facility and animals were periodically sampled

for vigour and haemolymph. Blood parameters of 10 animals maintained for two weeks in the

stocking tanks were considered as the control group.

Between capture and landing at the harbour 0.43 % mortalities (30 kg out of 7000 kg) were

registered, while during transport to Portugal mortalities rose to 0.92 % (60 kg of out of 6500

kg), though this value further increased to 20 % after 4 days in the recovery tanks, of which 11

% were crabs transported at the top and 9 % were crabs transported at the bottom. The vigour

pattern in the recovery tanks suggests that among the crabs transported at the bottom

mortalities occur preferentially within the first 48 h (13 %), while crabs transported at the top

only started to die after 48 h (19.0 %) (Figure 11).

Figure 11. Vitality index of crabs transported at the top (n=26) and at the bottom (n=30) of tanks after transport (0 h) and at the recovery tanks after 6, 24, 48, 72 and 96 h. Vitality index: 0, dead crabs (cumulative); 1, moribund crabs; 2, weak crabs; 4, healthy crabs; 5, very healthy.

Vigour of top crabs

46.2 46.238.5

46.2 46.246.2

3.8 3.8 3.811.5 11.5

15.423.1

0 h 6 h 24 h 48 h 72 h 96 h

Vigour of bottom crabs

43.333.3

43.350.0

6.7 10.0 13.3 13.3 16.7

0 h 6 h 24 h 48 h 72 h 96 h

4 3 2 1 0

During the first 48 h of recovery in the stocking tanks, vitality index was maintained at stage 4

and 3, whereas between 48 and 72 h the percentage of weak (stage 2) and moribund crabs

(stage 1) increased mostly in top crabs, resulting in 11 % mortalities in the following 24 h

against only 4 % in bottom crabs. Overall, most crabs considered as moribund died (67 %) in

opposition to only 8 % of crabs classified as weak. Comparing the vitality index of female crabs

in respect to males the results suggest that the latter have a healthier vigour after transport

(stage 4; 84 %) than females (stage 4; 37 %) (Figure 12). This apparently influences the

recovery process, especially after 48 h and, in the end, 26 % of females died against 16 % of

males.

Figure 12. Vitality index of male (n=37) and female (n=19) crabs after transport (0 h), and at the recovery tanks after 6, 24, 48, 72 and 96 h. Vitality index: 0, dead crabs; 1, moribund crabs; 2, weak crabs; 4, healthy crabs; 5, very healthy.

As far as blood parameters are concerned, top crabs had generally more lactate and

haemocyanin concentrations but lower pH than bottom crabs. After 58 h transport differences

were only observed in pH values. During recovery, differences were only observed after 48 h

with bottom crabs having higher lactate concentrations. Changes in haemolymph parameters of

all crabs during the experimental period are shown in Figure 13.

A wide variation in glucose concentration was detected most probably due to transport

conditions. After 58 h transport, glucose concentration increased from 0.26 to 1.30 mM and

values were kept high even after 24 h (2.03 mM) in the recovery tanks with strongly aerated

Vigour of female crabs

36.847.4

57.9 42.1

5.3 5.3 10.5 15.8 15.826.3

10.55.3

0 h 6 h 24 h 48 h 72 h 96 h

Vigour of male crabs

43.232.4

45.959.5 43.2

5.4 10.8 10.8 16.2

0 h 6 h 24 h 48 h 72 h 96 h

4 3 2 1 0

seawater and appropriate quality. There was a decrease tendency in glucose concentration in

the following 72 h but it never reached values similar to the control group (0.36 mM).

Significant lactate increase was observed during 58 h transport (from 2.19 to 5.86 mM) and

lactate values were maintained high (5.25 mM) in the subsequent 6 h recovery (Figure 13).

After 24 h recovery in the tanks, no statistical differences were found in comparison with the

control group (2.02 and 1.40 mM, respectively).

Blood pH was kept acidic during the entire experiment, never reaching control values, which

might explain mortalities observed even after lactate had reached resting levels (Figure 13).

Haemocyanin concentration was very wide between individuals, but statistically higher values

were observed after transport and in the subsequent 6 h recovery compared to the control

group (Figure 13).

Figure 13. Glucose (mM), lactate (mM), haemocyanin (mg/mL) and pH variations in haemolymph of crabs (n=30) throughout the experiment, prior to transport (0 h) after 58 h of transport, and during recovery at 6, 24, 48, 72, 96 h and in the control group (C). Values are presented as average ± standard deviation. Symbols: * significantly different from control mean (p<0.05).

Overall, the major critical points identified able to induce C. pagurus stress were: (a) poor

handling during capture (e.g. nicking), transport and storage at deposit facilities; (b) air

exposure and temperature fluctuations during unloading to vivier trucks and deposit facilities;

0 h 58 h 6 h 24 h 48 h 72 h 96 h Control

Transport

Recovery

Transport

Recovery

Transport

Recovery

Transport

Recovery

and (c) deficient transport conditions mainly due to poor water quality (e.g. low oxygen and low

pH) and high animal densities. These factors promoted physical damages, such as limb loss

and the impairment of aerobic metabolism with a shift to anaerobic metabolism. Consequently,

glucose concentration increased as a source of fuel necessary for anaerobic metabolism,

resulting in the production and accumulation of lactate.

There was also an acidification of haemolymph that might explain crabs‟ mortalities even after

lactate had reached control levels. Moreover, vigour though subjective was a good approach to

predict mortalities, since most crabs considered as moribund died (67 %) in opposition to only 8

% of crabs classified as weak. These results clearly indicate that transport conditions should be

improved. In fact, the transportation of immersed crabs requires better seawater quality through

efficient filtration systems specifically designed for vivier trucks. Minimum handling should be

implemented by designing mechanical unloading processes. Still, such improvements must be

cost effective and need to be tested. The current immersed transport conditions do not seem to

be effective and is rather costly since it requires the transportation of similar amounts of water

and crabs, and it costs just as much money to transport water as it does for crabs. Additionally,

it is fundamental to grade crabs prior to transport.

4.4 Use of anaesthetic and effect of temperature during transport

Considering the transport conditions of edible crab to Southern European countries, an

experiment was carried out to simulate wet transport conditions and to test for alternatives.

Refrigerated truck transport (8, 12 and 16 ºC) was simulated during 48 h in a refrigeration unit

with tanks. Ninety-five crabs were exposed to four treatments: 1) immersion with strong

aeration; 2) immersion with strong aeration and using anaesthetic (Aqui S®; 40 mg/L); 3)

emersion in humid conditions; and 4) emersion in humid conditions using anaesthetic (Aqui S®;

40 mg/L). Animal stress was periodically monitored, by sampling haemolymph and determining

animal vitality index at 0, 3, 8, 24 and 48 h. The haemolymph stress parameters analyzed were

glucose, lactate, protein concentration, pH and hemocyanine concentration.

Results in Figure 14 and 15, referring to long-term transportation (after 48 h), indicate that

edible crab‟s mortality was generally higher at 16 ºC in all treatments, and particularly higher in

immersed animals (100 %) compared to emersed crabs not treated with the anaesthetic (50 %).

The lowest mortalities were obtained at 12 ºC for immersed animals (0 % and 12.5 %) and

anaesthetised/emerged crabs (25 %), while at 8 ºC lowest mortalities were obtained in non-

anaesthetised emerged animals (12.5 %). Results obtained in short-term transportation (after

24 h) indicate that the highest mortalities were obtained in immersed animals without

anaesthetic (75 %) at 16 ºC, in emerged animals without anaesthetic (25 %) at 12 ºC, and in

emerged animals with anaesthetic (25 %) at 8 ºC.

In contrast, the lowest mortalities after 24 h were registered in immersed animals with

anaesthetic (12.5 %) at 16 ºC, in immersed crabs with and without anaesthetic and emerged

animals with anaesthetic (0 %) at 12 ºC, and in emerged animals without anaesthetic and in

immersed crabs with anaesthetic (0 %) at 8 ºC. Crabs treated with anaesthetic had generally

higher proportion of moribund animals and higher mortalities at 8 ºC than at higher

temperatures. These results indicate that the anaesthetic has a negative effect at low

temperatures that seems to counteract at higher temperatures. This might indicate that the

combination of low temperature and the use of anaesthetic lower too much the crab‟s

metabolism.

Figure 14. Vitality index (%) for each treatment: crabs exposed to air (AIR) and crabs anaesthetised with AQUI S® and exposed to air (AQS); experiments were carried out at 8 ºC, 12 ºC and 16 ºC.

Regarding haemolymph stress parameters, during the 48 h period, glucose greatly fluctuated,

while no significant changes were observed in proteins and hemocyanine concentration. In all

treatments, pH was mostly constant at 8 ºC and 12 ºC, while this parameter decreased at

16 ºC, particularly in air exposed crabs (Figure 16). Lactate was mostly constant at 8 ºC, but

increased at 12 ºC and 16 ºC, particularly in air exposed crabs (Figure 16). This seems to reflect

the higher metabolism of animals subjected to higher temperatures.

Overall, it seems that the use of anaesthetic in short-duration transport (24 h maximum) at

higher temperatures (16 ºC) is the best approach to minimize stress and mortalities of edible

crab compared to the remaining transportation systems (Table 5). The animals should not be

subjected to long-duration transport (more than 24 h) with any of the tested systems at 16 ºC. In

contrast, wet edible crab transportation without anaesthetic should preferably be employed at

12 ºC either in short and long-term transportation to minimize edible crab stress and mortalities,

while edible crab transportation at 8 ºC should preferably employ dry systems to minimize stress

and mortalities.

From a commercial perspective it is important to retain that it is possible to transport live

European edible crab without the costs of transporting seawater in humid chilled air if

temperature is kept low (less than 8 ºC) and crabs are not pilled. Transportation of live

crustaceans in humid chilled air is already a common practice in homarid and palinurid lobsters

12 ºC

16 ºC

n = 8 n = 8

n = 7 n = 8

n = 8 n = 8

AIR AQUI-S

0 h 3 h 8 h 24 h 48 h

0 (dead)

1 (moribund)

2 (weak)

3 (healthy)

4 (very healthy)

AIR AQUI-S

0 h 3 h 8 h 24 h 48 h

0 (dead)

1 (moribund)

2 (weak)

3 (healthy)

4 (very healthy)

50.037.5

37.550.050.0

50.037.5

13 12.5

0 h 3 h 8 h 24 h 48 h

Air AQUI-S

25.025.037.5

0 h 3 h 8 h 24 h 48 h

37.550.0

0 h 3 h 8 h 24 h 48 h

50.037.5

25.050.0

0 h 3 h 8 h 24 h 48 h

28.614.3

28.642.9

0 h 3 h 8 h 24 h 48 h

75.0 75.0

62.525.0

25.075.0

25.0 25.0

0 h 3 h 8 h 24 h 48 h

12 ºC

16 ºC

n = 8 n = 8

n = 7 n = 8

n = 8 n = 8

AIR AQUI-S

0 h 3 h 8 h 24 h 48 h

0 (dead)

1 (moribund)

2 (weak)

3 (healthy)

4 (very healthy)

AIR AQUI-S

0 h 3 h 8 h 24 h 48 h

0 (dead)

1 (moribund)

2 (weak)

3 (healthy)

4 (very healthy)

50.037.5

37.550.050.0

50.037.5

13 12.5

0 h 3 h 8 h 24 h 48 h

Air AQUI-S

25.025.037.5

0 h 3 h 8 h 24 h 48 h

37.550.0

0 h 3 h 8 h 24 h 48 h

50.037.5

25.050.0

0 h 3 h 8 h 24 h 48 h

28.614.3

28.642.9

0 h 3 h 8 h 24 h 48 h

75.0 75.0

62.525.0

25.075.0

25.0 25.0

0 h 3 h 8 h 24 h 48 h

(Paterson and Spanoghe, 1997). It is also important to bear in mind that transport duration

affects the crabs‟ quality and mortality. It was also feasible to have zero mortality during 48 h

transport of immersed crabs at 12 ºC.

Figure 15. Vitality index (%) for each treatment: crabs immersed in seawater (WT) and crabs immersed in seawater with AQUI S® (WQS)); experiments were carried out at 8 ºC, 12 ºC and 16 ºC.

12 ºC

16 ºC

n = 9 n = 8

n = 7 n = 8

n = 8 n = 8

AIR AQUI-S

0 h 3 h 8 h 24 h 48 h

0 (dead)

1 (moribund)

2 (weak)

3 (healthy)

4 (very healthy)

AIR AQUI-S

0 h 3 h 8 h 24 h 48 h

0 (dead)

1 (moribund)

2 (weak)

3 (healthy)

4 (very healthy)

44.433.3

33.322.2

11.122.2

0 h 3 h 8 h 24 h 48 h

Water AQUI-S

87.550.0

0 h 3 h 8 h 24 h 48 h

100.085.7

71.4 71.4

28.614.3

0 h 3 h 8 h 24 h 48 h

62.550.0

25.037.5

25.012.5

12.5 12.5

62.550.0 75.0

0 h 3 h 8 h 24 h 48 h

25.062.5

12.512.5

37.525.0

12.550.0

0 h 3 h 8 h 24 h 48 h

Figure 16. Changes in lactate concentration (mM) and pH values of C. pagurus during the experiments at 8, 12 and 16 ºC in crabs exposed to air (■; Air), immersed in seawater (●; WT), immersed in seawater with AQUI-S® (○; WQS), and exposed to air with AQUI-S® (□; AQS). The p value is shown for each column and different letters for each column represent significant changes between treatments. Control values shown as C (*).

0 h 3 h 8 h 24 h 48 h C

12 ºC

0 h 3 h 8 h 24 h 48 h C

16 ºC

0 h 3 h 8 h 24 h 48 h C

WT a b b c b

WQS a a a ab a

Air a a a a a

AQS a b b bc ab

p 0.347 0.000 0.000 0.001 0.006

WT a a ab a a

WQS a a a a b

Air a a b b b

AQS a a ab a a

p 0.058 0.260 0.028 0.003 0.000

WT a b ab ab -

WQS a a b b a

Air a b ab ab a

AQS a b a a -

p 0.171 0.000 0.026 0.041 0.083

WT a b b b -

WQS a a ab a a

Air a a a ab b

AQS a ab b b -

p 0.044 0.005 0.009 0.002 0.021

WT a a a a a

WQS a a a ab ab

Air a a b b b

AQS a a ab b b

p 0.204 0.218 0.006 0.000 0.001

WT ab b c b b

WQS b a bc b b

Air a a ab a a

AQS ab a a a a

p 0.011 0.001 0.001 0.000 0.001

This study represents a first step in the elaboration of a body of knowledge required to

understand the physiological responses of C. pagurus to live transport handling procedures.

However, further studies are still required to test such solutions in industrial operating conditions

and to verify the economical feasibility of the proposed solutions for each company, i.e.

retailers, importers and wholesalers.

Table 5. Transportation types that should be implemented and avoided according to storage temperature in order to diminish mortalities.

Transportation systems 8 ºC 12 ºC 16 ºC

Wet OK Preferably Wet + AQUI S® OK OK Preferably

Dry Preferably

Dry + AQUI S®

Long-T

Preferably

Wet + AQUI S® OK OK

Dry Preferably

Dry + AQUI S®

4.5 Effect of acclimation and recovery at at different temperatures in stocking tanks

Authors INRB I.P./L-IPIMAR: António Marques, Sara Barrento, Maria Leonor Nunes

The industry is interested in transporting Cancer pagurus exposed to air instead of immersed

due to economical constraints. However, there are species specific physiological limitations that

must be addressed. C. pagurus occupies subtidal biotopes where temperature changes from 4

to 16 ºC and rarely experiences air exposure, except during live marketing (Chartois et al.,

1994; Lorenzon et al., 2008). Previous studies simulating air shipment of C. pagurus proved to

be efficient, however it is crucial to determine the best recovery temperature after transport at

low temperatures in order to avoid changes that impair homeostasis and lead to poor survival.

Most crab‟s mortality is known to occur during recovery at reception facilities. The response of

crabs under these circumstances is essential considering that holding facilities of most

importers do not have the necessary infrastructures to lower the temperature below 16 ºC. In

this context, the present work aimed to assess the physiological changes during simulated live

shipment of immersed and air exposed crabs, and subsequent acclimation and recovery at

different temperatures in stocking tanks. The experimental design is shown in Figure 17.

Figure 17. Diagram summarizing the experimental procedure of treatments A, B, C and D.

Thirty two intermoult crabs were obtained in Autumn from a local importer, where they were

maintained without feeding in a well aerated recirculating seawater system at 16 ºC during 6

days prior to the experiment. Transport was simulated during 48 h with animals exposed to

humid air conditions (with a cloth soaked with seawater) or immersed in aerated seawater, in

order to compare with the current industrial procedures. The remaining crabs were put in

rescaled polystyrene tanks with aerated seawater (35 ‰; 1kg crab/L seawater).

Several haemolymph parameters were periodically measured, including glucose, lactate,

haemocyanin, pH and vigour. After 48 h of simulated transport conditions one box with animals

exposed to air and crabs transported at 12 ºC in aerated seawater were immediately put in a

recirculated stocking tank with aerated and filtrated seawater at 16 ºC during 72 h recovery

period (treatments A and B). The remaining crabs subjected to emersed transport had and

acclimation period of 6 h, where they were put in tanks with aerated seawater at 6 ºC (treatment

D) and 11 ºC (treatment C). Water was renewed every hour and temperature was gradually

increased up to 16 ºC (approximately 1.5 º C increases per hour in animals at 6 ºC, and 1 ºC

increases per hour in animals at 11 ºC). After this acclimation period all crabs were put in the

recovery stocking tanks at 16 ºC during 72 h. Blood parameters of 10 animals maintained for

two weeks without feeding in the stocking tanks of the importer were analyzed and considered

as the control group.

Both emersed and immersed simulated transport systems promoted physiological stress

culminating in two dead crabs in the immersed system and only one in the air exposed one

(Figure 18). During recovery of animals not subjected to acclimation, one crab died in each

treatment (A and B), while most crabs were able to recover (Figure 18). The majority of animals

subjected to temperature acclimation in seawater were healthy (treatments C and D) with only

one animal dead in treatment D (Figure 18). During the recovery of crabs subjected to

acclimation only one animal died in each treatment (C and D) and one became moribund in

treatment C (Figure 18).

Aclimation for 6 h

Simulated emersed transport (48 h) at 4 ºC

Samples: 0, 8, 24 and 48 h Recovery stocking tanks

H2O=16 ºC during 72 h

(n=16)

H2O=11 ºC

H2O=6 ºCD (n=8)

B (n=8)

C (n=8)

Samples: 8, 24, 48 and 72 h

Samples: 0, 8, 24 and 48 h A (n=8)

Sample: 6 h

Directly to stocking tanks for recovery

H2O=16 ºC during 72 h (n=16)

Simulated immersed transport (48 h) at 12 ºC

Samples: 8, 24, 48 and 72 h

Figure 18. Vigour of crabs during experimental conditions. Abbreviations: T0 – T48, transport at

0, 8, 24 and 48 h; A6, acclimation after 6 h; R8 – R72, recovery after 8, 24, 48 and 72 h; AC,

Acclimatation.

Regarding haemolymph parameters, both transports elicited anaerobic responses marked by

hyperglycaemia and increased lactate concentration in haemolymph. Air exposed crabs during

simulated transport (treatments B, C and D) had similar concentration of lactate, glucose and

haemocyanin, while crabs immersed in seawater at 12 ºC showed lower lactate and glucose

concentration, but higher pH values after 48 h of simulated live transport (Figure 19).

Haemolymph acidosis occurred mainly in air exposed crabs and was most likely due to

respiratory difficulties (hypercapnia) than to lactate accumulation, as blood lactate values

remained high even after re-immersion. Lactate concentration never reached values similar to

those of control during simulated transport, while glucose concentration only resumed control

values in immersed crabs after 48 h. Values of pH were similar to control in immersed crabs

since 24 h. Haemocyanin concentration decreased in the last hours of transport with a negative

significant correlation with the duration of both transport systems and low values remained

during recovery.

At 4 ºC, which is the lower thermal limit for this species, the metabolic rate might be lowered

due to temperature induced dormancy. Therefore, crabs submitted to a sudden increase of 12

ºC (from 4 to 16 ºC, treatment B) of 7 ºC (from 4 to 11 º C, treatments C) would be expected to

suffer heat shock and show signs of stress. In contrast, crabs submitted to a 2 ºC increase

followed by a progressive increase to 16 ºC (treatment D) would be expected to show a minor

stress response and a quicker recovery. Such events were not observed and no major

differences were perceived between the different treatments after transport. In fact, during

acclimation no major differences were detected between treatments C and D for all

haemolymph parameters, and only pH was similar to the control.

Throughout the 72 h recovery in seawater at 16 ºC no major differences were found between

treatments in lactate, glucose and haemocyanin concentrations.

87.5 87.5 87.575.0 50.0

37.525.0

12.5 12.5

12.5 12.525.0 25.0 25.0

37.5 37.5

2512.5

T0 T8 T24 T48 R8 R24 R48 R72

12.5 12.5 12.5 12.5

87.5100.0

75.062.5

87.575.0

62.5 62.575.0

0.012.5 12.5 12.5

25.0 25.025.012.537.5

T0 T8 T24 T48 A6 R8 R24 R48 R72

Immersed transport 12 ºC

Recovery 16 ºC Aerial exposure 4 ºC

Recovery 16 ºC AC

100.0 100.087.5

75.0 75.0

37.5 37.5

12.5 12.5 12.525.0 25.0 25.0

25.037.537.5

T0 T8 T24 T48 R8 R24 R48 R72

12.525.0

25.012.5

100.0 100.0 100.087.5

75.037.5

12.5 12.5

T0 T8 T24 T48 A6 R8 R24 R48 R72

100.0 100.087.5

75.0 75.0

37.5 37.5

12.5 12.5 12.525.0 25.0 25.0

25.037.537.5

T0 T8 T24 T48 R8 R24 R48 R72

12.525.0

25.012.5

100.0 100.0 100.087.5

75.037.5

12.5 12.5

T0 T8 T24 T48 A6 R8 R24 R48 R72

Aerial exposure 4 ºC

Recovery 16 ºC Aerial exposure 4 ºC

Recovery 16 ºC AC

R8 R24 R48 R72

4 (very healthy) 3 (healthy) 2 (weak) 1 (moribund) 0 (dead)

Figure 19. Blood parameters (lactate, pH, glucose and hemocyanine) of crabs throughout the

experiment. Abbreviations: A (acclimation); C (control).

Control values were only reached after 72 h for lactate (treatments C and D) and pH (treatments

B and D). The lack of differences during recovery can be due to several reasons: (1)

haemolymph parameters were not sensitive to evaluate stress recovery; (2) too fast acclimation

and insufficient recovery time; (3) thermal tolerance limits of this species were not exceeded (4-

16 ºC; Chartois et al., 1994); or (4) recovery was more dependent on the similar magnitude of

the inflicted stress than on the recovery temperature amplitude per se.

Since in this study both transport systems caused a similar stress response, it can be argued

that recovery was also alike, taking into consideration that crabs had a similar initial vigour. This

suggests that C. pagurus can be put directly in stocking tanks to recover at 16 ºC (upper limit of

seawater temperature in natural conditions) without being acclimated, as long as seawater in

well aerated and filtered. However, such conclusion can be hasty and should not be regarded

as a generalization, especially considering that thermal tolerance of crustaceans is seasonally

dependent (Cuculescu et al., 1998; Hopkin et al., 2006). Moreover, crustaceans‟ thermal

tolerance temperatures are measured considering a progressive increase in temperature (0.2

ºC per minute) instead of sudden increases, which is rarely seen in nature, except in

anthropomorphic situations (Jiang et al., 2008).

Transport in humid air conditions is certainly the recommended practice considering that only

crabs under these conditions reached lactate and pH values similar to control at the end of the

recovery period, as well as this transport system promotes a strong reduction in economical and

ecological costs. Nonetheless, further studies are still required to evaluate the feasibility of this

transport system in industrial operating conditions.

0 15 30 45 60 75 90 105 120 135 150

0 8 24 48 6 8 24 48 72 C

Transport A Recovery

0 8 24 48 6 8 24 48 72 C

5. Norway Lobster (Nephrops norvegicus) – physiological changes related to stress

Authors VRC

5.1 Knowledge from literature

5.1.1 Live market Nephrops

The Norway lobster, Nephrops norvegicus is fished throughout its range in northern European

waters. Traditionally sold as either a frozen or fresh product, recent years have seen the

development of a high-value live market. Delivery of live Nephrops from source to sale is a

complex process including a particular catch handling protocol, use of holding facilities and

specialised transportation. Transport is either in tanks on refrigerated vivier-trucks or by air.

Vivier containers usually have 10 tanks of seawater which are filled at the animals‟ landing port

or holding facility. During the journey, which can take up to 72 hours, the seawater is aerated

but not renewed or filtered. Each tank contains a single species stocked at a maximum density

of 1kg per litre of water. Transport of live crustaceans is generally in cardboard or polystyrene

boxes (in refrigerated and moist conditions) with separation between individual animals. Small

refrigerated trucks are used to convey the animals between the airport and the holding facilities

(Barrento et al, 2008).

Stressors imposed on animals during the whole process are responsible for huge economic

losses due to high levels of mortality. These have been investigated by several authors (Spicer

et al. 1990, Uglow 1990, Schmitt and Uglow 1997b and 1998, Stentiford and Neil 2000,

Ridgway et al. 2006 a and b, Barrento et al. 2008, Albalat et al. 2009, Lund et al. 2009). Those

especially associated with a high mortality rate include physical damage such as compression

caused by trawling (Albalat et al. 2009, Lund et al. 2009, Milligan et emersion (Spicer et al.1990,

Ridgway et al.2006a, Schmitt and Uglow 1997a), hypoxia (Hagerman and Uglow 1985, Schmitt

and Uglow 1998), low salinity (Harris and Ulmstrand 2004), high ambient ammonia level (Hosie

et al. 1991, Schmitt and Uglow 1997b), high temperature (Ridgway 2007, Bernasconi and

Uglow 2008) and simply handling stress (Spicer et al. 1990, Schmitt and Uglow 1997a). The

effects of these conditions tend to be cumulative which is reflected in increased mortality rates

at holding facilities (Barrento et al, 2008). al. 2009),

5.1.2 Emersion

During commercial handling of Nephrops, animals are exposed to air a number of times.

However, this species is fully aquatic and thus not adapted for emersion. Consequently, during

the times when the animal is not in water, gill function is impaired resulting in reduced oxygen

uptake and the inability to excrete or metabolise toxic waste products such as ammonia and

lactate. Further, exposure may lead to a collapse of some gill lamellae (Danford et al, 2001)

which impedes the correct functioning of the organs upon re-immersion. Inefficient gas

exchange by the gills in air is a primary cause of stress (Taylor et al. 1997). During emersion

lobsters double their ventilation rate. However their oxygen uptake is approximately halved

relative to that in water (Taylor and Waldron, 1997). This causes their metabolism to switch to

the use of anaerobic pathways in order to maintain function which in turn leads to the elevation

of internal lactate levels and metabolic acidosis. Further, ammonia is the main nitrogenous

excretory product of lobsters, and failure of ammonia excretion by the gills in air leads to

elevated blood ammonia. This is, believed to be toxic to lobsters (Taylor et al. 1997).

Emersion also impacts severely on animals‟ immune function as exemplified by low haemocyte

counts, increased clotting times and high bacteraemia levels (Fotedar, 2001, Ridgway et al,

2006 a). This indicates increased susceptibility to infection and can impact on the survivability of

animals at the holding stations. Ambient temperature during emersion is a very important factor.

Ridgway et al. (2006 a) investigated the effects of aerial exposure at three different

temperatures and found that mortality rate increased concurrently with temperature. By

comparison, Spicer et al. (1990) found that the survival of lobsters held on ice was significantly

greater than those at an ambient air temperature of 10oC.

Currently, a variety of procedures are used onboard Nephrops fishing boats although these can

be broadly classified into two categories: aerial storage and seawater tank storage. Animals

stored in air are protected from desiccation by covering with an absorbent material (frequently

old pieces of carpet or hessian sacks) which is regularly drenched with seawater and heavily

iced to keep their temperature as low as possible. Schmitt and Uglow (1997a) investigated this

feature. They subjected Nephrops to 8 hours of emersion, either between layers of seawater-

soaked hessian (sprayed with seawater every 20 minutes) or to unprotected emersion. The

ability of Nephrops to cope with emersion appears to be little influenced with high humidity

conditions probably because of gill collapse (Schmitt and Uglow 1997a). Air transport practices

usually see the brief application of low temperatures to anaesthetize animals and to reduce their

metabolic rate.

Other than this, there is a paucity of data in the literature referring to Nephrops transported by

air. But the effect of such transport on Nephrops can be predicted from the data we have on

emersion. Danford et.al. (2001) investigated long-haul international transport on the American

lobster, Homarus Americanus, and transported the lobsters from Halifax to Hull, a journey

lasting for almost 50 hours. Journey temperature varied between 1,5 and 2,0°C. The study

involved groups (n = 10) of freshly caught and 127 days stored lobster. All lobsters survived the

period of aerial exposure. However, three of the stored lobsters died within 4 hours of re-

immersion. Hemolymph acidosis (-0,5 pH units) developed during the consignment, and

hemolymph levels of metabolic end products (ammonia, lactate and urate) increased. Blood

protein levels showed a progressive decrease during consignment an recovery and had the

lowest concentration in those lobsters that died (Danford et.al. 2001).

5.1.3 Low salinity

Nephrops is a marine stenohaline which means that it is physiologically incapable of tolerating

large fluctuations in salinity; across their distribution, animals are found within the salinity range

29-34‰. Nephrops lose blood electrolytes and gain water rapidly by osmosis when transferred

to low-saline water. Body mass increases have been reported in Nephrops when transferred to

low salinities suggesting a large and rapid uptake of water (Harris and Ulmstrand 2004).

Experimentally, the lowest salinity which animals can compensate for has been determined as

approximately 28‰ (Harris and Ulmstrand 2004).. Below this, water uptake causes swelling and

the rupturing of organs. Consequently, if vivier tanks are filled with low saline water, animals will

be exposed to these adverse conditions for prolonged periods leading to high mortality rates

(Kristensen and Lund 2008). In addition, Chen and Lin (1991) found that the lethal

concentration of ammonia for shrimps was 80% lower when at salinity levels of 25‰ that at

34‰. This indicates that low salinity compounds the toxic effects of ammonia which is important

in the transport procedure where ammonia levels can build up in the tanks.

5.1.4 Ammonia

Aquatic crustaceans are ammoniotellic which means that they excrete their nitrogenous waste

products in the form of soluble ammonia (Freire et al, 2008). Rate and volume of ammonia

production and excretion is influenced by several factors including nutritional status, moult

stage, activity level, salinity and temperature (Schmitt and Uglow 1997b). The vast amount of it

is removed from the organism via the gills by passive diffusion so efficient expulsion depends

on a low external concentration. When large numbers of Nephrops are kept in the same mass

of seawater the levels of ammonia can increase. When the external ammonia levels rise, the

animal cannot maintain their ammonia gradient causing it to build up in the blood, often

reaching a toxic level. This can also occur during periods of emersion as gill function is severaly

impaired (Schmitt and Uglow 1997a, Danford et al.2001). Specific toxic effects of ammonia on

the internal system of crustaceans are related to pH changes affecting enzyme activity (ref).

Animals transported in poorly designed vivier tank systems in which water quality control is

absent may be exposed to accumulating ammonia. Uglow (1990) found ammonia levels of 2000

µmol per litre in some tanks with a simultaneous increase in haemolymph levels of animals held

there. Nevertheless, Schmitt and Uglow (1997b) observed that animals held in ammonia-

enriched environments for period of nine hours appeared to implement some regulatory

mechanisms to prevent ammonia build up within the haemolymph since internal levels never

reached that of the external conditions. Upon return to normal seawater, a rapid recovery to pre-

exposure levels was observed.

5.1.5 Holding duration

During the process chain, lobsters may be stored for various lengths of time in special holding

facilities. This can take place either prior to transport (known as „conditioning‟ a period that

allows animals to acclimate to storage) or when they arrive at their destination (where they may

await favourable market conditions). This imposes certan stresses on the animals which affect

mortality rates. For example, whilst storage temperature within the range of 5-10°C does not

appear to affect survival, sudden increases in temperature have been found to be detrimental

although decreases were not (Kristensen and Lund, 2008). The condition in which animals

arrive at holding facilities is critical, in particular, damage to the integrity of the cuticle as

experienced during trawling or as a result of fighting.

A large and rapid stress leading to death may be experienced by animals during re-immersion

following transport especially in animals which had been previously stored for prolonged

periods. Emersion causes mobilization of glucose reserves as a response to crustacean

hyperglycaemic hormone (CHH) which is released at this time. In animals which have

experienced extensive starvation, such reserves may already be depleted and thus efficient

metabolism may be impaired (Danford et al. 2001). This is exemplified in work by Kristensen

and Lund (2008) who found that lobsters stored for 1 month in vivier tanks survived „dry‟ storage

better than those stored for 2 months. In addition, the immune function of animals experiencing

prolonged storage may be impaired. Jussila et al. (2001) and Fotedar et al. (2006) observed

fluctuations in the amount and type of haemocytes, and the clotting time of haemolymph.

5.1.6 Lactate

When insufficient oxygen reaches respiring cells, animal metabolism usually switches to using

anaerobic pathways in order to continue function. This results in an inefficient consumption of

reserves compared to aerobic mechanisms, and the production of waste products including

lactic acid or lactate. Through the processing chain, there are several instances in which

Nephrops may be caused to respire anaerobically. In some cases lactate levels can be high

enough to disrupt the acid-base status of the lobster and force it into respiratory acidosis.

Capture of lobsters for the live market is still predominantly undertaken using traps rather than

trawls. Creel-caught animals experience lower overall stress (as measured by CHH

concentration) and in particular, lower concentrations of haemolymph lactate compared to trawl-

caught animals (Ridgway et al, 2006 b). Further, Bernasconi and Uglow (2006) found that creel-

caught animals recovered from acid-stress faster than trawled Nephrops. Nevertheless, Lund et

al. (2009) showed that these animals have a powerful capacity to recover from high lactate

levels if provided with good storage conditions, although this is dependent to some extent on

season.

In instances where animals do not have suitable facilities in which to recover, for example,

during prolonged emersion (Bernasconi and Uglow, 2008), long trawls (Ridgway et al, 2006 b),

or storage in low oxygen conditions (Schmitt and Uglow 1998), the production of lactic acid can

have toxic effects. These include loss of vigour (Harris and Andrews 2005) and in severe cases,

localized or extensive cell death (Ridgway et al, 2006 a). Thus, it is vital that the recovery of

acid-stressed animals be facilitated by the provision of a suitably oxygen-rich environment.

5.1.7 Hypoxia

Nephrops are a burrowing species which inhabit soft, muddy clay, an environment which often

experiences low oxygen saturations to which the animals show some adaptation. For example,

in the Kattegat and Skagerrak region Nephrops may be subjected to strong hypoxia for several

weeks due to eutrophication in the area (Hosie et al, 1991). This adaptation is important since

onboard vivier trucks Nephrops may also experience hypoxia since the vehicles are not always

provided with aeration systems or those that are may be inadequate to supply all the animals

being tranported (Schmitt and Uglow, 1998). Stress from hypoxia induces parallel physiological

and behavioural changes including an increase in haemolymph haemocyanin concentration

(Baden et al, 1990) and reduced activity. It is known that Nephrops catabolise haemocyanin

during periods of prolonged starvation (Hagerman and Uglow 1985) so animals which had

previously been stored in holding facilities may be less able to compensate for hypoxia.

5.1.8 Crustacean Hyperglycaemic Hormone (CHH)

Crustacean Hyperglycaemic Hormone (CHH) is responsible for regulating the concentration of

glucose in the haemolymph (Lorenzon et al, 2005). It is considered an extremely sensitive

measure of general stress in crustaceans and has been used in many research areas to

quantify the effects of exposure to various stressors. Those which have been shown to promote

release include acute hypoxia, elevated temperature and altered salinity (Chang et al, 2005),

disease and pollutants (Lorenzon 1997), emersion (Paterson and Spanoghe 1997), and trawling

(Ridgway et al, 2006b).

5.1.9 Idiopathic Muscle Necrosis (IMN)

IMN in lobsters is a muscle condition which was first detected in animals destined for export as

a live product. Gross symptoms include a progressive loss of vigour, the appearance of

characterisitc white lesions which are apparent through the thin ventral membrane and

ultimately death. At a microscopic level, the muscle tissue has a loss of z-line material,

condensation of myofibrils and infiltration of necrotic regions by granulocytes (Stentiford and

Neil 2000). The cause of this condition has not been fully determined to date, however, it is

considered likely to be a result of combined stressors in addition to damage to the integument

integrity. Due to the inevitable fatality of this condition, it is extremely important to implement

good handling practices throughout the processing chain to minimize occurrence. The

progression of the condition is rapid once initiated and so current export practices are to screen

for early clinical symptoms prior to transportation or during storage in order to minimize losses.

Following appearance of the first necrotic lesions, death usually occurs within a few days

(Ridgway et al, 2007).

5.1.10 Summary

Throughout the supply chain there are several points during which lobsters are exposed to

stressors. In particular, times of emersion are seen to be critical although fluctuation in

temperature and the quality of water in holding stations is also very important. The effects of

these stressors on product quality ranges from a reduced lobster vigour through to increased

mortality rates of the shipment, which in turn affects the value of the consignment considerably.

It is therefore essential that care be taken to understand these critical points and to reduce the

extent and duration of applied stress.

5.2 Experiments on storage and transport by air

A series of experiments were carried out to investigate the physiological changes experienced

by Nephrops destined for the live market. The aim was to determine tolerance limits of storage

in the holding facility, emersion during shipping and capacity for recovery at point of sale. This

would enable any further critical points in the supply chain to be indentified and for the process

to be optimised such that maximum product quality is achieved.

5.2.1 Storage

In the live Nephrops industry, animals are stored in a holding facility for periods ranging from

three days through to three weeks prior to dispatch. This allows for recovery from the stress of

fishing and boat-to-shore transport as well as acclimation to holding facilities. Two different

types of system are employed in the industry, flow-through and closed. Flow-through systems

make use of water pumped directly from the sea; it is passed through the storage tanks and

expelled at the other end. It may be roughly filtered and aerated but is generally not

temperature-controlled, i.e. the intake temperature is determined by the sea temperature. In a

closed system, the same body of water is circulated between the holding tanks and reservoir.

Water quality is maintained by the use of powerful pumps that force water through filtration,

sterilising and aerating modules. An important component of the system is a chilling unit which

removes the excess heat introduced by the pumps and which can be used to control the tank

temperature.

This study followed the journey of a Nephrops

catch from the trap right through the storage

process. The traps were deployed in fleets of fifty

and set for a period of 48 hours. On board the

boat, the animals were placed in crates or „tubes‟

(Figure 20) and held in static water tanks for the

duration of the trip (a maximum of 8 hours). Upon

arrival to the holding station, the catch was

randomly allocated to one of the two systems,

flow-through or closed. The water temperature of

the flow-through system was approximately 8oC

whilst that of the closed system was 5oC as is

current practice. Samples were taken on board

the boat, upon arrival at the holding facility eight

hours later, following twenty four hours, forty eight

hours, one week and three weeks of holding. Lobsters were weighed whole before a volume of

3ml haemolymph was withdrawn. The hepatopancreas was removed and weighed then divided

in two with half being fixed for histological assessment and the remaining half frozen for

biochemical analysis. Sections of tail muscle were stored cryogenically for analysis of

nucleotide composition and protein content.

Results of biochemical and nucleotide analysis indicated that Nephrops recover quickly from the

stresses imposed by fishing and transporting to the holding station. In most of the animals

measured prior to entering the system, the lactate level circulating in the haemolymph was

below the detection concentration of 0.8 mmoll-1

. In some animals, an increased concentration

was observed; this was considered a result of vigorous tail-flipping activity following exposure to

air whilst being moved from the boat to the station. After 24 hours of storage, this had returned

to negligible levels in animals from both systems (Figure 21a). A small accumulation of

ammonia in the haemolymph was detected in animals between point of capture and arrival at

the holding station (Figure 21b). However, ammonia concentration of the water in which the

Figure 20. Prawns are placed vertically in compartmentalised crates for transport and storage.

animals had been held for the duration of the trip was found to be insignificant indicating that

ammonia was built up in the localised region of the tubes. Further, the increase observed was

below the threshold which can be detrimental to animals‟ health and had disappeared within 24

hours of storage.

A small difference was observed between systems in the rate of recovery from fishing stress. In

the flow-through system, removal of circulating ammonia was faster over a 48 hour period.

Analysis of the nucleotide composition showed that those animals also regained an AEC of 0.8

within 24 hours whilst the animals in the closed system took 48 hours to attain this level (Figure

22). It is possible that these responses are a result of the difference in temperature of the two

systems as the water entering the flow-through tanks was on average 8°C compared to the 5°C

of the closed circulation aquaria. Animals at the slightly higher temperature may be metabolising

a little faster which could enable them to expel breakdown products and mobilise energy

reserves. However, in these instances the difference in temperature is very small and

accordingly the difference in recovery rate is also low. It is unlikely to have a measureable effect

on the quality of the live lobster product.

Sea Rec +24Hr +48Hr

Closed

Sea Rec +24HR +48HR

Closed

Figures 21a&b. Concentration of haemolymph metabolites lactate an ammonia in animals at point of capture, arrival to the reception, twenty four and forty eight hours of storage.

Over the duration of the study, the relative weight of the hepatopancreas decreased

considerably. The hepatosomatic index (HSI), an indicator of energy reserves, was calculated

as the weight of hepatopancreas divided by total body weight. Over the first 48 hours, no

significant decrease was observed after which time it decreased by a total of 35% (Figure 23).

Histological examination revealed that after three weeks of storage, the R-cells of the

hepatopancreas tubules (which have a storage function) had shrunk causing an expansion in

the size of the tubule lumens (Figure 24). This indicated that the lipid reserves of the

hepatopancreas had begun to be mobilised. There was no significant difference in the rate of

HSI decrease between the two systems despite the slight difference in holding temperature.

Figure 22. AEC measured at point of capture, arrival to reception and selected storage times (5°C and 8°C).

Sea Rec 24 h 48 h 1 week 3 weeks

Figure 23. Hepatosomatic Index measured at selected storage times.

6HR 24HR 48HR 1WK 3WK

Closed

This confirms that the small difference in holding temperature has no perceivable effect on use

of energy reserves by stored lobsters.

The consumption of reserves was not only found to affect the hepatopancreas. The

concentration of haemocyanin in the haemolymph animals in both storage systems was found

to decrease over storage time (Figure 25a). However, this reduction was only found to be

significant in animals stored in the flow through system (F = 3.292, p < 0.05). The effects of

storage on muscle protein were negligible (Figure 25b) in either system indicating that the

quality of the ultimate meat product would not be reduced.

Figure 24. Narrow lumen to tubule at start of storage and expanded lumen due to shrinkage of R cells after 3 weeks storage (arrows point to lumen).

0Hr 24Hr 48Hr 1Wk 3Wk

Closed

0HR 24HR 48HR 1WK 3WK

Closed

Figures 25a&b. Haemolymph haemocyanin concentration and muscle protein content measured at selected storage times.

The difference in recovery from fishing stress and mobilisation of energy reserves between the

two storage systems was found to be small. However, an important distinction in the health

status of stored animals was observed. The number of circulating haemocytes was measured at

point of capture, arrival to the holding station, and following twenty four hours, forty eight hours,

one week and three weeks of storage (Figure 26). Animals arriving at the holding station had

increased the number of haemocytes relative to that of freshly-caught lobsters. When they were

stored in the closed system, this figure returned to original levels within forty eight hours.

However, in the flow-through system the number remained high indicating that the immune

system was actively engaged throughout the duration of storage.

Histological examination revealed further very important differences in the hepatopancreatic

condition of the animals. After three weeks of storage, those held in the flow-through system all

had melanised tubules and bodies located throughout the organ (Figure 27). In some animals,

it could be seen by gross visual examination that entire lobes had hardened. By comparison,

those held in the closed system maintained healthy organs with no evidence of melanosis.

Such granulomas are formed by the aggregation of haemocytes when the immune system

attempts to isolate substances perceived as foreign by which are not possible to eliminate from

the body. In the following figure, haemocytes can be seen swarming to the infection site. Typical

examples instigating such a response include infectious agents such as bacteria and fungi. In

crustaceans and other arthropods, melanising such granulomas is brought about by the

prophenoloxidase activating cascade which leads to the synthesis and deposition of melanin.

This step is intimately involved in the stimulation of cellular defence by aiding phagocytosis and

encapsulation reactions.

The presence of these melanised granuloma indicates the presence of a pathogenic agent.

Seawater is known to contain bacteria, in particular many Vibrio species which could account

for the observations. This could also explain the fact that no melanised nodules were recorded

from animals stored in the closed system; all water circulating there is treated with UV light to kill

bacteria. Total cell counts were carried out on water samples taken from both systems

throughout the experiment.

Figure 26. Total haemocyte count was measured at point of capture and selected storage times.

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

0HR 6HR 24HR 48HR 1WK 3WK

Closed

These did not reveal significantly higher levels of bacteria in the flow through than closed

system; whilst those in the closed system were zero, in the flow through were very low.

However, sustained challenging by even low levels of bacteria could lead to the formation of

such nodules. This theory is supported by the fact the nodules were only detected at the end of

the experiment suggesting low infection rates and slow progression of disease.

Summary

Using traps to catch Nephrops means that animals are unstressed at point of capture. Careful

handling on board the boat and the use of appropriate water tanks so that animals are emersed

for the minimum amount of time post-capture ensures that they arrive to the storage facility in a

good condition. Any small increases in the concentration of circulating metabolites and reduction

of AEC is resolved within forty eight hours irrespective of storage system. Extended storage

(three weeks) has the effect of mobilising energy reserves as seen by a decrease in relative

hepatopancreas size and haemocyanin concentration. However, the protein content of muscle is

not reduced indicating no loss of quality.

The type of storage system in which animals are held, particularly when stored for prolonged

periods, impacts considerably on their health. Those in the flow-through system exhibited

evidence of disease by the end of the experiment. In addition to this, they maintained high levels

of circulating haemocytes throughout indicating that their immune systems were constantly

engaged. These conditions present a significant source of stress on stored animals. This is very

likely to affect the survival of lobsters both during transport to the market or customer, and during

recovery at the destination holding facility.

5.2.2 Transport by Air

The movement of live lobsters from the storage location to the market destination can be

undertaken by vivier vehicle or by air. In the first, lobsters are carried in specially designed tanks

which circulate filtered seawater ensuring that animals can both respire and excrete

metabolites. In the latter means, lobsters are packed into polystyrene boxes into which the

separators are fitted to keep individuals apart. Frozen gel packs are placed into the boxes to

reduce the temperature and a damp cloth used as a liner to maintain humid conditions within.

Thus, animals are emersed for the duration of their journey after which time they may be sold

for immediate consumption or for further storage. In Iceland, the option for the export of live

animals is the use of air transport so our experiments focused on this.

Figure 27a&b. Melanised tubule (a) and granulomas surrounded by swarming haemocytes (b).

Lobsters were caught using baited traps (creels) and stored for a period of three weeks in one

of two systems. The first was a closed system where the water was recycled between the

holding tanks and a reservoir. The quality maintenance of circulating water involved a protein

skimmer, sand filtration, UV sterilisation and cooling to 5oC. The second system was a flow-

through design where the intake was supplied by a bore in the harbour. Clean water was

pumped into a tower which was aerated and allowed to pass through the tanks at a rate fast

enough to flush out detritus (determined visually). The water was not additionally cooled and so

entered the tanks at the ambient sea temperature from the bore, 8oC. After the designated

storage time, the industry established protocol was followed for packing the animals into boxes

in order to simulate transport to market. At times of 24, 36, 48 and 72 hours of emersion,

samples were taken for analysis.

A volume of 3ml haemolymph was withdrawn from each animal and assessed for haemocyte

number, haemocyanin concentration, pH, lactate and ammonia. The vigour of each animal was

recorded according to the industry-standard index (Table 6).

Vigour Index Abdomen Claws Mouthparts &

Antennae

1. Vigorous

Rapid tail-flipping sustained

for at least 60 flips

Raised in defensive position

above plane of body

Strong

2. Strong

Rapid tail-flipping

Raised to plane of body; may

be lowered quickly

Strong

3. Weak

One-two tail-flips only

Unable to raise claws to plane

of body

Strong

4. Moribund

No tail-flip response

Slack, unresponsive claws

Slow movement of

scaphognathites and

antennae

5. Dead No movement No movement No movement

The tail muscle was investigated for evidence of idiopathic muscle necrosis and the muscle

stored cryogenically for nucleotide analysis. Results were examined to determine both the effect

of prolonged emersion and whether the prior storage facilities caused a difference in quality

(vigour and survival).

It was found that during the first 24 hours of emersion, animals from both systems remained

vigorous, although the proportion at peak vigour was lower in the group from the flow through

system was lower than the closed system group. By 36 hours, many more from the flow-through

group were moribund than the closed system which had increased the proportion assessed as

simply „poor‟. By 48 hours, however, the two groups were equal in that approximately one third

of animals had died and one third was moribund. By 72 hours almost all of the lobsters had

died, irrespective of the system from which they had come.

Table 6. Industry standard vigour index

Emersion stress has a significant impact on haemolymph chemistry; acidosis from a build up of

lactic acid and CO2 has been found to cause a reduction in pH in Homarus during aerial

exposure. In our experiment, the pH decreased in both groups of lobsters (Figure 29a). The rate

at which this occurred was faster in animals from the flow-through system during the first 36

hours. By 48 hours, the pH measurements were equal indicating that perhaps a lower limit had

been reached. At this stage many animals from both groups had become moribund. Lactic acid

levels increased with emersion time, being higher and more variable in animals from the flow-

through tanks (Figure 29b).

The effect of emersion stress on the two groups of lobsters was particularly interesting. It was

found that animals from the closed system increased the number of circulating haemocytes

during the first 24 hours of aerial exposure. After this they decreased progressively during the

second 24 hour period. By comparison, animals from the flow-through system began the

experiment with a high number of circulating haemocytes. This stayed high for the first 36 hours

of emersion then they decreased significantly at the 48 hour emersion point (Figure 30).

Figure 28. Lobster vigour at selected periods of emersed transport time.

0HR 24HR 36HR 48HR 72HR

Flow-through

System

Vigorous

Strong

Moribund

0HR 24HR 36HR 48HR 72HR

Closed System

Vigorous

Strong

Moribund

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

45000000

Control 0Hr +24Hr +36Hr +48Hr

Closed

Figure 29a&b. Lobster haemolymph pH and lactate concentration at selected times of emersed transport.

Control 0Hrs +24Hrs +36Hrs +48Hrs

pH Closed

Flow-through

24Hr Cl 24 Hr Fl 36hr Cl 36Hr Fl 48Hr Cl 48Hr Fl

Figure 30. Total haemocyte count measured at selected periods of emersion.

In the earlier experiment on storage, it was found that lobsters in the flow-through system were

chronically stressed by exposure to low numbers of passing bacteria which resulted in the

development of granulomas in the hepatopancreas. Clearly, this condition impacts on the

animals‟ capacity to respond to emersion stress. By comparison, lobsters from the closed

system began the emersion period in the same condition as though they had just been captured

and mounted a normal immune response. Nevertheless, 48 hours of aerial exposure is the limit

for Nephrops survival and by that time both groups showed a reduction in circulating

haemocytes.

The haemocyanin concentration of the two groups of lobsters was also found to be considerably

different throughout the experiment. Based on work by Baden et al (2003), it was predicted that

animals would exhibit a progressive reduction in haemocyanin concentration as a result of

extreme hypoxia. We found that animals from the closed system followed the expected model.

However, lobsters from the flow-through system showed a rapid and dramatic decrease in the

amount of haemocyanin in the haemolymph within 24 hours. This figure stayed low throughout

the experiment and the quantities measured in animals from the closed system matched it by 48

hours (Figure 31). We suggest that prolonged exposure to low quantities of bacteria in the flow-

through system has caused systemic disease development which has compromised the ability

the organs to function normally.

The final analysis performed on the lobsters was the AEC which is a measure of available

metabolic energy for the animal stored in the nucleotide adenine pool. It is a particularly useful

parameter in the measure of stress since animals often show quantifiable effects in AEC before

other features. In the experiment, animals from the closed system showed a rapid and

progressive decrease in AEC which reached a lower limit of 0.25 after 48 hours of emersion. By

comparison, animals from the closed system maintained a high AEC of 0.9 for the first 24 hours

of aerial exposure. By 36 hours of emersion, the AEC began to drop and continued to fall until

also reaching a low figure, in this instance 0.20 (Figure 32). The different response from the two

groups of animals supports the theory that animals from the flow-through system are

compromised and less able to cope with the stress of emersion

Control 24Hr 36Hr 48Hr

Closed

Fig 31. Haemocyanin concentration at selected points during emersion.

Summary

It was found that during the first 24 hours of emersion lobsters from both groups remain

vigorous. However, during the next 24 hours, there is a considerable increase first in the

number which measure „poor‟ and then in the number of „moribund‟ animals. This progression is

more rapid in animals which had previously been stored in the flow-through system than those

from the closed system. Investigation of various health and stress parameters produced results

which indicate that animals from the flow-through system are less able to cope with emersion as

experienced during transport from the storage facility to the market. This supports the earlier

findings from the storage experiment that these animals are compromised by chronic exposure

to bacteria. Thus, in order to ensure a high quality product in terms of low levels of mortality and

prolonged vigour, animals need to be stored in a closed system prior to transport and emersion

should last a maximum of 24 hours.

5.2.3 Recovery at destination

Lobsters which are exported live are destined for either immediate consumption or further live

storage in the destination country. Typically, those aimed at the further storage market are

transported by vivier container whilst those for immediate consumption are transported by air.

The only transport option available to producers in Iceland is air freight and so we wanted to

investigate the capacity for animals to recover following different periods of emersion. This

would enable the transport time to be optimised such that the further storage market could be

accessed too.

Lobsters were caught by creels and stored in a closed circulatory system for a period of three

weeks. They were then packed as though for aerial transport following industry protocols and

stored in a cool room for a period of 12, 24 or 36 hours. After this time the lobsters were re-

introduced to holding tanks; the flow-through system was chosen for this as it most closely

reflected the type of systems used by the customers in Spain where the majority of live lobsters

go. At times 0, 12, 24, 48, and 72 hours after re-immersion, groups of lobsters were removed

and samples taken for analysis.

One of the initial findings was that the emersion time had a serious effect on survival. Table 7

shows the cumulative mortality in the periods following re-immersion. It can be seen that

0 24 36 48

Closed

Fig 32. AEC of lobsters from either the closed or flow-through system measured at selected hours of emersion.

Table 7. Cumulative mortality (%) in lobsters emersed for periods of 12, 24 or 36 hours.

lobsters emersed for only 12 hours experienced no deaths during the 72 hours after, however

animals emersed for both 24 and 36 hours died despite re-introduction to water.

It was found that during emersion, lactate built up in the haemolymph as animals switched to

anaerobic metabolism. In both the groups emersed for 24 and 36 hours, the average

concentration was approximately 5mmoll-1

whilst the group emersed for only 12 hours was

much lower, at 2mmoll-1

. Within 12 hours of re-immersion, all groups showed a significant

decrease with that of the 12 hour emersion group returning to pre-emersion levels. This took

longer in the 24 and 36 hour emersion group, but nevertheless within 24 hours their

haemolymph lactate concentration was no different to the control group which had not been

emersed at all (Figure 33.)

Haemolymph chemistry also varied between groups with respect to haemocyanin concentration

(Figures 34-36). In the group emersed for 12 hours, the concentration did not change

significantly throughout the experiment. In the group emersed for 24 hours, the concentration

began to increase within 24 hours and continued to do so for the duration of the study. The

group emersed for 36 hours initially showed an increase after 24 hours of emersion, however,

by 48 hours this had begun to decrease again. This is because by this stage animals had begun

to die as a consequence of prolonged aerial exposure. Nucleotide analysis revealed that in

animals emersed for 36 hours, the AEC dropped as low as 0.3 whilst those in air for 12 hours

only went as low as 0.8 (Figure 37). All recovered within 24 hours of re-immersion.

Emersion time

(Hours)

Re-immersion time (Hours)

0Hrs 12Hrs 24Hrs 48Hrs 72Hrs

12 0 0 0 0 0

24 0 2 3 3 5

36 5 8 12 12 17

Control 0 12 24 48 72

12 Hrs Em

24 Hrs Em

36 Hrs Em

Figure 33. Haemolymph lactate concentration at selected times following re-immersion.

tress indicators direct impact on sensory food quality

Figures 34-37. Haemocyanin concentration and AEC at selected times following re-immersion from lobsters emersed for 12, 24 or 36 hours.

Control 00 12 24 48 72

12 Hrs

Control 00 12 24 48 72

24 Hrs

Control 00 12 24 48 72

36 Hrs

Control Emersion 12Hr Re 24Hr Re 48Hr Re 72Hr Re

0 12 24 48 72

Hours re-immersion

12Hr Emersion

0 12 24 48 72

Hours re-immersion

24Hr Emersion

0 12 24 48 72

Hours re-immersion

36Hr Emersion

6. Brown crab - sensory, microbiological and nutritional quality

Cancer pagurus is much appreciated in Southern European countries where crabs are

consumed all year round, but particularly in Summer months and in December (Barrento et al.,

2008). The muscle, hepatopancreas and gonads are consumed separately or as a mixture.

Also, larger males are usually more expensive than smaller males and females. Considering

consumers‟ habits, INRB I.P./L-IPIMAR tested the impact of edible crab wet transportation on

food quality, particularly the organoleptical, microbiological and nutritional characteristics. In this

way, different parameters were considered, such as animal condition, season, sex, edible

tissue, transport duration and use of anaesthetic.

6.1 Sensory quality

Authors INRB I.P./L-IPIMAR: António Marques, Sara Barrento, Patrícia Anacleto, Bárbara

Teixeira, Maria Leonor Nunes

Sensory quality analyses were carried out with an expert panel composed of 10 persons. For

each parameter edible crabs were evaluated live (vitality, aspect and odour) and boiled (odour,

aspect, texture and taste) (n=24).

Animal condition (or vitality index; VI) is a critical food quality parameter for trade of live edible

crab. Results in Figure 38 indicate that 57 % weak (VI= 3), 60 % moribund (VI=4) and 100 %

dead crabs (VI=5) were rejected by the panel, while only 17 % reactive (VI=1) and 0 % strong

(VI=2) animals were rejected (Figure 38). Additionally, the aspect of animals is also a very

important food quality parameter for live edible crab (lack of appendages, fractures, presence of

epibionts).

Figure 38. Correlation between the organoleptical scores of live edible crabs and their vitality

index.

As to boiled animals, the most important food quality parameters were the overall animal

aspect, and meat taste and odour. Interestingly, no correlation was found between the animal

Sensorial index

y = 0,6694x + 0,8021

R2 = 0,5009

1 2 3 4 5

Vitality index

Reject

Accept

vitality/aspect and its meat taste/odour, thus meaning that meat of animals in poor condition or

with bad aspect but showing good meat yield can be commercialized.

Generally, season plays an important role in edible crab‟s metabolism. In fact, the edible crab‟s

meat content deeply varies with season, being generally higher in Autumn and lower in Winter

(Table 8; Barrento et al., 2009c). This issue certainly affects crab resistance to stressful

conditions, as well as the edible crab‟s food quality, since consumers dislike animals with low

meat content.

Table 8. Cancer pagurus average meat content percentage of female and male crabs sampled during Spring, Summer, Autumn and Winter (adapted from Barrento et al., 2009c).

Season Sex Meat content (%)

Spring F 21,6

M 25,6 Summer F 22,5

M 23,5 Autumn F 26,7

M 26,1 Winter

F 17,6

M 21,2

The influence of animal sex to the edible crab‟s condition was also taken into account after wet

transportation at 16, 12 and 8 ºC. Results in Figure 39 indicate that in general male crabs had

higher mortalities than females, particularly at lower temperatures, thus meaning that males can

withstand fewer temperature fluctuations than females during wet transportation.

Figure 39. Results obtained for survival of edible crab after 48h wet transportation at 16, 12 and

8 ºC.

Experiments were also carried out to evaluate the effect of the anaesthetic Aqui-S to the edible

crab‟s condition. Results presented in Figure 39 indicate two patterns: a) less crab‟s mortalities

when transported with Aqui-S at 12 ºC and 8 ºC (males); b) higher crab‟s mortalities when

transported with Aqui-S at 16 ºC (males) and 8 ºC (females). These results indicate that male

Aqui-S

Males Females Males Females Males Females

16ºC 16ºC 12ºC 12ºC 8ºC 8ºC

crabs have higher metabolism than females, and therefore at 8 ºC the metabolism decrease to

critical levels in females, while at 16 ºC Aqui-S probably becomes toxic to male crabs.

Figure 40. Sensorial characteristics of muscle and hepatopancreas of boiled edible crabs transported in water with and without Aqui-S. The edible tissues (muscle and hepatopancreas) of boiled crabs transported in water with Aqui-

S had generally worst organoleptical characteristics than animals transported in water without

Aqui-S, mostly due to changes in taste and odour (Figure 40). Generally, muscle of crabs from

both treatments showed better organoleptical quality than hepatopancreas, since

hepatopancreas spoils faster than muscle, likely due to the higher fat content characteristic of

this tissue.

The organoleptical characteristics of muscle and hepatopancreas of processed/boiled edible

crabs from seven origins available at retailers were investigated, namely: 1 – Live crabs

immediately cooked and analysed; 2 – Overnight dead crabs cooked and analysed; 3 – Chilled

crabs processed in Portugal; 4 – Chilled crabs processed in Ireland; 5 – Frozen crabs

processed in Portugal; 6 – Frozen crabs processed in Ireland; and 7 – Frozen crabs processed

in Scotland.

Results in Figure 41 indicate that origin plays an important role in edible crab quality, particularly

in chilled products, as products processed in Portugal generally had better organoleptical quality

than products processed abroad, likely due to logistic constraints. Yet, as far as frozen

processed edible crabs are concerned, apparently the best quality was obtained crabs

processed in Ireland compared to Portugal. Therefore, processing edible crab soon after

capture seems the best strategy to improve food quality. Nonetheless, the quality of crabs is

certainly very important, as the worst results were obtained with frozen crabs processed in

Scotland (producer country).

Regarding edible tissues, processed hepatopancreas showed higher rejection by the expert

panel than muscles of the same animals (Figures 40 and 41), since hepatopancreas spoils

faster than muscle, likely due to the higher fat content characteristic of this tissue.

Excellent

Medium

Rejected

Figure 41. Sensorial characteristics of muscle and hepatopancreas of boiled edible crabs from

different origins.

6.2 Microbiological quality

Authors INRB I.P./L-IPIMAR: Sónia Pedro, Sara Costa, Sara Barrento, Bárbara Teixeira,

António Marques, Maria Leonor Nunes

The influence of wet transportation on microbiological quality of edible crab was also analysed

taking into account several aspects: tissue, sex, animal condition, origin/transport duration.

Samples were collected from animals in aseptic conditions. The presence of aerobic

heterotrophic bacteria was estimated using spread plate dilution technique. Proteolytic,

pseudomonads and chitinolytic bacteria were assessed as separate physiological groups of

heterotrophic bacteria.

Sex apparently influenced microbial contamination, as 92 % of males showed higher levels of

total bacteria than females (Figure 42), while no significant differences were found for

proteolytic, pseudomonads and chitinolytic bacteria. Among males total bacteria levels ranged

from 102 to 10

5 cfu/g, whereas in females those levels ranged from non-detected to

approximately 104 cfu/g.

Concerning edible tissues, the microbiological data obtained showed that 67 % and 83 % of

muscle samples presented higher levels of total and proteolytic bacteria, respectively, than

brown meat (hepatopancreas plus gonads). In muscle, total bacteria levels ranged from 103 to

105 cfu/g, while proteolytic bacteria ranged from non-detected to 10

4 cfu/g (Figure 42). In brown

meat levels of total bacteria showed a wider range, between 10 and 105 cfu/g while proteolytic

bacteria levels ranged from non-detected to 103 cfu/g (Figure 43). Proteolytic bacteria

- Live

- Chille

Chille

Ireland

Scotla

Excellent

Medium

Rejected

contributed to 15-40 % of muscle total bacterial counts and to 17-48 % of brown meat total

bacterial counts (Figure 44). Differences in the content of chitinolytic bacteria were also

observed between tissues (muscle vs. brown meat), with muscle samples presenting higher

contamination than brown meat, the former with levels ranging from non-detected to 103 cfu/g

(Figure 44). No major differences were detected at the level of pseudomonads bacteria.

Figure 42. Levels of total bacteria in females and males (brown meat – V and muscle – M) expressed by log of colony formation unit (cfu) per g.

Dead crabs generally showed higher levels of total bacteria than reactive specimens, while no

major differences in total bacteria were detected in weak and moribund animals. No significant

differences were found for proteolytic, pseudomonads and chitinolytic bacteria.

Total bacteria

V M V M V M V M V M V M

Female Male

Figure 43. Percentage of total and proteolytic bacteria in edible tissues (brown meat and muscle).

Proportion of proteolytic bacteria in two tissues

Total bacteria Proteolytic

Viscera Muscle

Figure 44. Levels of chitinolytic bacteria in edible tissues (brown meat and muscle) expressed by log of colony formation unit (cfu) per g.

No clear trends were observed in the microbiological levels of animals captured at different

seasons and origins. The results support that microflora abundance and composition changes

in Cancer pagurus mainly with tissue, sex and animal condition.

6.3 Nutritional quality

Authors INRB I.P./L-IPIMAR: António Marques, Sara Barrento, Patrícia Anacleto, Bárbara

Teixeira, Rogério Mendes, Narcisa Bandarra, Maria Leonor Nunes

Cancer pagurus is much appreciated in Southern Europe, where muscle, hepatopancreas and

gonads are regularly consumed with peaks in Summer and December (Barrento et al., 2008).

The nutritional quality of C. pagurus (twenty animals per variable) was evaluated after wet

transportation according to different variables: season (Spring, Summer, Autumn and Winter),

sex (Males and Females), tissue (Muscle, Hepatopancreas and Gonads) and origins (Scottish

coast and English Channel). Several parameters were analysed, namely the proximate

chemical composition (moisture, protein, fat, ash and carbohydrates), amino acids and fatty

acids profile, cholesterol, energy, essential elements (S, Cl, K, Ca, Fe, Cu, Zn, Se, Br, Sr, Na,

Mg and Mn) and chemical contaminants (As, Cd, Pb and Hg) (part of the results were adapted

from Barrento et al., 2009a,b,c).

Seasonal effect

The edible crab Cancer pagurus is subjected to a large number of environmental variables

following their annual and daily cycles (e.g. migration and habitats) that influence behaviour,

feeding, metabolism and ultimately the nutritional quality.

The protein content of muscle and hepatopancreas did not strongly varied throughout seasons

(except for Summer in both sexes muscle), whereas male gonads had lower amount of proteins

than females (except in Winter).

Chitinolytic bacteria

Viscera Muscle

The fat content of muscle was significantly lower in Winter males (0.1 %), while in

hepatopancreas fat decreased in Autumn (males) and Winter (both sexes), and fat content of

female gonads (3.1 – 5.6 %) was always higher than male gonads (0.9 – 1.3 %) with maximum

values obtained in Winter in both sexes.

Cholesterol content in hepatopancreas was constant throughout seasons, while muscle crabs

sampled in Spring showed the highest values (46-47 mg/100g tissue). The cholesterol content

increased in female gonads from Spring (121 mg/100 g) to Autumn (200.8 mg/100 g), followed

by a strong decrease in Winter (94 mg/100 g), while in males there was a decrease in Winter

(65 mg/100 g).

Regarding fatty acids, in muscle the lowest and highest levels of saturated fatty acids (SFA)

were obtained for females in Winter (17.4 %) and Summer (13.9 %), respectively;

Monounsaturated fatty acids (MUFA) values were generally lower in Autumn (30.4 %) and

Winter (26.6 %), and higher during Spring (30.7 %) and Summer (31.9 %), particularly in

females; and polyunsaturated fatty acids (PUFA) levels were constant throughout the year

(43.5-49.2 %). Hepatopancreas had proportionally more SFA in Autumn (both sexes; 25.5-27.0

%) and less in Winter (females; 16.3 %); MUFA was lower in Winter (females; 26.6 %) and

Autumn (males; 27.6 %); and PUFA levels were lower during Summer (females; 24.2 %) and

Autumn (both sexes; 20.9 %). Concerning gonads, in both sexes SFA values were lower in

Spring (15.3-17.2 %) and Summer (16.6-17.6 %); no seasonal variations were detected in the

proportion of MUFA (32.2-39.4 %); and PUFA content was lower in Winter (both sexes; 35.4-

36.6 %). The main SFA in all tissues was palmitic acid (16:0), which was fairly constant in

muscle during the four seasons independently of sex, while in hepatopancreas and gonads it

predominated during Autumn and Winter, respectively. Among MUFA, oleic acid (18:1n-9) was

the prevailing fatty acid in all tissues, with particularly higher values found in Spring (all tissues),

Summer (muscle and hepatopancreas) and Winter (gonads). The main n-3 PUFA was

eicosapentaenoic (EPA, 20:5n–3; muscle and gonads) and docosahexaenoic (DHA, 22:6n–3;

hepatopancreas and gonads) acids. In muscle, the proportion of EPA was fairly constant during

the four seasons independently of sex (19.1-22.2 %), while in hepatopancreas lower values

were detected in Summer (females; 5.5 %) and Autumn (males; 4.6 %), and in gonad males

had higher proportion during Summer (13.9 vs. 19.7 %) and Autumn (13.1 vs. 20.9 %) than

females. DHA in female muscle had higher proportion during Winter (13.8 %) and lower in

Spring (11.2 %) and Summer (10.9 %); in hepatopancreas DHA was predominant in Winter

(12.6-14.6 %) and Spring (11.3-12.0 %) in both sexes, while in gonads there was a tendency to

decrease DHA levels from Spring (13.4-16.3 %) to Winter (8.7-10.7 %) in both sexes.

PUFA/SFA was higher in Winter in muscle of females (3.5) and in hepatopancreas of both

sexes (1.9-2.2), while in gonads higher values were found in Spring (2.6) and Summer (2.5-2.9)

for both sexes.

Regarding amino acids, among female gonads the lowest amino acid values were observed in

Winter (due to threonine, leucine, arginine and alanine; 20.5 g/100 g tissue) compared to the

remaining seasons, while in male gonads the highest values occurred in Winter (due to

threonine, isoleucine, leucine, histidine, lysine, aspartic acid, glutamic acid, tyrosine, proline and

hypoxanthine; 15.5 g/100 g tissue). Hepatopancreas of male crabs had higher concentration of

amino acids during Winter (14.4 g/100 g tissue), particularly of threonine, valine, isoleucine,

leucine, phenylalanine, histidine, arginine, aspartic acid, serine, glutamic acid, glycine, alanine,

tyrosine, and hypoxanthine. On the other hand, during Autumn female hepatopancreas had

higher concentrations of amino acids (13.3 g/100 g tissue) than male hepatopancreas due to

lysine, serine, alanine and hypoxanthine, while the lowest amino acid content was detected in

Spring (due to threonine, valine, isoleucine, leucine, phenylalanine, lysine, serine, glutamic acid,

glycine, hypoxanthine and taurine; 8.8 g/100 g tissue). As far as muscle is concerned, generally

no significant differences were detected between seasons (15.8-18.2 g/100 g tissue).

In terms of essential elements and chemical contaminants, no seasonal changes were

observed in the content of some elements in muscle (Na, Fe, Cu and Se), hepatopancreas (Cu,

Se and Hg) and gonads (K, Ca, Zn, Br, Fe and Se) (Table 9-11). It is likely that these elements

are vital for edible tissues all year long, while others like contaminants are bio accumulated in

edible tissues.

In contrast, Mg, S, Cl, K, Ca and Hg were more concentrated in muscle in Autumn and Winter,

while hepatopancreas showed higher concentrations of Cu (Autumn, females), Zn (Autumn and

Winter, both sexes), As (Spring, females), Ca (Winter, males), Mg and Sr (Winter, males)

(Tables 9-11). Generally, season affected most elements‟ concentration in gonads. The

concentration of Br and Sr (Spring), Cl and K (Summer), and Mg and Ca (Winter) in male

gonads was statistically higher than in females (Tables 9-11). In contrast, females had

statistically higher Fe, Zn, As and Se levels than males during all seasons (Tables 9-11). The

seasonal differences observed are most probably related with the spawning cycle and

metabolism that influence feeding, reproductive state and weight. As far as consumers are

concerned, muscle is an excellent source of Cu and Se (as values can reach the

Recommended Daily Allowance/Action limits and are below the Upper Level), and a good

source of Na and Zn in both sexes all year long (as values are higher than 20 % of

recommended values) (Tables 9-11). Hepatopancreas is an excellent source of Ca (males all

year long and females in Winter), Cu, Zn, and Se (all year long), Mg and Mn (Winter males), Cl

(Winter) and Fe (Autumn and Winter). Gonads are excellent sources of Na (Winter), Cu

(Summer) and Se (all year long), and good sources of Mg (Winter), Cl (Summer males), Fe

(Summer/Autumn/Winter females), Zn (females all year long and Spring/Summer males) and

Mn (Winter males).

As far as contaminants are concerned, As concentration was higher in Summer (in all tissues),

while Cd (gonads) and Hg (all tissues) were more concentrated in Winter (Tables 10-11).

Lead concentration did not varied in muscle and gonads but had a peak in female

hepatopancreas during Autumn. The higher concentrations of Cd and Hg coincided with Winter,

which is a period of lower seawater temperatures and salinities, factors that have been reported

to be inversely related with Cd uptake by crabs (Legras et al., 2000; Rainbow et al., 2001).

Concerning the potential hazards of C. pagurus consumption, only Cu, Cd and Hg levels were

above the limits set by authorities. Copper was above the UL (Upper Level) in hepatopancreas

of Autumn females. Cadmium was above the AL (Action Limit) and MPC (Maximum Permissible

Concentration) in hepatopancreas all year round and in male gonads in Winter, and was also

above the MPC of female gonads‟ in Winter. Mercury was above the MPC in muscle and

hepatopancreas of most Autumn crabs, but always below the AL limit. It is important to

remember that MPC is only set for muscle and not for other tissues.

Table 9. Mean macro elemental composition (mg per 100 g-1

wet weight; ± standard deviation) in female and male C. pagurus edible tissues during the four seasons. In each column different letters indicate significant differences in the elemental concentration per tissue (p<0.05). Abbreviations: F – female; M – male. Adequate Intakes (AI), Recommended Dietary Allowances (RDA) and Tolerable Upper Intake Levels (UL) set by authorities are also shown.

Tissue Season Sex Na

AI=1500 UL=2300

Mg RDA = 400

RDA=2300 K

RDA=4700

Ca RDA=1000 UL=2500

Spring F 351±46 a 36±4 ab 117±20 b 243±25 b 164±26 b 48±16 a

M 241±54 a 25±7 b 162±38 ab 244±30 b 172±24 b 47±11 a

Summer F 275±54 a 30±7 b 107±31 b 283±40 b 165±19 b 20±6 b

Muscle M 278±98 a 21±10 b 135±63 b 244±33 b 179±21 b 24±5 b

Autumn F 301±54 a 39±5 a 247±51 a 430±48 a 305±25 a 38±1 a

M 354±56 a 39±4 a 251±45 a 465±96 a 286±30 a 35±4 a

Winter F 306±78 a 31±6 b 240±21 a 515±56 a 294±21 a 38±8 a

M 272±11 a 29±1 b 224±61 a 508±61 a 303±12 a 35±3 a

Spring F 223± 75 a 30±2 c 164±34 b 417±64 c 162±8 b 285±58 c

M 224±67 a 50±10 b 181±45 b 326±68 c 167±12 b 631±157 b

Summer F 307±79 a 33±7 bc 206±68 b 567±64 b 185±10 b 260±79 c

Hepato M 334±118 a 42±17 bc 234±37 b 469±64 b 201±10 b 943±79 b

Autumn F 253±27 a 53±5 b 387±81 a 600±55 b 287±36 a 642±165 b

M 279±68 a 57±23 ab 340±81 a 668±83 b 281±48 a 919±717 abc

Winter F 281±77 a 51±7 b 345±49 a 780±34 a 288±23 a 781±214 b

M 228±53 a 79±5 a 392±92 a 664±54 b 318±25 a 1926±368 a

Spring F 309±155 c 7.2±0.6 c 126±51 b 154±67 d 108±49 d 8.5±3.4 e

M 110±2 d 56±1 b 152±128 ab 673±19 bcd 377±34 b 59±6 b

Summer F 220±22 c 17±2 c 306±61 a 380±11 c 187±7 d 20±1 d

Gonads M 281±1 c 12±1 d 291±37 a 1114±39 a 488±18 a 57±3 b

Autumn F 167±56 cd 23±4 c 366±69 a 333±72 c 179±16 d 21±2 d

M 502±118 b 26±4 c 161±35 b 601±60 b 293±32 bc 33±4 c

Winter F 754±246 b 65±14 b 293±61 ab 490±53 b 274±12 c 21±1 d

M 1879±7 a 90±1 a 210±16 b 673±19 b 348±2 b 85±2 a

Table 10. Mean trace elemental composition (mg per 100 g-1

wet weight; ± standard deviation) in female and male C. pagurus edible tissues during the four seasons. In each column different letters indicate significant differences in the elemental concentration per tissue (p<0.05). Abbreviations: F – female; M – male; bdl – below detection limit. Adequate Intakes (AI), Recommended Dietary Allowances (RDA), Tolerable Upper Intake Levels (UL) and Action Limits (AL) set per day by authorities are also shown.

Tissue Season Sex Fe

RDA = 8

Cu RDA = 0.9 UL = 10

Zn RDA = 11

As AL=7.6

Spring F 0.37±0.06 b 0.86±0.13 a 5.5±0.2 b 4.2±1.1 ab 2.0±0.3 b 1.6±0.3 a

M 0.48±0.14 ab 0.93±0.28 a 6.0±1.0 ab 3.6±1.1 b 2.3±0.3 b 1.1±0.3 ab

Summer F 0.48±0.30 ab 0.93±0.21 a 5.5±0.4 b 4.3±0.9 a 2.2±0.2 b 0.72±0.29 b

Muscle M bdl 0.86±0.25 a 5.9±0.3 ab 4.0±0.8 a 2.1±0.3 b 1.0±0.5 ab

Autumn F 0.68±0.10 a 1.0±0.2 a 7.0±0.9 a 2.4±0.3 c 2.2±0.1 b 0.82±0.30 b

M 0.38±0.15 b 0.67±0.25 a 7.4±1.5 ab 3.5±2.1 b 2.4±0.2 b 0.73±0.37 b

Winter F 0.44±0.09 b 0.90±0.18 a 6.6±0.4 a 2.7±0.5 c 2.6±0.2 ab 1.1±0.4 ab

M 0.32±0.04 b 1.1±0.2 a 6.1±0.4 ab 1.8±0.4 d 2.9±0.3 a 0.59±0.10 b

Spring F 1.6±0.3 b 3.1±0.2 b 2.2±0.2 b 4.3±0.4 a 5.5±0.8 b 6.8±1.1 c

M 1.7±0.1 b 1.8±1.1 b 1.7±0.2 b 2.4±0.6 bc 6.4±1.2 b 10±2 b

Summer F 1.8±0.2 b 4.7±1.9 ab 2.6±0.5 a 4.9±1.1 ab 8.1±0.3 a 6.0±0.9 c

Hepato M 2.3±1.0 ab 1.7±0.6 b 2.0±0.2 b 2.9±0.4 b 8.3±2.4 ab 12±5 ab

Autumn F 4.5±1.1 a 10±4 a 2.7±0.5 a 2.6±0.2 b 7.0±0.5 b 9.1±1.3 b

M 3.2±0.8 a 3.3±3.9 ab 2.5±0.8 a 2.0±0.2 c 11±4 a 11±8 ab

Winter F 3.0±0.7 a 2.9±2.0 b 2.6±0.3 a 2.6±0.5 bc 6.5±1.0 b 8.9±1.2 b

M 4.3±0.5 a 3.9±1.7 b 2.7±0.4 a 2.0±0.3 c 7.7±0.8 b 17±2 a

Spring F 1.2±0.4 a 0.56±0.28 bc 4.7±0.8 b 3.0±1.8 ab 1.9±0.9 c 0.25±0.11 d

M bdl 0.75±0.03 b 2.8±0.1 c 2.1±0.0 c 8.2±0.2 a 1.6±0.0 a

Summer F 1.9±0.1 a 1.2±0.1 a 9.0±0.4 a 4.7±0.2 a 3.8±0.2 b 0.41±0.03 d

Gonads M 0.45±0.06 b 1.4±0.0 a 3.3±0.1 c 4.3±0.1 b 8.8±0.2 a 1.2±0.0 b

Autumn F 1.9±0.2 a 0.96±0.15 b 9.0±1.3 a 1.8±0.2 c 2.7±0.3 c 0.31±0.04 d

M bdl 0.44±0.02 c 1.8±0.1 d 1.5±0.0 d 4.2±0.1 b 0.61±0.01 c

Winter F 2.7±0.5 a 0.83±0.07 b 5.7±1.3 b 2.5±0.2 c 3.3±0.3 c 0.39±0.04 d

M 0.32±0.03 b 0.70±0.02 b 2.0±0.1 d 1.1±0.0 d 4.1±0.1 b 0.78±0.01 c

Table 11. Mean ultra-trace elemental composition (mg per 100 g-1

wet weight; ± standard deviation) in female and male C. pagurus edible tissues during the four seasons. In each column different letters indicate significant differences in the elemental concentration per tissue (p<0.05). Abbreviations: F – female; M – male; bdl – below detection limit. Adequate Intakes (AI), Recommended Dietary Allowances (RDA), Tolerable Upper Intake Levels (UL), Maximum Permissible Concentrations (MPC) and Action Limits (AL) set per day by authorities are also shown.

Tissue Season Sex Mn AI = 2.3

Se RDA=0.055

UL=0.4

Cd MPC=0.05

AL=0.3

Hg MPC=0.05

AL=0.1

Pb MPC=0.05 AL=0.15

Spring F 0.022±0.003 b 0.11±0.02 ab 0.003±0.002 a 0.018±0.001 b bdl

M 0.027±0.005 b 0.09±0.03 ab 0.002±0.001 a 0.016±0.004 b 0.002±0.000 a

Summer F 0.027±0.003 b 0.09±0.02 ab 0.002±0.001 a 0.016±0.001 b 0.002±0.000 a

Muscle M 0.024±0.004 b 0.09±0.02 ab bdl 0.016±0.001 b 0.002±0.000 a

Autumn F 0.062±0.012 a 0.13±0.01 a bdl 0.061±0.018 a bdl

M 0.026±0.006 b 0.10±0.06 ab bdl 0.049±0.015 a bdl

Winter F 0.024±0.001 b 0.083±0.17 b 0.002±0.001 a 0.038±0.008 a 0.003±0.001 a

M 0.029±0.006 b 0.11±0.02 ab 0.003±0.003 a 0.021±0.004 b 0.002±0.000 a

Spring F 0.11±0.01 c 0.19±0.04 a 0.80±0.70 b 0.021±0.003 b 0.002±0.001 b

M 0.21±0.02 b 0.15±0.04 a 0.60±0.30 b 0.015±0.001 b 0.002±0.001 b

Summer F 0.20±0.13 bc 0.16±0.05 a 2.0±1.2 ab 0.023±0.006 b 0.003±0.002 b

Hepato M 0.17±0.11 bc 0.11±0.04 a 2.8±1.1 a 0.018±0.004 b 0.003±0.002 b

Autumn F 0.31±0.05 b 0.20±0.06 a 0.70±0.30 b 0.051±0.013 a 0.010±0.004 a

M 0.33±0.32 abc 0.14±0.07 a 1.6±1.4 ab 0.039±0.014 a 0.003±0.002 b

Winter F 0.22±0.06 b 0.18±0.04 a 2.7±1.6 ab 0.033±0.011 a 0.004±0.002 b

M 0.47±0.06 a 0.17±0.02a 0.80±0.30 b 0.030±0.008 a 0.006±0.003 b

Spring F 0.10±0.00 d 0.20±0.10 ab 0.013±0.003 e 0.006±0.004 b bdl

M 0.11±0.00 d 0.13±0.01 b 0.006±0.000 f 0.006±0.000 b bdl

Summer F 0.24±0.01 b 0.26±0.02 a 0.018±0.001 d 0.006±0.001 b 0.002±0.000 a

Gonads M 0.11±0.03 d 0.14±0.01 b 0.072±0.020 c 0.007±0.002 b 0.002±0.000 a

Autumn F 0.24±0.06 b 0.29±0.06 a 0.004±0.002 e 0.007±0.002 b 0.004±0.002 a

M 0.17±0.02 c 0.070±0.008 b 0.032±0.001 d 0.014±0.000 a 0.002±0.000 a

Winter F 0.41±0.07 b 0.34±0.06 a 0.20±0.11 b 0.017±0.004 a bdl

M 0.58±0.00 a 0.12±0.00 b 0.43±0.01 a 0.009±0.001 b bdl

Generally, males are more expensive than females mostly due to the higher muscle content. In fact,

males had more muscle (10.3 – 16.2 %) than females (7.9 – 8.5 %), less gonads (males: 0.9 – 2.2 %;

females: 1.8 – 7.3 %), while in hepatopancreas the only difference was observed in Winter females

(lower values; 7.8 %).

Female gonads showed significantly lower ash and moisture than male gonads (1.5-1.7 % vs. 2.7-2.9

% in ash and 56.5-68.2 % vs. 77.0-80.4 % in moisture), whereas no major differences were found in

hepatopancreas and gonads.Male gonads had lower amount of proteins than females except in

Winter (13.1-14.7 % vs. 19.2-25.5 %).

No major differences were found between males and females in the fat content of muscle, while

generally females had higher fat content in hepatopancreas (in Autumn and Winter; 7.2-10.2 % vs.

2.9-6.5 %) and gonads (3.1-5.6 % vs. 0.9-1.1 %).Overall, females had more cholesterol in gonads

than males throughout the seasons (94-201 mg/100g tissue vs. 65.0-89.4 mg/100g tissue).

Statistical variations in the energy content were only observed in gonads, with female gonads

showing higher energy content than males regardless of season (153-201 Kcal/100g tissue vs. 76-93

Kcal/100g tissue).Regarding fatty acids, the ratio n-3/n-6 was generally higher in females than in

males, particularly in muscle (3.8-5.2 vs. 3.5-3.9) and gonads (4.2-5.8 vs. 3.1-3.9).

Figure 45. PCA analysis considering the content of all amino acids in the edible tissues. Solid lines represent clusters related to tissue, while dashed lines represent clusters related to sex. Abbreviations: M (Males); F (Females).

Factor 1 (83 %)

r 2 (8

Muscle

Hepatopancreas

Gonads-10 -4 2 8

As far as amino acids are concerned, significant differences were only detected in gonads, where

females had higher amounts of most amino acids during all seasons compared to males. These

differences influenced the cluster separation observed between sexes in the PCA analyses with

amino acids data (Figure 45).

Concerning essential elements and chemical contaminants, sex differences were mainly found in

gonads, where in general female gonads had more Fe, Zn, As and Se than males but less Ca, Cl, Br

and Sr (Tables 9-11). Additionally, females usually had higher contents in the muscle (Fe, Zn and Se)

and hepatopancreas (Cu and Se), while in males the most relevant amounts were found in

hepatopancreas (Na, K, Ca, Mn and Fe).

Tissue

Generally, significant differences were found in most parameters between tissues of C. pagurus,

suggesting distinct physiological roles of each tissue. Hepatopancreas is a midgut multifunctional

organ that among several functions acts as a temporary reservoir for energy and minerals, regulating

physiologically important cations and detoxifying dietary contaminants like Cd, Ca, Zn and Cu. Muscle

is a structural organ mostly involved in neuromuscular functions and in the mechanical movements of

appendages and claws. Gonads are the reproductive organs where high nutritional investments are

made to produce the reproductive cells.

In general, female gonads and muscle (both sexes) were rich in proteins (16.4-25.5 %), while

hepatopancreas had more fat (2.9-16.6 %). Female gonads and hepatopancreas (both sexes) had

higher energy and cholesterol content compared to muscle.In terms of fatty acids, muscle and gonads

had a similar pattern dominated by PUFA, followed by MUFA and SFA; while hepatopancreas had

proportionally more MUFA, followed by PUFA and SFA. PUFA/SFA ratio was higher in muscle (2.6-

3.5) followed by gonads (1.7-2.9) and hepatopancreas (0.8-2.2). Hepatopancreas had generally

higher values of atherogenic (AI) and thrombogenic indices (TI) (AI: 0.2-0.5; TI: 0.1-0.4) followed by

gonads (AI: 0.1-0.4; TI: 0.1-0.2) and muscle (AI: 0.1-0.4; TI: 0.1-0.2).

Generally, hepatopancreas had the lowest amounts of amino acids compared to muscle and gonads

(Table 12). The only exceptions were taurine (lower in muscle than in the remaining tissues) and

tryptophan (no differences between tissues).

As far as essential elements and chemical contaminants are concerned, hepatopancreas had more S,

Cl, Ca, Br, Sr, Fe, Cu, Cd and Pb, gonads had higher concentration of Na and muscle was richer in

Zn. No statistical differences between tissues were observed in the concentration of K and As. These

results lead to muscle cluster separation in PCA analyses (Figure 46). Regarding contaminants for

human consumption, the levels of As, Hg and Pb found in all edible tissues pose minimal risks to

consumers, while Cd concentration in hepatopancreas was always above the action limits. Therefore,

it is recommended to moderate hepatopancreas consumption.

Origin

C. pagurus has different market prices according to the fishing ground. Crabs caught off in the English

Channel are usually more expensive than those caught off Welsh, Scottish and Irish coasts,

presumably due to the intrinsic quality of these populations (e.g. bigger animals, more resistant to

transport, better taste and higher meat content). In the present study no major differences were

observed in the proximate chemical composition of muscle, gonads and hepatopancreas of crabs

caught in the English Channel and Scottish Coast.

The fatty acids profile of muscle showed market differences related to location, as crabs from the

Scottish Coast had higher proportion of 14:0, 18:1n-9 and 18:2n–6, and lower 18:1n-7 and 16:4n–3.

In contrast, hepatopancreas and gonads did not reveal any clear pattern related with origin in respect

to individual fatty acids. The ratio n-3/n-6 in hepatopancreas from crabs of the English Channel was

lower in females compared to males. In contrast, PUFA/SFA in hepatopancreas from specimens of

the Scottish coast was lower in females than in males. The atherogenic index was higher in

hepatopancreas of Scottish coast females than English Channel females. In gonads, the n-3 and n-6

fatty acids were more concentrated in Scottish coast males than in other crabs, while PUFA/SFA was

higher in gonads of Scottish coast males and lower in English Channel males. The atherogenic index

of gonads from Scottish coast crabs was higher in females than in males. These results lead to the

fatty acids cluster separation in PCA analyses (Figure 47).

Table 12. Statistical differences between tissues with the respective p values. Each amino acid was tested independently of season and sex. Abbreviations: ND (no differences); THR (Threonine), VAL (Valine), MET (Methionine), ILE (Isoleucine), LEU (Leucine), PHE (Phenylalanine), HIS (Histidine), LYS (Lysine), ARG (Arginine), ASP (Aspartic acid), SER (Serine), GLU (Glutamine), GLY (Glycine), ALA (Alanine), TYR (Tyrosine), PRO (Proline), HYP (Hydroxyproline), TAU (Taurine), EAA/NEAA (Essential Amino Acids/Non Essential Amino Acids), Total AA (Total Amino Acids).

Tissues p

THR (Muscle=Gonads)>Hepatopancreas <0,01

VAL (Muscle=Gonads)>Hepatopancreas <0,01

MET (Muscle=Gonads)>Hepatopancreas <0,01

ILE (Muscle=Gonads)>Hepatopancreas <0,01

LEU (Muscle=Gonads)>Hepatopancreas <0,01

PHE (Muscle=Gonads)>Hepatopancreas <0,01

HIS Muscle=Gonads>Hepatopancreas 0,01

LYS (Muscle=Gonads)>Hepatopancreas <0,01

ARG Gonads>Muscle>Hepatopancreas <0,01

TRP ND 0,21

ASP (Muscle=Gonads)>Hepatopancreas <0,01

SER (Muscle=Gonads)>Hepatopancreas <0,01

GLU (Muscle=Gonads)>Hepatopancreas <0,01

GLY Muscle>Gonads>Hepatopancreas <0,01

ALA (Muscle=Gonads)>Hepatopancreas <0,01

CYS (Muscle=Gonads)>Hepatopancreas <0,01

TYR (Muscle=Gonads)>Hepatopancreas <0,01

PRO Gonads>Muscle>Hepatopancreas <0,01

HYP Muscle=Gonads>Hepatopancreas <0,01

TAU (Gonads=Hepatopancreas)>Muscle <0,01

EAA/NEAA (Gonads=Hepatopancreas)>Muscle <0.01

Total AA (Muscle=Gonads)>Hepatopancreas <0.01

Figure 46. Result of four PCA analyses considering the concentration of all elements in: muscle, hepatopancreas and gonads

Regarding amino acid composition, all edible tissues had similar amino acid profiles with few

differences detected between origins. Yet, generally, the ratio of EAA/NEAA was higher in crabs from

the Scottish coast than in specimens from the English Channel.

In general, crabs caught off in the Scottish coast had statistically higher values of Cl, K, Zn and Se in

hepatopancreas, and Na and Cu in gonads, while specimens harvested in the English Channel

showed higher Mn content in hepatopancreas and gonads. These results indicate that Scottish

coastal waters have less Mn and more Na, Cl, K, Cu, Zn and Se in a bio available form. Crabs from

both fishing grounds are excellent sources of macro (Na, Cl and Ca) and trace elements (Fe, Cu, Zn

and Se), while crabs harvested in the Scottish coast were better sources of most elements studied

than animals from the English Channel, except Mg and Mn (Table 13).

As far as chemical contaminants are concerned, crabs caught off in the English Channel had higher

levels of Cd (gonads) and Br (muscle), while crabs from the Scottish Coast had higher content of S

and As in gonads. Particularly, male crabs from the Scottish Coast had more As and Hg than male

crabs from the English Channel (Table 14). Such differences likely reflect distinct availability of

contaminants in both locations. Yet, the values were below the maximum permissible concentrations

and action levels, except Cd in hepatopancreas of crabs from both origins.

Tissues

Muscle

Hepatopancreas

Gonads

Tissues

Muscle

Hepatopancreas

Gonads

Figure 47. Results of PCA analyses considering the concentration of fatty acids: (A) in all tissues; (B) in gonads depending on sex and season; and (C) in hepatopancreas depending on season. Abbreviations: SF (Spring Females); SM (Spring Males); SUF (Summer Females); SUM (Summer Males); AF (Autumn Females); AM (Autumn Males); WF (Winter Females); WM (Winter Males).

Gonads

Tissues

Músculo

Hepatopâncreas

Gónadas

Tissues

Muscle

Hepatopancreas

Gonads

Hepatopancreas

Spring

Summer Autumn

Winter

Gonads

Hepatopancreas

Spring

Summer Autumn

Winter

Table 13. Average percentage of macro and trace elements per 100 g portion of crabs caught off Scottish Coast (SC) and English Channel (EC), considering the Dietary Reference Intakes (DRI) set per day by the USNAS. Values in brackets for Mg, Mn, Fe and Zn refer to genders DRI: the first and second values correspond to adult females and males aged between 19 and 50 years, respectively.

Elements DRI Origin Muscle Hepatopancreas Gonads

♀ ♂ ♀ ♂ ♀ ♂

Macro elements

Na 1500 mg* SC 20 18 18 25 15 -

EC 16 17 17 12 12 -

Mg (413-317) mg SC (7.0-9.1) (4.6-6.0) (8.2-11) (11-14) (4.1-5.4) -

EC (7.3-9.5) (4.4-5.7) (7.0-9.1) (15-19) (3.4-4.4) -

Cl 2300 mg* SC 12 11 82 66 17 48

EC 13 14 20 20 13 20

K 4700 mg* SC 3.5 3.8 13 12 4.0 11

EC 3.7 3.8 3.7 5.0 3.4 5.1

Ca 1000 mg* SC 2.2 2.4 79 231 2.0 5.6

EC 2.8 2.2 42 125 1.5 3.2

Trace elements

Mn (2.3-1.8) mg* SC (1.3-1.7) (0.87-1.1) (6.1-7.8) (10-13) (10-13) -

EC (1.3-1.7) (1.3-1.7) (8.7-11) (15-19) (13-17) -

Fe (8-18) mg SC (4.3-1.9) (3.9-1.7) (73-32) (76-34) (24-11) (5.6-2.5)

EC (5.4-2.4) (2.8-1.2) (14-6.1) (48-21) (16-7.2) (3.0-1.3)

Cu 0.9 mg SC 103 83 1843 594 130 151

EC 116 75 644 149 90 53

Zn (11-8) mg SC (50-69) (54-74) (73-100) (52-71) (82-113) (30-41)

EC (51-70) (47-65) (19-26) (16-23) (65-90) (15-20)

Se 0.055 mg SC 178 168 966 633 467 262

EC 197 120 207 85 323 154

Table 14. Ratio between the content of As, Cd and Hg in the edible tissues (muscle, hepatopancreas, gonads and whole crab) of C. pagurus and the maximum permissible concentration (MPC) set by the European Commission for the crustaceans‟ muscle, or the action level (AL) set by the USFDA for the food commodity Crustacea, data labelled with *. Values are expressed as percentage. Abbreviations: Scottish Coast (SC), English Channel (EC).

Muscle (%) Hepatopancreas (%) Gonads (%)

Elements MPC/AL SC EC SC EC SC EC

♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂

Arsenic (As) 76 ppm* 57 53 46 21 64 38 46 18 57 50 43 11

Cadmium (Cd) 0.5 ppm 4.0 2.0 4.0 2.0 4120 6004 2400 3400 36 - 60 -

Cadmium (Cd) 3 ppm* 0.67 0.33 0.67 0.33 687 1001 400 567 6.0 - 10 -

Mercury (Hg) 0.5 ppm 32 32 33 11 46 36 39 19 14 12 10 3.0

Mercury (Hg) 1 ppm* 16 16 16 5.3 23 18 20 10 6.8 6.0 5.0 1.5

7. The effect of stress on final product quality

7.1 Dry transport brown crab - sensory quality, stressed vs unstressed)

Authors Møreforsking Marin: Astrid K. Woll & Wenche E. Larssen, Introduction Brown crab can survive out of water in good condition depending of factors such as air temperature

and humidity. This property is utilized during the catching process, on-board storage, live

transportation in inshore fisheries, and live export to over-seas markets. Dry transport has the benefit

that water is not transported from one region to another, thus avoiding the risk for being a carrier of

infections or spreading introduced species. Also the cost of transport can be reduced by transporting

them dry as opposed to vivier trucks (Robson et al. 2007).

Several biomarkers have previous been used to evaluate health and stress in brown crab condition.

These have been compared with vitally indexes during laboratory experiments simulating dry storage,

both being negatively influenced during emersion depending of time and air temperature. Knowledge

of critical limits during the air exposure in all steps of the value chain is essential to secure the

animals‟ well-being, survival, and condition.

Aim of the study

Considering that parts of the landing may be weak or moribund after dry storage and transport the

aim of this study was to examine if difference were apparent in sensory traits (taste, smell, texture)

between stressed due to dry transport and unstressed crabs taken directly from tanks with flow

through water of good quality.

Methods and material

Pre-treatment and stressing

Two tests were conducted, the first in week 51 (2007) and the second in week 43 (2008). In 2007

female crabs were put in tanks with water temperature 6 -7 °C and during one week fed three times

with saithe in order to reduce individual differences in taste and color of brown meat (Berge and Woll

2006). After the last feeding the crabs were starved in three days before the pre-treatment started on

17 December. In 2008 the crabs were held 3-4 days in tanks with water temperature 11-12°C the

treatment started on the 21 October. The crabs were not fed.

Stressing procedure. Females judged to be of good quality and without any damages were

stacked in a polystyrene box covered with wet sacking. The box was placed in a warm room.

Air temperature was logged to be between 17 – 20 °C during the 17 hours long storage.

Unstressed crabs. During the same time interval, the rest of the crabs stayed in the tanks.

After the treatment vitality index, carapace width and shell condition were recorded (Table 15).

Hemolymph was sampled and pH and hemolymph protein was measured immediately at ambient

temperature using a micro-electrode and pH-meter (EBRO phx 1495) for pH measurements and a

clinical refractometer (no. 300005) for the hemolymph protein (Ozbay & Riley 2002). Hemolymph

protein together with the shell hardness indicates the meat yield assessed after moulting (Ozbay &

Riley 2002, Paterson et al. 1999).

Table 15. Morphology and physiology of the stressed and unstressed crabs that were sorted out and

used in the sensory duo trio tests. Several more were treated, but not used in the tests.

Date Treatment Crabs

Vitality

condition

Blood protein

(g/dl)

Dec 2007 Unstressed 8 1.0 ± 0 3.1 ± 0.1 9.32 ± 1.5 154 ± 13 7.97 ± 0.1

Stressed 7 3.9 ± 0.1

3.0 ± 0

11.6 ± 1.5

152 ± 7.5 7.22 ± 0.1

Oct 2008 Unstressed 16 1.0 ± 0 3.1 ± 0.4 8.5 ± 1.7 142 ± 9 7.90 ± 0.08

Stressed 16 3.6 ± 0.7 3.3 ± 0.5 9.3 ± 1.7 145 ± 11 7.25 ± 0.13

Cooking

After the treatment the crabs were cooked in 25 minutes. Stressed and unstressed groups were

cooked separately and water changed between the groups. After cooking the crabs were chilled

separately at air temperature 2 – 3 °C in order to get a quick cooling process.

Self-amputation of appendages

In 2008 all crabs were spiked both in the thorax ganglion and the brain ganglion before cooking. Just

a few legs and claws were self amputated during the cooking and these crabs were not used in the

sensory test. In 2007 the crabs were put directly into the boiling water. No self-amputation occurred

for the stressed crabs as they were weak or moribund. However, several of the unstressed crabs

amputated legs and claws, and some of these had to be used in the test in order to get enough test

material.

Choosing and picking samples for the test

After 3 hours cooling the crabs were opened and meat content evaluated. Crabs with high meat

content and liver of approximately same color were sorted out for the sensory test. The meat was

picked and differentiated in the following fractions, gathered from all crabs in the respective group:

- Liver

- The skin covering liver and roe

- Claw meat (from the chelae and carpus)

- Roe (not used in the test)

Figure 48. Good quality crab full of liver and roe. The skin can be seen at the sides.

The liver and the skin were mixed in the ratio 65% (liver) and 35% (skin), hereafter called brown meat,

homogenized and pH measuerd (Figure 49).

In 2008 great effort was put into making color and consistent alike for the stressed and unstressed

brown meat. A Minolta Chromameter CR300 (Minolta, Osaka, Japan) was used to ensure that the

lightness had the same value. For the claw meat the same homogenizing procedure was conducted

as for the brown meat. The ready-made samples were stored in the fridge over night.

Figure 49. Brown meat consisting of homogenized liver and skin from the stressed and unstressed

groups in 2008.

Duo-trio test

Each of the persons in the sensory panel was given a plate with three samples, one marked reference

and two others marked with a number randomly chosen. One of these two samples was from the

same group (stressed or unstressed) as the reference sample. Each of the brown meat samples was

10 gram and the claw meat 7 grams.

The brown meat was first served. After this there was a pause, and then the white meat was judged.

In 2007 a sensory panel of 10 persons was used served in two rounds, i.e. 20 judges. In 2008 the

panel consisted of 17 persons and was only used in one round.

Statistics

The number of right and wrong answers was counted and compared with the binomial distribution in

order to find the probability if the hypothesis “no difference in taste and texture of cooked stressed

and unstressed crabs” should be kept or rejected.

Results and discussion Measurement of pH of the cooked homogenized claw meat was 8.02 ± 0.01 for unstressed crabs and

7.89 ± 0.01for stressed. For the brown meat pH was 6.98 for unstressed and 6.82 ± 0.01for stressed

crab (Table 16).

Table 16. pH in claw and brown meat of the cooked homogenized samples of unstressed .

pH Color

Claw meat REF 8.02 ± 0.01 - - -

Stress 7.89 ± 0.01 - - -

Brown meat REF 6.98 ± 0.01 62.0 ± 0.21 3.3 ± 0.26 32.4 ± 0.57

Stress 7.89 ± 0.01 65.6 ± 0.55 3.5 ± 0.27 30.3 ± 0.82

Probability from the binomial distribution showed significant difference on a 5 % level for stressed and

unstressed white meat in 2007 and 2008 and brown meat in 2007 (Table 17). Several in the sensory

panel had comments that indicated a sharper crab taste of the stressed crab which also gave the

meat a more appetizing taste.

Table 17. Results from duo-trio test for taste and smell of brown and white meat of brown crab, probability from the binomial distribution.

Date Sample Right Wrong Sum P

Dec 2007 Brown meat 19 1 20 < 0.05 *)

White meat 15 5

Oct 2008 Brown meat 11 6 17 Ns

White meat 13 4 17 < 0.05

*) Probably due to lighter and softer brown meat for stressed crabs.

Possible biases

The killing and cooking procedure in 2007 resulted in about 90 % of the unstressed crabs self-

amputated one or more leg or claw while the stressed crabs had no amputation. The amputation

resulted in hemolymph loss for the unstressed crabs which might have affect the quality (positive or

negatively) of the cooked meat.

The homogenized brown meat from the unstressed crabs in 2007 was slightly lighter and softer then

the brown meat from the stressed crabs probably overshadowing the parameters smell and taste.

Conclusions 1. Differences in taste and smell were observed between the claw meat of crabs stressed by dry

storage until weak or moribund compared with strong unstressed crabs.

2. The consumer observed differences but the difference was not negative.

3. Difference in brown meat was observed in one of the two tests, but was most probably due to

differences in color and texture.

4. Objective parameters should be identified in order to measure the quality (smell, taste) after

cooking.

References Berge, G. M. and A. K. Woll (2006). "Feeding saithe fillet or a formulated moist feed to the Brown crab

Cancer pagurus: Effects on yild, composition and sensory quality of medium filled captured crabs." Aquaculture 258: 496-502.

Ozbay, G. and J. G. Riley (2002). "An analysis of refractometry as a method of determining blood total protein concentration in the American lobster Homarus americanus (Milne Edwards)." Aquaculture Research 33: 557-562.

Paterson, B. D., G. W. Davidson, et al. (1999). "Identifying stress when western rock lobsters are stored out of water: the average and individual blood lactate concentration." Proceedings. International symposium on lobster health management: 35-41.

Robson, A. A., M. S. Kelly, et al. (2007). "Effect of temperature on the spoilage rate of whole, unprocessed crabs: Carcinus maenas, Necora puber and Cancer pagurus." Food microbiology 24: 419-424.

7.2 Norway lobster: transport-induced stress and product quality

The quality of Nephrops as a food is determined by the parameters of taste, texture, appearance and smell. At the optimum end of the spectrum, the taste is fresh and delicate, the meat is firm and translucent when raw, bright white when cooked, the shell is orange-red and unmarred by black or brown spots and no strong fishy smell can be detected. Lobster quality begins to deteriorate rapidly post-mortem and so the obvious means of maintaining high quality is to ensure the animals reach their destination alive. Nevertheless, whilst there is a paucity of research specifically directed to the sensory implications of stress, a review of the literature allows some inferences to be made. Shell Appearance Under healthy conditions, the Nephrops shell is a bright orangey-red, becoming pale and translucent on areas where the carapace is thin and membranous. However, both live and particularly dead animals are prone to a condition known as melanosis, characterised by the appearance of dark brown or black spots expanding over the surface of the shell. Melanosis is caused by the deposition of dark pigments as a result of a natural biomechanism usually associated with defence. Thus, melanotic regions are often first detected around the site of wounds or injury and one key means to avoid them is to prevent mechanical stress being imposed on the lobsters. For this reason, creel-caught animals are often preferred to trawl-caught ones. However, the cascade of enzymatic reactions leading to the development of melanosis can also be triggered by other stressors, most importantly, aerial exposure. In addition to initiating the cascade, the presence of oxygen in air stimulates the rapid spreading of melanotic spots. Tail Meat Appearance and Texture Nephrops tail meat is firm and white when cooked, translucent when uncooked. However, certain conditions induced by stress can bring about changes which considerably detract from the product quality. In particular, anaerobic respiration either from emersion hypoxia or an over-stimulated escape response (for example during trawling) can lead to the build up of lactic acid which has been posited as a cause of idiopathic muscle necrosis (IMN). This chronic and fatal disease was first detected in live Nephrops being shipped to Europe from Scotland. The animals can be identified by a lack of vigour and lethargy. The tail muscle features patches of white which range from a few lesions through to complete abdominal opacity; these are regions where the muscle fibres have begun to break down. As such, the appearance and texture of the abdominal muscle is likely to be greatly affected. Taste & Smell The combination of free amino acids and the ratio of nucleotides at the time of consumption determine the taste and smell of fish and shellfish products. It is widely accepted that IMP confers a pleasant taste to fresh meat whilst its degradation to hypoxanthine leads to the loss of desirable flavour. Post-mortem, the normal process is for ATP to degrade along the following pathway: ATP→ADP→AMP→IMP→Inosine(HxR)→Hypoxanthine(Hx). Under stressful conditions, ATP reserves can be mobilised leading to a shift in equilibrium towards ADP. This has the effect of speeding up the deterioration of quality upon death of the lobster and can therefore reduce the shelf-life of the product.

8. The effect of stress on product shelf-life

8.1 Shelf life related to stress parameters - Brown crab

Møreforsking Marin: Astrid K. Woll, Wenche E. Larssen, Trygg Barnung, & Grete H. Aas A study was conducted to examine whether stress by air storage prior to processing influences product quality and shelf life. Crabs were stressed (treatment) by being emersed at 17°C (20 hrs) and at 4°C (3 days) before cooking and were compared with crabs that were revitalized in flow through water before cooking. The cooked crabs were stored at 4 C and samples taken after 5, 8 12 and 20 days:

- microbiological growth was examined in the claw meat - sensory parameters to evaluate quality of claw meat and meat in carapace during storage

was developed and described.

- correlations between microbiolagical growth and sensory parameters of claw meat was

studied.

Conclusions of the study.

- No correlation was found between treatment, storage, and the other parameters.

- When all treatment groups was assessed together, smell (both from claws and body) had the

strongest effect according to storage, second the bacterial counts.

- Smell appears before microbiological growth.

- Spoilage for human consumption between day 5 and 8 of storage both for claw meat and

carapace.

Reference

Astrid K. Woll, Wenche E. Larssen, Trygg Barnung, & Grete H. Aas Kvalitet og holdbarhet på hel kokt

krabbe med ulik forbehandling. Møreforsking report no. MA 10/01. 28 pp.

8.2 Shelf life related to stress parameters – Norway lobster

The ultimate shelf life of lobster products depends on two factors: progression to spoilage (decomposition) and appearance of dark melanotic spots on the shell. Spoilage is generally associated with microbial activity although autolysis is also important. The rate of spoilage is reduced by processing fresh animals, for example, freezing, treating with antimicrobial agents or rapid chilling. „Black-spot‟ or melanosis is a harmless but unsightly condition which is caused by a naturally occurring chemical reaction which creates dark pigments in the cuticle. Black spot is not necessarily an indicator of spoilage, however, it reduces the visual quality of the product and thus results in loss of commercial value. Prevention is by treatment with anti-melanotic agents such as sodium metabisulphate and rapid chilling. Both of these factors are the result of post-mortem changes; the general shelf-life of a lobster product is determined by whether it is live, fresh or frozen. A live product only retains its value as long as it is alive because without further processing, the rapid progression to spoilage or development of blackspot precludes its use as a fresh or frozen product. Consequently, when investigating the delivery of a live crustacean product from source to sale, it is vital to focus on aspects that impact on the animals‟ survival. Most significantly, these include mode of transport (i.e. wet – viviere- or dry – emersed in air for prolonged periods) and the effects of other physiological stresses.

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