1 Interactions between environmental copper, microbial community structure and histamine levels in edible crustaceans
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Interactions between environmental copper,
microbial community structure and histamine levels
in edible crustaceans
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Thesis 2011-06 of the Department of Ecological Science, VU University Amsterdam. ISBN-13: 978-94-6191-069-1 Cover photograph by Budi Widianarko and Adi Santosa, showing a traditional Javanese tambak in the northern coast of Central Java, with pak Yatin showing a live shrimp.
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VRIJE UNIVERSITEIT
Interactions between environmental copper,
microbial community structure and histamine levels
in edible crustaceans
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad Doctor aan de Vrije Universiteit Amsterdam, op gezag van de rector magnificus
prof.dr. L.M. Bouter, in het openbaar te verdedigen
ten overstaan van de promotiecommissie van de faculteit der Aard- en Levenswetenschappen
op vrijdag 2 december 2011 om 13.45 uur in de aula van de universiteit,
De Boelelaan 1105
door
Bernadeta Soedarini
geboren te Yogyakarta, Indonesië
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promotoren: prof.dr. N.M. van Straalen
prof.dr.ir. B. Widianarko
copromotoren: dr.ir. C.A.M. van Gestel
dr. W.F.M. Röling
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Contents
Summary 9 Samenvatting 15 Ringkasan 23 Chapter 1. General introduction 31 Chapter 2. Copper contamination in coastal pond
sediments of Semarang – Indonesia
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Chapter 3. Copper toxicokinetics in marbled crayfish (Procambarus sp.): implications for food quality and safety
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Chapter 4. Effect of copper exposure on histamine concentrations in the marbled crayfish (Procambarus sp.)
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Chapter 5. Interactions between accumulated copper, bacterial community structure and histamine formation in stored crayfish meat after copper exposure
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Chapter 6. General discussion 113 References 125 Acknowledgements 151
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The Irish inspiring prayer
God, give me
the serenity to accept what I can’t change;
the courage to change what I can;
and the wisdom to know the different.
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Summary
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Interactions between environmental copper,
microbial community structure and histamine levels
in edible crustaceans
Water pollution, particularly metal and microbial, is a global problem. When
the pollution occurs in aquacultural areas, it may negatively impact the
edible species and eventually threaten food quality and safety. Indonesia, one
of the top ten aquaculture producers in the world, also experiences severe
water pollution. Aquaculture is economically importance throughout
Indonesia, like for Semarang – a major city on the Northern coast of Central
Java – known as one of aquaculture producers. One of the most important
aquaculture activities in Semarang is shrimp culture. Many ponds used for
shrimp and fish culture are situated along the coast line and are influenced by
metal pollution from a variety of sources and microbial pollution from city
drainage. For that reason, we studied the influence of metal pollution and
microbial contamination in an edible crustacean species. The main aim was
to determine the interaction between copper and microorganisms in the
aquatic environment and their impact on crustaceans. To deal with the
problem, several approaches were selected, i.e. metal bioaccumulation,
histamine formation in the animal triggered by metals exposure, bacterial
community structure under the influence of metal contamination and
histamine accumulation in the crustacean’s edible tissue during storage.
The metal of interest in this study was copper, referring to the situation found
in shrimp ponds along the Semarang coast line (Chapter 2). In three
sampling sites, copper concentrations were significantly higher than those of
the other sampling sites, up to twice the local background concentration,
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which is approximately 40 µg/g dry sediment. Cadmium concentrations in
sediment samples taken from different aquacultural ponds in the Semarang
coastal area were below the detection limit (0.03 μg/g dry weight), while
zinc, nickel and iron concentrations varied among locations but were
generally also low. Sediments taken from two shrimp ponds located close to
the intercity highway of Semarang had lead (Pb) concentrations that were
significantly higher than at the other locations. In addition to copper and
lead, at some sites nickel and chromium also showed elevated sediment
concentrations. According to the Sediment Quality Guidelines (SQGs)
applied in several countries, the Semarang coastal pond sediments can be
classified as mid-range (medium range) contaminated. The contamination
may partly be attributed to anthropogenic inputs, such as intensification of
agricultural and aquacultural practices, and metal-contaminated waste from
industrial and household activities.
The interaction between copper, microbial community structure and
histamine level was studied by performing a set of experiments. Because of
its accessibility for biological experiments, i.e. ease of culturing and
producing high numbers of genetically identical offspring, the freshwater
marbled crayfish (Procambarus sp.) was chosen as a model of edible
crustacean species (Chapters 3, 4 and 5). Our study on copper toxicokinetics
in marbled crayfish (Procambarus sp.) showed there was no clear uptake in
animals exposed to 0.031 mg Cu/L, suggesting that the animals at low
copper exposure levels are able to regulate copper concentrations in their
body to a fairly constant level. However, at higher exposure levels (0.38 mg
Cu/L) the internal copper concentration was not regulated at the same level,
at least not in all organs. The exoskeleton, gills and muscle tissues
accumulated copper relatively fast and reached equilibrium within 10 days of
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exposure. Copper accumulation was highest in the hepatopancreas as uptake
in this storage organ steadily increased with time and did not reach
equilibrium within the 14-day exposure period (Chapter 3). The copper
accumulation levels in the marbled crayfish found in this study were
hepatopancreas > gills > exoskeleton > muscle. In terms of food safety, the
highest copper concentrations measured in the marbled crayfish muscles
(meat) were 40 µg/g dry weight (~10 µg/g wet weight). This level does not
exceed the recommendations set by the Australian National Health and
Medical Research Council (ANHMRC) for seafood, which is 10 µg/g wet
weight.
In Chapter 4, we observed that metal accumulation in marbled crayfish
organs affected the concentrations of histamine, an important indicator of
food spoilage used in food safety research. The higher the copper exposure
concentrations, the higher the histamine levels were in the hepatopancreas. A
rapid built-up of histamine in the hepatopancreas started right from the
beginning of the copper exposure of the crayfish. Copper exposure to
average concentrations of 0.031 and 0.38 mg Cu/L did not affect histamine
concentrations in the crayfish muscle. In contrast, histamine concentrations
in the hepatopancreas of crayfish exposed to 0.38 mg Cu/L was significantly
higher than in crayfish exposed to 0.031 mg Cu/L and reached approximately
10 mg histamine/kg fresh weight. Histamine is a well-known
neuromodulator (besides dopamine) in the animal nervous system. Histamine
is specifically associated with modulation of muscle action in intestinal
tissues. Why histamine levels in the crayfish would increase with copper
accumulation is unknown. This is an interesting phenomenon but so far has
not been reported in the literature. Two proposed mechanisms are (1) a stress
response of the animal, or (2) up-regulation of histidine production followed
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by decarboxylation reactions. Histamine concentrations in the muscle in all
cases never exceeded 2 mg histamine/kg fresh weight, which is much lower
than the maximum level of histamine in seafood of 50 mg histamine/kg fresh
weight set by the United States Food and Drug Administration (US FDA).
Our experiment on histamine formation in the marbled crayfish triggered by
copper exposure therefore indicated that in terms of the meat, copper
exposure itself did not pose any threat for seafood safety.
In Chapter 5, the bacterial community structures under the influence of
copper contamination and their effect on the histamine accumulation in the
marbled crayfish meat during storage are described. Cluster analysis of 16S
rRNA gene-based microbial community fingerprints revealed copper toxicity
to the freshwater bacterial community. Further, we observed a relation
between bacteria in the water and bacteria playing role in deteriorative
process in crayfish meat during storage. Histamine concentrations in the
meat of the marbled crayfish exposed to 0.5 mg Cu/L upon storage were
significantly lower and did not increase as rapidly compared to those in the
control marbled crayfish. After 10 days of storage, meat from crayfish
exposed to 0.5 mg Cu/L contained approximately 7.5 mg histamine/kg fresh
weight, significantly less than the meat of animals incubated in copper-free
water, which approximated 22 mg/kg fresh weight. We suggest that copper
exposure can slow down histamine accumulation in crayfish meat during
storage through affecting the composition of bacterial communities and the
associated histamine production.
According to our findings on the copper accumulation in the marbled
crayfish and by extrapolating of these results to other crustaceans (Chapter
3), it is recommended to consume only muscle tissue (meat) rather than the
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whole body of crustaceans. Consuming small size edible crustaceans, where
it is hard to avoid eating the gills and hepatopancreas, which usually contain
the highest metal concentrations, indeed may pose a hazard for food safety.
Histamine levels induced by copper exposure, however, generally are much
lower than the threshold level for food safety.
Furthermore, copper concentrations in the water up to 0.5 mg Cu/L have
been shown to have a large impact on the microbial community structure in
both the culturing water and in the stored marbled crayfish meat. Copper
exposure can decrease histamine levels in crayfish meat during storage
through affecting the bacterial community-associated histamine production
(Chapter 5). In spite of these findings and recommendations, still several
uncertainties remain. Future research therefore might focus e.g. on (1) the
mechanism(s) of histamine formation triggered by copper exposure, (2)
copper uptake and internal distribution in different species of crustaceans,
especially species living in brackish water, (3) determination of sources of
bacterial contamination – from the aquatic environment or from seafood
handling – that play a major role in histamine accumulation, (4) impact of
other chemicals of concern for the coastal areas of Java, such as Semarang
on microbial communities in crustaceans, especially on the histamine-
producing microorganisms.
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Samenvatting
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Interaktie tussen koper in oppervlaktewater, de
structuur van microbiële levensgemeenschappen en
histamine concentraties in eetbare kreeftachtigen
Vervuiling van oppervlaktewater, vooral met metalen en microorganismen,
is een algemeen probleem. Wanneer de vervuiling plaatsvindt in gebieden
waarin op commerciële schaal waterorganismen voor consumptie worden
geproduceerd (zogenaamde aquacultuur), kan dit leiden tot een belasting van
deze organismen en zo de kwaliteit en veiligheid van het voedsel bedreigen.
Ook in Indonesië, dat behoort tot de top-tien van landen met aquacultuur, is
sprake van vervuiling van oppervlaktewateren. Aquacultuur is van groot
economisch in heel Indonesië. Dit geldt ook voor Semarang, een stad langs
de noordkust van Centraal Java. Een van de belangrijkste activiteiten op het
gebied van aquacultuur in Semarang betreft de kweek van kreeftachtigen,
met name van garnalen. Om die reden is in dit onderzoek de invloed van de
belasting met metalen en microbiële contaminatie in een eetbare
kreeftachtige onderzocht. Het voornaamste doel van dit onderzoek was de
interactie te bepalen tussen koper en microorganismen in oppervlaktewater
en hun invloed op kreeftachtigen. Voor dit onderzoek zijn verschillende
aspecten onderzocht, zoals de bioaccumulatie van metalen, de vorming van
histamine in de dieren onder invloed van metaalvervuiling, en de invloed van
metaalvervuiling op structuur van de bacteriële levensgemeenschap en de
accumulatie van histamine in de eetbare delen van de kreeft gedurende
opslag.
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Met het oog op de situatie die werd gevonden in aquacultuur vijvers langs de
kust bij Semarang (Hoofdstuk 2), werd koper als metaal voor dit onderzoek
gekozen. Cadmium concentraties in sediment monsters van verschillende
garnalenkweekvijvers in dit gebied lagen beneden de detectiegrens (0,03
μg/g drooggewicht), terwijl de concentraties aan zink, nikkel en ijzer
varieerden maar over het algemeen laag waren. Sediment van twee garnalen-
kweekvijvers dicht bij de snelweg van Semarang bevatte hoge gehalten aan
lood (Pb), die significant hoger waren dan op de andere locaties. Op drie
lokaties waren de kopergehalten in het sediment significant hoger dan in de
andere vijvers, tot tweemaal de locale achtergrondconcentratie van 40 µg/g
droog sediment. Naast koper en lood werden op sommige locaties ook
verhoogde concentraties nikkel en chroom in het sediment aangetroffen.
Volgens de Sedimentkwaliteitscriteria (SQGs) die in verschillende landen
worden gehanteerd zijn de garnalenkweekvijvers langs de kust bij Semarang
matig vervuild. De vervuiling kan deels worden toegeschreven aan
menselijke invloeden, zoals intensieve landbouw en aquacultuur en de lozing
van metaalvervuild afval van industriële en huishoudelijke activiteiten.
De interactie tussen koper, structuur van de microbiële levengemeenschap en
histamine gehalten werden onderzocht door middel van een serie
experimenten. Vanwege de geschiktheid voor biologische experimenten,
zoals goede kweekbaarheid en de productie van grote aantallen genetische
identieke nakomelingen, werd de zoetwater marmerkreeft (Procambarus sp.)
gekozen als model voor eetbare kreeftachtigen (Hoofdstukken 3, 4 en 5). Het
onderzoek naar de toxicokinetiek van koper in de marmerkreeft
(Procambarus sp.) liet zien er nauwelijks sprake is van koperopname in de
dieren bij blootstelling aan 0,031 mg Cu/L. Dit suggereert dat de dieren bij
lage blootstellingsconcentraties in staat zijn het kopergehalte in hun lichaam
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op een vrij constant niveau te houden (regulatie). Bij een hogere
blootstellingsconcentratie (0,38 mg Cu/L) kon het interne kopergehalte niet
meer worden gereguleerd, tenminste niet in alle organen. Het exoskelet, de
kieuwen en het spierweefsel vertoonden een snelle accumulatie van koper,
waarbij binnen ongeveer 10 dagen een evenwicht werd bereikt. De koper-
accumulatie was het hoogst in de hepatopancreas, en het kopergehalt in dit
opslagorgaan bleef toenemen met de tijd; ook na 14 dagen blootstelling was
geen evenwicht bereikt (Hoofdstuk 3). De kopergehalten in de verschillende
organen van de marmerkreeft namen af in de volgorde hepatopancreas >
kieuwen > exoskelet > spierweefsel. Het hoogste kopergehalte dat werd
gemeten in het spierweefsel (vlees) van de marmerkreeft bedroeg 40 µg/g
drooggewicht (~10 µg/g versgewicht). Met het oog op voedselveiligheid kan
dus worden geconcludeerd dat de kopergehalten in het vlees van de
marmerkreeft onder de grens bleven, die wordt aanbevolen door de
Australian National Health and Medical Research Council (ANHMRC) voor
visproducten, die 10 µg/g versgewicht bedraagt.
In Hoofdstuk 4 werd onderzocht hoe de metaalaccumulatie in de organen
van de marmerkreeft de vorming van histamine beïnvloeden. Histamine is in
de monitoring van voedselveiligheid een belangrijke indicator voor bederf
van voedsel. Hoe hoger de blootstellingconcentraties aan koper, des te hoger
waren de histaminegehalten in de hepatopancreas. Een snelle toename van
histamine werd waargenomen in de hepatopancreas kort na het begin van de
koperblootstelling. Blootstelling aan gemiddelde concentraties van 0,031 en
0,38 mg Cu/L had geen invloed op de histaminegehalten in het spierweefsel
van marmerkreeft. In de hepatopancreas van kreeften blootgesteld aan 0,38
mg Cu/L waren de histaminegehalten significant hoger dan in dieren
blootgesteld aan 0,031 mg Cu/L, met waarden van ongeveer 10 mg
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histamine/kg versgewicht. Histamine is een van de belangrijkste
modulatoren van het zenuwstelsel (naast dopamine). Histamine wordt
specifiek geassocieerd met modulatie van de werking van spieren in het
maagdarmkanaal. Het is echter onbekend waarom koperopname leidt tot een
toename van de histaminegehalten in kreeften. Dit fenomeen is voor zover
bekend niet eerder beschreven in de literatuur. De mechanismen die dit
fenomeen zouden kunnen verklaren zijn (1) een stress reactie van het dier, of
(2) een stimulering van de histidineproductie gevolgd door decarboxylerings-
reacties. Histaminegehalten in het spierweefsel van de marmerkreeften
waren nooit hoger dan 2 mg histamine/kg versgewicht, wat veel lager is dan
het maximale gehalte van 50 mg histamine/kg versgewicht in visproducten
dat is vastgesteld door de United States Food and Drug Administration (US
FDA). Het onderzoek naar de histaminevorming in marmerkreeften laat dus
zien dat de blootstelling aan koper geen invloed heeft in termen van
voedselveiligheid.
In Hoofdstuk 5 wordt het effect van koperbelasting op de structuur van de
bacteriële levensgemeenschap in het water en de marmerkreeft beschreven,
en het effect hiervan op de accumulatie van histamine in het vlees van de
marmerkreeft tijdens bewaren. Clusteranalyse van 16S rRNA-gebaseerde
‘fingerprints’ toonde aan dat koper een effect had op de samenstelling van de
bacteriële levensgemeenschap in het water waarin de kreeften werden
gehouden. Ook werd een relatie gevonden tussen de bacteriën in het water en
de bacteriën die een rol spelen in de afbraakprocessen die leiden tot bederf
van het vlees van de kreeften tijdens opslag. Histaminegehalten in marmer-
kreeften blootgesteld aan 0,5 mg Cu/L bleven tijdens bewaren significant
lager en namen veel minder snel toe dan in kreeften die niet aan koper waren
blootgesteld. Na 10 dagen bewaren bevatte het vlees van kreeften
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blootgesteld aan 0,5 mg Cu/L ongeveer 7,5 mg histamine/kg versgewicht,
hetgeen significant lager is dan dat in vlees van dieren gehouden in koper-
vrij water (ongeveer 22 mg/kg versgewicht). Deze resultaten suggereren dat
blootstelling aan koper de histamine-accumulatie in het vlees van de kreeften
tijdens bewaren kan vertragen doordat het een effect heeft op de bacteriële
levensgemeenschap die verantwoordelijk is voor de histamineproductie.
Op grond van onze bevindingen met betrekking tot de accumulatie van koper
in de marmerkreeft (Hoofdstuk 3) wordt aanbevolen om alleen het
spierweefsel (vlees) te consumeren in plaats van de hele kreeft. Consumptie
van kleine kreeftachtigen, waarbij het moeilijk is het eten van kieuwen en
hepatopancreas-weefsel, kan wel leiden tot een risico voor voedselveiligheid
omdat deze organen de hoogste gehalten aan metalen bevatten.
Histaminegehalten geïnduceerd door koperblootstelling blijven over het
algemeen ruim beneden de grenzen voor voedselveiligheid.
Kopergehalten tot 0,5 mg Cu/L blijken een grote invloed te hebben op de
microbiële levensgemeenschap, zowel in het kweekwater als in de het
opgeslagen vlees van de marmerkreeft. Blootstelling aan koper kan de
histaminegehalten in het vlees van de kreeften verlagen, doordat het de
microbiële gemeenschappen beinvloedt die verantwoordelijk zijn voor de
vorming van histamine (Hoofdstuk 5). Ondanks deze bevindingen blijven
nog vele onzekerheden bestaan. Verder onderzoek is nodig naar (1) de
mechanisme(n) van de vorming van histamine onder invloed van
koperbelasting, (2) de opname en interne verdeling van koper in
verschillende soorten kreeftachtigen, met name ook in soorten die in brak
water leven, (3) bepaling van de bron van bacteriële contaminatie – vanuit
het aquatische milieu of tijdens het verwerken van de kreeften en garnalen –
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die een belangrijke rol spelen in de vorming en accumulatie van histamine,
(4) de invloed van andere potentieel gevaarlijke stoffen in het kustgebied van
Semarang op de microbiële levensgemeenschap in kreeftachtigen, en de
daarmee samenhangende histamineproductie.
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Ringkasan
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Interaksi antara paparan tembaga, struktur
komunitas mikroba dan kadar histamin
dalam udang
Pencemaran air, khususnya logam dan mikroba, merupakan permasalahan
global. Apabila pencemaran tersebut terjadi di area budidaya perikanan,
dampak negatif terhadap spesies yang dibudidayakan, yang mengancam
kualitas dan keamanan pangan hasil perikanan yang dihasilkan,
kemungkinan besar terjadi. Budidaya perikanan memiliki peranan yang
penting dalam perekonomian Indonesia, termasuk juga bagi Semarang – kota
utama di pesisir utara Jawa Tengah – yang dikenal sebagai salah satu sentra
perikanan. Udang dipilih sebagai obyek dalam studi ini karena udang
merupakan salah satu produk budidaya perikanan yang terpenting di
Semarang. Tujuan utama studi ini adalah untuk menelaah interaksi antara
cemaran logam dan mikroba yang ada dalam air serta dampaknya terhadap
udang, ditinjau dari kualitas dan keamanan pangan. Pendekatan ilmiah yang
dipilih dalam studi ini antara lain meliputi bioakumulasi logam,
pembentukan histamin dalam tubuh udang akibat paparan logam, serta
pengaruh pencemaran logam terhadap stuktur komunitas bakteri dalam
daging udang serta pengaruhnya terhadap tingkat akumulasi histamin selama
penyimpanan.
Tembaga (Cu) dipilih sebagai fokus dalam studi ini berdasarkan hasil
analisis logam yang terkandung dalam sedimen-sedimen yang diambil dari
tambak-tambak udang di sepanjang pantai Semarang (Bab 2). Sedimen di
tiga lokasi pengambilan sampel menunjukkan cemaran tembaga yang jauh
lebih tinggi dibandingkan lokasi-lokasi lain, yang mencapai hampir dua kali
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lipat dari kadar tembaga normal untuk lingkungan Semarang (the local
background concentration ) yaitu berkisar 40 μg/g sediment kering.
Konsentrasi cadmium dalam semua sampel sedimen sangat rendah, lebih
rendah dari tingkat kepekaan alat analisis logam yang digunakan (0,03 μg/g
sampel kering), sedangkan konsentrasi seng, nikel dan besi bervariasi antar
lokasi, meskipun secara umum kadarnya rendah. Berbeda dengan tambak di
lokasi-lokasi lain, sedimen yang diambil dari dua tambak yang terletak di
dekat Jalan Arteri Utara Semarang menunjukkan adanya cemaran logam
timbal. Selain tembaga dan timbal, beberapa lokasi tercemar nikel dan krom
pada kadar yang cukup tinggi. Mengacu pada pedoman-pedoman penilaian
kualitas sedimen (sediment quality guidelines ) yang berlaku di beberapa
negara, sedimen tambak di perairan Semarang dapat diklasifikasikan “cukup
tercemar”. Pencemaran tersebut mengindikasikan adanya pembuangan
limbah dari berbagai sumber yang mengandung logam, sebagai akibat dari
intensifikasi pertanian dan perikanan serta pembuangan limbah pabrik
maupun limbah rumah tangga ke badan-badan air.
Dalam studi ini, serangkaian percobaan telah dilakukan. Udang-karang air
tawar (Procambarus sp.) dipilih sebagai hewan model dengan pertimbangan
antara lain kemudahannya untuk dikembang-biakkan serta kemampuannya
menghasilkan keturunan yang identik secara genetik serta dalam jumlah
yang besar (Bab 3, 4 dan 5). Studi toksiko-kinetika logam tembaga dalam
udang-karang air tawar (Procambarus sp.) menunjukkan tidak adanya pola
penyerapan yang jelas ketika udang mendapat paparan tembaga pada
konsentrasi 0,031 mg Cu/L. Hal tersebut dapat diartikan bahwa pada kadar
paparan relatif rendah, hewan masih mampu mengatur stabilitas kadar
tembaga dalam tubuhnya. Namun demikian, pada tingkat paparan yang lebih
tinggi, kadar tembaga dalam tubuh hewan meningkat, meskipun penyerapan
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tidak terjadi pada tingkat yang sama di setiap organ tubuhnya. Cangkang
luar, insang, daging dan telur relatif menyerap tembaga dengan cepat, dan
segera mencapai titik keseimbangan (stabil) kurang dari 10 hari dari durasi
paparan. Akumulasi tembaga tertinggi terjadi di jaringan hati dan pankreas
(hepatopancreas), hal tersebut tampak dari penyerapan yang terjadi terus-
menerus hingga hari ke-14 masa paparan, serta belum tercapainya titik
keseimbangan pada akhir masa paparan tersebut (Bab 3). Studi ini
menunjukkan tingkat akumulasi tembaga dalam tubuh udang-karang dari
mulai dari yang tertinggi hingga terendah adalah hepatopancreas > insang >
cangkang luar > daging. Ditinjau dari aspek keamanan pangan, kadar
tembaga tertinggi dalam jaringan daging udang-karang rerata berkisar 40
μg/g berat kering (~10 µg/g berat basah). Kadar tersebut tidak melampaui
kadar tembaga maksimum yang direkomendasikan oleh Dewan Nasional
Penelitian Kesehatan dan Medis Australia (ANHMRC) untuk makanan hasil
laut, yaitu 10 µg/g berat basah.
Di Bab 4 dibahas mengenai peningkatan kadar histamin dalam udang-karang
air tawar akibat paparan tembaga. Histamin merupakan salah satu indikator
kerusakan bahan pangan yang umum digunakan sebagai acuan keamanan
pangan. Kadar histamin dalam hewan meningkat seiring dengan peningkatan
kadar tembaga, dan peningkatan tertinggi terjadi di hepatopancreas.
Histamin terbentuk dengan segera setelah hewan mendapat paparan tembaga.
Paparan tembaga pada konsentrasi 0,031 dan 0,38 mg Cu/L tidak secara
signifikan berpengaruh terhadap peningkatan histamin dalam jaringan daging
udang. Rerata kadar histamin dalam hepatopancreas udang-karang setelah
mendapat paparan tembaga 0,38 mg Cu/L adalah 10 mg histamin/kg berat
basah. Histamin dikenal sebagai neuromodulator utama (selain dopamin)
yang berperanan dalam sistem saraf hewan. Histamin terutama berperan
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dalam perangsangan kerja otot jaringan usus. Fenomena peningkatan kadar
histamin seiring dengan peningkatan akumulasi tembaga ini merupakan
fenomena baru yang menarik, dan belum pernah dipublikasikan sebelumnya.
Mekanisme yang terjadi dibalik fenomena tersebut kemungkinan adalah (1)
hewan mengalami stress and peningkatan kadar histamine merupakan respon
fisiologis dari stress tersebut, (2) terjadi perubahan pengaturan produksi
asam amino histidin yang diikuti dengan reaksi dekarboksilasi sehingga
menghasilkan peningkatan kadar histamin. Kadar histamin yang terukur
dalam sampel jaringan daging tidak pernah lebih dari 2 mg histamin/kg berat
basah, jauh lebih rendah dibandingkan standar histamin maksimum yang
diijinkan ada dalam makanan hasil laut seperti yang ditetapkan oleh Food
and Drug Administration Amerika Serikat (US FDA) tahun 2011.
Di Bab 5 dijelaskan mengenai pengaruh cemaran tembaga terhadap struktur
komunitas bakteri serta akibat-lanjutnya terhadap akumulasi histamin dalam
daging udang selama penyimpanan. Analisis klaster terhadap komunitas
bakteri yang didasarkan pada 16S r-RNA gene (teknik molekuler)
menunjukkan adanya toksisitas tembaga terhadap komunitas bakteri air
tawar. Selain itu, nampak bahwa bakteri dalam air memiliki keterkaitan
dengan bakteri penyebab pembusukan daging udang selama penyimpanan.
Kadar histamin dalam daging udang yang sebelumnya mendapat paparan 0,5
mg Cu/L tidak hanya lebih rendah tetapi juga meningkat secara lambat
dibandingkan daging udang kontrol. Setelah penyimpanan 10 hari,
kandungan histamin dalam daging udang yang ketika masih hidup mendapat
paparan tembaga 0,5 mg Cu/L hanya berkisar 7,5 mg histamin/kg berat
basah, jauh lebih rendah daripada daging udang yang ketika masih hidup
diinkubasi dalam air bebas tembaga yang mencapai sekitar 22 mg
histamin/kg berat basah. Mempertimbangkan bahwa paparan tembaga
28
terhadap udang hidup ternyata memperlambat akumulasi histamin dalam
daging udang selama penyimpanan, nampaknya peningkatan histamin dalam
daging udang sangat tergantung pada komunitas bakteri yang berada di
dalam air tempat hidup udang tersebut.
Mengacu pada fakta hasil penelitian mengenai akumulasi tembaga dalam
tubuh udang-karang air tawar (Bab 3), disarankan konsumsi udang sebaiknya
dibatasi hanya pada bagian dagingnya saja. Konsumsi udang berukuran
kecil, akan memunculkan kerumitan untuk memisahkan bagian insang dan
hepatopancreas dari bagian dagingnya, tentu memberikan risiko keamanan
pangan yang lebih tinggi. Peningkatan kandungan histamin yang diakibatkan
paparan tembaga, bagaimanapun jauh lebih rendah dari batasan terendah
(threshold) untuk keamanan pangan (Bab 4).
Lebih lanjut, kadar tembaga dalam air yang mencapai 0,5 mg Cu/L
memberikan pengaruh nyata terhadap struktur komunitas mikroba baik
mikroba dalam air aquarium maupun mikroba dalam daging udangnya.
Paparan tembaga dapat menekan kadar histamin dalam daging udang selama
masa penyimpanan karena tembaga mempengaruhi komunitas bakteri,
khususnya bakteri penghasil histamin (Bab 5). Meskipun dalam studi ini
beberapa penemuan, fenomena baru dan rekomendasi telah berhasil
diungkapkan, masih terdapat beberapa pertanyaan ilmiah yang dapat diteliti
lebih lanjut. Beberapa topik penelitian yang dapat dikembangkan di masa
mendatang antara lain (1) mekanisme pembentukan histamin yang dipicu
oleh paparan tembaga, (2) penyerapan tembaga dan distribusinya dalam
beberapa spesies crustacea, khususnya yang hidup di air payau, (3)
penentuan sumber kontaminasi utama – apakah dari lingkungan air ataukah
dari penanganan pasca panen – yang memainkan peran utama dalam
29
akumulasi histamin, serta (4) pengaruh cemaran-cemaran lain yang terdapat
di perairan pantai utara Pulau Jawa, seperti halnya Semarang terhadap
komunitas mikroba dalam crustacea, khususnya mikroba penghasil histamin.
30
31
Chapter 1
General introduction
32
Food security is a global issue of continuous concern. The Food and
Agriculture Organization of the United Nations (FAO, 2010) has defined
food security as “an existence when all people, at all times, have physical,
social and economic access to sufficient, safe and nutritious food that meets
their dietary needs and food preferences for an active and healthy life”. This
definition covers three main aspects of the food security concept: (1) food
sufficiency, (2) food safety and (3) nutritional value. These three aspects of
concern are sometimes arranged in order of importance, but the fundamental
message is that in terms of global food security, all three aspects should be
taken into account.
Providing food for the global population in a sufficient amount is not only a
matter of optimal performance of the food production sector, but also a
concern because of the still growing global human population. In the 20th
century, the world population increased 3.7-fold to reach 6.3 billion people
in 2003 and currently is expected to grow to 8.9 billion by 2050 (Cohen,
2003). The ever increasing number of people definitely is a challenge to
agriculture and aquaculture – two main food production sectors besides wild-
fishery and terrestrial wildlife hunting. The green revolution, which took
place between 1950 and 1970, has greatly reduced food shortage through
increasing crop yields as well as livestock production, using high-yielding
plant and animal varieties, synthetic fertilizers and biocides (Evenson and
Gollin, 2003). In addition, the rapidly growing aquaculture sector has applied
intensification methods to boost production. Especially between 1987 and
1997, aquacultural production increased more than twofold in weight and
value of its products (Naylor et al., 2000). The so-called “rapid-aquaculture”
by means of semi-intensive and intensive practices was highly needed
33
because of the declining worldwide fishery stocks that became clear since
1985 (Naylor et al., 2000).
Year Figure 1. The average number of undernourished people in the world over the period 1969-1971 to 2010, based on the Food and Agriculture Organization (FAO, 2010). The X-axis represents the year and the Y-axis represents the number of undernourished people (in millions). Data for 2009 and 2010 are presented in dashed lines as they are estimated by FAO with input from the United States Department of Agriculture, Economic Research Service.
FAO (2006) stated that global aquaculture has grown tremendously during
the last 50 years, from a production level of less than a million tonnes in the
early 1950s to 59.4 million tonnes by 2004. Due to the growth of aquaculture
production, its products now contribute to at least 15% of the global average
animal-protein consumption (Smith et al., 2010).
34
The three main reasons for the global hunger problem are the low
productivity of agriculture (especially in tropical Africa and remote parts of
Asia as well as Latin America), poverty, and unemployment (especially in
South and East Asia, Latin America, Central Asia, and the Middle East).
From an economical point of view, hunger decreases labour productivity
leading to losses of 6 to 10% of the Gross National Product (Sachs et al .,
2004 in Sanchez and Swaminathan, 2005). Considering that small-scale
agriculture and aquaculture farming families represent about half of the
hungry population worldwide, increasing the productivity of their crops,
livestock, fish, and trees is a major way to fight the global hunger problem.
Actions to restore soil fertility and to increase agricultural productivity might
include, among others, the use of appropriate combinations of mineral and
organic fertilizers, leguminous green manures, agro-forestry fertilizer trees
and returning crop residues to the soil as well as improving water
management. In terms of aquaculture, culturing of fast-growing species is a
progressive strategy to produce more seafood in less time (Brummett and
Williams, 2000). Considering that malnutrition weakens immunity, making
humans more susceptible to diseases (Kadiyala and Gillespie, 2003), fighting
global malnutrition is very important to improve human health and the
quality of life. Providing adequate nutrition in the diet via food fortification,
clean drinking water, protein-rich foods (beans and seafood) and
micronutrient-rich foods (vegetables and fruits) are major elements of the
program of the Millennium Development Goals targeted by the United
Nations for 2015 (Sanchez and Swaminathan, 2005). Since 2003 the World
Health Organization (WHO) issued a recommendation for most countries to
regularly consume seafood (Sioen et a l., 2007). Indeed, seafood contains
high levels of protein and omega-3 polyunsaturated fatty acids that are
essential for brain development and beneficial for human health in
35
comparison to meat which has a high content of saturated fatty acids
(Nettleton, 1991). Furthermore, compared to cheese, eggs, beef, chicken or
any legumes, seafood has a balanced content of proteins with all 20 amino
acids being present (Sosulski and Imafidon, 1990).
The favourable nutritious profile of food is unfortunately often counteracted
by factors that may have adverse health effects. Legumes, especially peanuts,
frequently cause allergy, sometimes with laryngeal oedema and asthma
reactions that can be fatal (Ewan, 1996). Besides, peanuts also may contain
aflatoxins – produced by the fungus Aspergillus flavus – that may cause liver
damage (Timbrell, 1989). The wild mushrooms Cortinarius specio sissimus
and C. orellanus , which have the characteristic orange gills and thick stipe,
were first recognized to cause renal failure in 1972 in Finland (Short et al .,
1980). Orelline and orellanine, the toxic compounds of C. orellanus , are
highly resistant to heat, freezing, and drying. Ohlson and Anjou (1979)
observed that rapeseed and mustard (Brassica napus , B. campestris and B.
juneea), although containing 40-45% oil and 25% high nutritive quality
proteins, also contain 4% glucosinolates –compounds that cause the thyroid
to increase in weight (goitrogenic effect).
Three classes of disease are associated with seafood consumption, i.e.
intoxication, infection and allergy (Eastaugh and Shepherd, 1986). There are
several examples of natural toxins in seafood, among others phycotoxins
(Van Egmond, 2004) and tetrodotoxin (Watters, 1995; Daly, 2004).
Phycotoxins are produced by dinoflagellate algae that are accumulated by
planktivorous marine animals, especially shellfish. The most important
marine phycotoxins are the shellfish-poisons that accumulate in mussels,
oysters and clams, and the ciguatera toxins that accumulate in finned fish.
36
Two types of shellfish poisoning are known: diarrhetic shellfish poisoning
(DSP), which involves severe gut convulsions and diarrhea due to effects of
okadaic acid on intestinal cells, and paralytic shellfish poisoning (PSP),
which is due to saxitoxin and involves confusion, lack of coordination and
can be fatal in immunocompromised individuals. Tetrodotoxin is considered
as the most lethal seafood toxin and is especially found in the puffer fish
(Spheroides rubripes ) and the marine goby fish (Yongeichthys criniger )
(Daly, 2004). The approximate LD50 value of tetrodotoxin for human beings
is 0.1 mg/kg body weight, a thousand times more toxic than, for instance, the
organochlorine insecticide DDT (Timbrell, 1989). Tetradotoxin poisoning is
especially known in Eastern Asia. Fugu is a delicacy puffer fish in the
Japanese kitchen, which can only be prepared in restaurants where the cook
has a special licence to remove the highly toxic liver and ovary. Most
accidents related to puffer fish consumption happen due to lack of
knowledge and reckless behaviour of consumers. Ciguatera is a symptom of
seafood poisoning due to food-chain accumulation of ciguatoxin. This very
persistent compound, produced by dinoflagellate algae, accumulates in fish
and may reach high concentrations in predatory fish such as barracuda and
grouper. Ciguatera is especially known from the Caribean.
Seafood is also known as the vehicle for a variety of pathogens (Klaassen
and Watkins III, 2010; Wallace et al., 1999). Butt et al. (2004) distinguished
three groups of infectious agents, i.e. viruses, bacteria and other parasites
that may cause seafood-related illnesses. Several outbreaks of viral
gastroenteritis associated with clam and oyster consumption have been
reported by Morse et al . (1986). The existence of infectious human
caliciviruses and hepatitis A virus in seafood is a result of poor hygiene and
sanitation, either in aquaculture production or during seafood handling. The
37
existence of infectious bacteria in seafood, such as Vibrio parahaemolyticus,
Listeria monocytogenes , Aeromonas hydrophila and Campylobacter jejuni ,
is also of concern. Seafood consumption is estimated to cause an average 10-
19% of the 76 million food-borne illnesses reported in the United States
every year (Butt et al ., 2004). Bacterial pathogens implicated in seafood-
borne diseases may be indigenous to the marine or estuarine environment or
may involve enteric bacteria due to faecal contamination, or contamination
during seafood handling and processing (Feldhusen, 2000).
A special aspect of bacterial contamination in seafood is the accumulation of
histamine, especially in crustaceans (shrimp, prawn, crab and crayfish).
Histamine is a derivative of the amino acid histidine (Sobel and Painter,
2005). A group of so-called histamine-producing bacteria possess histidine
decarboxylase, an enzyme that transforms the free amino acid histidine into
histamine (Niven et al ., 1981; see Figure 2). Seafood containing levels
higher than 50 mg histamine/kg may cause histamine poisoning with
allergic-like symptoms such as nausea, vomiting, diarrhea, hives, itching, red
rash, and hypotension (Taylor et al ., 1989). Several histamine poisoning
outbreaks (known as scombrotoxism or scombroid poisoning) via seafood
consumption have been documented in the United States (Feldhusen, 2000),
South Africa (Auerswald et al ., 2006) and Denmark (Emborg et al ., 2005).
Especially in crustaceans, high concentrations of histamine may not only be
caused by bacteria, but may also be naturally present in the animal as
histamine acts as a neurotransmitter or neuromodulator (Cebada and Garcia,
2007). Histamine is often used as an indicator of food spoilage in studies
aiming to determine the safe consumption of stored seafood products. With
prolonged storage, even under cooled conditions, shrimps and prawns will
gradually degrade until consumption becomes hazardous. One of the first
38
indicators of health risk is a rise of the histamine concentration. Therefore
histamine is often measured routinely in shelf-life studies.
Figure 2. Conversion of histidine to histamine by histidine decarboxylase (http://en.wikipedia.org/wiki/File:Histidine_decarboxylase.svg).
In addition to the risk of poisoning, infection and allergy caused by natural
agents, consuming seafood is also endangered by chemical pollutants.
Availability of safe food is a basic human right as it improves human health
and contributes to the productivity of daily activities (WHO, 2002). But
ensuring the availability of safe food requires a continuous effort due to the
environmental quality degradation occurring in many areas of the world.
Although the green revolution and the rapid-aquaculture contributed
significantly to the world food supply, they also may have negative impacts
on the environment (Tilman et al ., 2001; Badgley et al ., 2006).
Contamination of surface and groundwater by pesticides, biocides and other
chemicals derived from intensified agricultural practices is taking place
globally. Moreover, the ever increasing industrial activity and the application
of a great variety of chemicals in industrial sectors definitely cause an extra
load of potentially toxic compounds to the environment. Also, chemicals
used in personal care products, pharmaceutical products and marine
antifouling agents are expected to contribute to environmental quality
degradation.
Many aquatic species are able to accumulate pollutants in their bodies
(Rainbow, 2002). The accumulation capacity is well-known for mussels,
39
which may contain environmental pollutants in concentrations several orders
of magnitude higher than in the water. Also crustaceans may accumulate
pollutants, which are retained in the hepatopancreas. The high concentrations
of pollutants in some representatives of the marine coastal environment may
form a basis for biomonitoring programmes which aim to document temporal
or geographic trends (Van Straalen, 2008). For example, in the 1980s a
“mussel watch” programme was set-up to monitor pollution along the West
coast of the US (Farrington et al., 1983).
Environmental pollution has increased worldwide due to industrial activity
but also due to intensification of agricultural and aquaculture practices. The
increased global population also produces an increasing amount of waste that
is released into the environment via wastewater, waste incineration processes
and controlled or uncontrolled dumping of solid wastes. Many toxic
pollutants present in the water may end up in animal organs through
bioaccumulation. Several researchers reported that persistent organochlorine
compounds, such as polychlorinated biphenyls (PCBs), dioxin-like
compounds and toxic metals like mercury and cadmium are found in seafood
(Smith and Gangolli, 2002; Bayen et al., 2005; Moon and Ok, 2006; Storelli,
2008). Depending on how aquatic animals are exposed to the pollutants, the
route of uptake in the animal body may be different. A distinction is made
between passive diffusion, facilitated transport, active transport and
endocytosis (Simkiss and Taylor, 1995 in Veltman et al., 2008).
In general, seafood that is cultured in polluted areas is contaminated by the
same pollutants that exist in water and sediment. Ahmed (1991) stated that
seafood quality always reflects the quality of the water from which it is
captured. The Minamata disease in the Minamata bay Japan is an example of
40
dramatic tragedies caused by the consumption of metal-contaminated
seafood (Takeuchi et al ., 1962). The Minamata disease is a toxic
encephalopathia without a primary inflammation as a result of consumption
of large amounts of fish and shellfish that were contaminated with methyl-
mercury. The Minamata bay was contaminated by mercury originating from
the effluent of a chemical factory producing artificial fertilizers, vinyl
chloride, acetaldehyde and its derivatives. In the production of especially
acetaldehyde, mercuric chloride and sulphate were used as catalysts. The
mercury was eventually bioaccumulated in shellfish and fish growing in the
bay, after being methylated by sediment-living microorganisms.
Although less dramatic, various reports dealing with metal contamination in
seafood are available worldwide including among others copper-
contaminated seafood in Taiwan (Han et al ., 1994), mussels contaminated
with cadmium, copper, lead and zinc in the Philippines (Dumalangan et al .,
2010), mercury-contaminated edible fish species in Malaysia (Hajeb et al .,
2009), trace metals in edible tissues of several aquatic animals in Australia
(Fabris et al., 2006), edible fish species contaminated with lead and cadmium
in Finland (Tahvonen and Kumpulainen, 1996), fish contaminated by
mercury and methyl-mercury in Brazil (Malm et al., 1995), intake of arsenic,
cadmium, mercury and lead through metal-contaminated seafood in Spain
(Falco et al ., 2006) and mercury-contaminated seafood in Indonesia
(Soegianto et al., 2010).
The health benefits and risks of consuming seafood to some extent cause a
conflict between the dietary recommendation to eat seafood regularly and the
seafood safety problem. Sioen et al. (2008) even mentioned that debating the
health benefits versus the risks of seafood consumption has reached a
41
scientific and social interest. In addition, pathogen contamination of seafood
may interact with environmental pollutant accumulation, as these types of
contamination often co-occur. Several aspects of this complex problem of
seafood safety are studied in this thesis, focused on seafood production in
Central Java, Indonesia.
Indonesia is one of the top ten aquaculture producers in the world (FAO,
2006). With regard to the international trading as well as local food supply,
assuring seafood quality and safety is therefore crucial. Among the coastal
cities of Indonesia, Semarang, Java Island, is known to have good resources
for aquaculture. Around 42% of the Semarang coast line is utilized for
aquaculture with milk-fish (Chanos chanos ) and tiger prawn (Penaeus
monodon) being the favourite seafood commodities cultured in the coastal
ponds (“tambaks”). From an ecological prespective, aquaculture in the
estuarine environments tends to be problematic, because of the tendency of
these areas to trap and accumulate particle-bound pollutants such as metals
(Fernandes et al ., 1994). Previous studies on metal concentrations in the
sediment of streams along the Northern coast of Central Java, including
Semarang, have shown that several spots contained especially high
concentrations of copper and lead (Widianarko et al ., 2000a; Widianarko et
al., 2000b; Takarina et al., 2004). Java is the most densely populated island
in Indonesia (Whitten et al ., 1996), and even in the World (Gillespie and
Clague, 2009). The fact that typhoid fever ranked fourth among the ten
commonest diseases in Semarang (Gasem et al., 2001) most likely also links
to the poor food hygiene (in addition to bad housing sanitation). In fact,
typhoid fever spreads through faecal contamination of water and food (Egoz
et al ., 1988; King et a l., 1989). Typhoid fever is caused by Salmonella
enterica and the outbreak spreads up easily via typhoid fever patients or
42
carriers of Salmonella-contaminated feces. In practice, shrimp obtained from
Java Island appeared to be contaminated with human bacterial pathogens
(Dewanti-Hariyadi et al., 2005). This underlines the seriousness of bacterial
contamination in the water but also in the food. It adds to the complexity of
environmental problems in the estuarine area of Semarang via poor hygiene
and sanitation.
Considering the complex pollution in the Semarang coastal area there is a
serious concern about quality and safety of seafood cultured in the
productive coastal area. Seafood may not only be contaminated with e.g.
metals and microbial agents, both actors may also have an effect on
indigenous agents like histamine that is produced by the organisms
themselves or may result from microbial activity upon storage of seafood. By
nature, the interaction between metals and (micro)organisms is very
complex. Some metals are essential to animals, plants or microorganisms and
therefore selectively retained from the food and often regulated in the body.
Others are non-essential but may still circulate through animal bodies. Both
essential and non-essential metals may be toxic at low concentrations. This
asks for a comprehensive study on the interaction between metal
contamination and microbial-related effects on seafood quality. This study
therefore focuses on the interaction between metal pollution of aquaculture
ponds, microbial community structure and histamine production in related
seafood species.
A conceptual model that links the three factors (metals, microorganisms and
seafood) is shown in Figure 3. According to this model, metals in the aquatic
environment will not only affect the community structure in the water but
may also affect the seafood directly through metal bioaccumulation. Metal
43
accumulation may also lead to toxic effects on the organisms cultured, with
histamine production being one element of concern in case of crustacean
seafood items like shrimp and crayfish. Histamine may be produced in
response to stress (Cebada and Garcia, 2007). Changes in the microbial
community structure due to metal exposure may not only occur in the water
but also in the seafood. As a consequence of these changes, the population of
histamine-producing bacteria in the seafood items may change, both in size
and activity. This may possibly affect seafood quality and safety, by
increasing histamine production. All these factors have to be studied as they
may affect food quality and safety individually as well as in combination.
In this study, we focused on crustaceans, which are the most economically
valuable seafood commodity after cyprinid fish (FAO, 2006). Edible
crustaceans, especially shrimp and prawn, are also cultivated world-wide
making that the study will have a broader impact. Crustaceans are known to
accumulate more metals than fish (Rainbow, 1992). Also, crustaceans
naturally produce histamine, a human allergenic substance (Mietz and
Karmas, 1978). The most common edible species of crustacean cultured in
Indonesia is the tiger prawn, Peneaus monodon. In European restaurants the
tiger prawn is commonly known as “gamba”. Live tiger prawns however, are
not available in The Netherlands and most likely also not obtainable in any
European laboratory. Therefore we chose a crustacean species that is more
accessible to biological experiments. The freshwater marbled crayfish
(Procambarus sp.; Malacostraca, Decapoda, Astacida) was chosen as a
model in this study, due to the ease of culturing and production of high
numbers of genetically identical offspring (Martin et al., 2007).
44
Figure 3. Conceptual model of the interactions between metals and microorganisms in the aquatic environment and their impact on crustaceans. Shown are several processes of concern for seafood quality and safety: (1) metal bioaccumulation in crustaceans, (2) histamine formation in the animal triggered by metal exposure, (3) bacterial community structure and functioning in the animals affected by metal contamination and its consequences for histamine accumulation in the edible tissues during storage.
Objectives of the thesis
The main purpose of this study is to determine the interaction between
metals and microorganisms in the aquatic environment and their impact upon
crustaceans. The study especially focused on food quality and safety aspects
related to metal and microbial contamination. Several approaches were
chosen in this study, i.e. metal bioaccumulation, histamine formation in the
animal triggered by metals, bacterial community structure as affected by
metal contamination and histamine accumulation in the crayfish edible tissue
during storage.
45
Outline of the thesis
In Chapter 2 , the profile of metal pollution in coastal shrimp pond
sediments along the Semarang coast line was investigated. It was concluded
that metal contamination in Semarang coastal pond sediments most likely is
due to anthropogenic input. Among the metals, copper was generally found
and at several sites the concentrations were up to twice the local background
concentration.
In Chapter 3 , copper uptake and elimination kinetics in the organs of
marbled crayfish (Procambarus sp.) were determined at two exposure levels
in the water. Copper showed fast uptake kinetics with – in most organs –
equilibrium was reached within 10 days of exposure. Copper accumulation
was highest in the hepatopancreas, which acts as a storage organ. Copper
concentrations in muscle (edible tissue) showed fast kinetics, even at 0.38
mg Cu/L in the water and as a result concentrations remained fairly low and
therefore do not pose any risk to food safety.
In Chapter 4 , the effect of copper exposure levels on triggering histamine
production in the hepatopancreas and the muscle of marbled crayfish was
examined. Histamine levels showed a significant increase under the
influence of increased copper exposure levels. Histamine production may be
linked to a stress response of the animal or could arise because of up-
regulation of histidine production (to scavenge excess copper) followed by
transformation to histamine.
In Chapter 5 , interactions between accumulated copper, bacterial
community structure and histamine formation in crayfish meat were studied
during storage of the meat at 5oC. Exposure of the crayfish to an aqueous
copper concentration of 0.5 mg Cu/L changed the bacterial community
structure in the water and significantly reduced the histamine accumulation
levels in crayfish meat during 10 days of storage.
46
Chapter 6 provides a general discussion of the thesis. Results and findings
from the field investigation as well as the scientific knowledge obtained from
the laboratory experiments are discussed and general conclusions with
respect to improvement of seafood quality and safety are drawn.
47
Chapter 2
Metal contamination in coastal pond sediments of
Semarang – Indonesia
48
Abstract
Semarang is one of the largest coastal cities in Indonesia with a long
tradition of aquaculture and seafood consumption. The rapid residential
growth, industrial expansion and agricultural intensification have contributed
to increasing metal pollution in the Semarang rivers and coastal areas. In this
study, we examined the metal concentrations of aquaculture pond sediments
along the Semarang coast line. Sediment samples were taken from ten
aquaculture ponds in spring 2009. Copper concentrations at several sites
were found to be up to twice the local background concentration, which is
approximately 40 µg/g dry weight. Some sites close to the intercity highway
and the Semarang harbor were polluted by lead. According to Sediment
Quality Guidelines (SQGs) applied in Australia and New Zealand, Hong
Kong and the Netherlands, Semarang coastal pond sediments can be
classified as mid-range (medium range) contaminated. Natural erosion of
Mount Ungaran as well as the use of copper-based chemicals in
manufacturing industries, agriculture, fisheries and household wastes are
possible sources of copper. Lead most likely originates from automobile
exhaust. Considering that concentrations of other trace metals (Cr and Ni)
were also increased in some of the sediments, the ecological risk and
possible consequences for aquaculture should be assessed further.
49
Introduction
Increasing industrial activities, large population density and agricultural
intensification may lead to increased levels of anthropogenic pollutants in the
environment. Coastal areas could be the most affected places among
potentially polluted sites as pollutants in any water will end up in the coastal
areas and the sea. Since coastal areas are also used for culturing several
edible species, pollution may affect these organisms. The effects of pollution
may not only include reduction of the productivity but the bioaccumulation
of pollutants in animal organs, especially in the edible parts, will also have
consequences for food quality and food safety.
Metals are naturally occurring elements whose concentrations and
bioavailability have been greatly influenced by human activities. Metals
cause bioaccumulation problems in many organisms (Luoma and Rainbow,
2008) which may lead to many health effects. The itai-itai disease, which
occurred in the Toyama Prefecture in Japan in the 1950, is an example of
dramatic metal (cadmium) poisoning that also involved metal-contaminated
food consumption (Inaba et al ., 2005). Another example is the Minamata
disease caused by mercury-contaminated fish eaten by Japanese fishermen
and their families around the Minamata bay in 1950s (Takeuchi et al., 1962).
There are many more, less dramatic, examples of metal bioaccumulation in
seafood (Pourang, 1995; Malm et al., 1995; Madany et al., 1995) that exceed
the legal safety limits defined by FAO (Nauen, 1983) and ANHMRC
(Maher, 1985).
Aquaculture plays an important role for the economy of Indonesia. Referring
to the state of aquaculture summarized by FAO (2006), Indonesia takes the
50
sixth position among the top ten aquaculture producers in the world.
Indonesian aquaculture products are essential not only for the local food
supply but also for export. Considering that aquaculture products are traded
as a food commodity, therefore quality and safety are important
considerations. Not only pathogenic microorganisms but also chemical
contaminants, such as persistent organic pollutants and potentially toxic
metals, are major concerns for seafood safety in that they may pose a risk to
consumer health.
Semarang is one of the coastal cities of Indonesia which has good resources
for aquaculture. About 42% of the Semarang coast line is utilized for
aquaculture. Milk fish (Chanos chanos) and tiger prawn (Penaeus monodon)
are the two favorite seafood commodities cultured in the coastal ponds. With
the city’s increasing economic development, metal pollution is also
increasing as was already found in the rivers of Semarang urban area some
10 years ago (Widianarko et al ., 2000a; Widianarko et al ., 2000b; Takarina
et al., 2004). Metal contamination in the aquaculture ponds of the Semarang
coast line is therefore of concern, but so far there are no data available on
metal concentrations in these ponds.
The objectives of the present study were (1) to determine physical
characteristics and metal concentrations in the Semarang coastal pond
sediments, (2) to compare the metal concentrations found in the sediments
with the local background concentration and internationally accepted
Sediment Quality Guidelines, and (3) to provide information on the possible
sources of the metals and their potential risk for food quality and safety.
51
Material and methods
Study Area
Semarang, the capital city of the province of Central Java, is the fifth largest
city of Indonesia. It is located at South Latitude 6o56’08’’ to 7o06’57’’ and
East Longitude 110o16’17’’ to 110o30’31’’ (Marfai and King, 2008). The
city has a typical coastal geography, consisting of a hilly area in the South
and a coastal lowland area in the North. In the South, Semarang is bordered
by Mount Ungaran (2050 m), an eroded stratovolcano. The Semarang
municipality covers an area of about 373.7 km2 in which the northern parts
are adjacent to the Java coast line (Lubis et al ., 2011). Semarang has a
tropical climate with alternating rainy and dry seasons. The annual rainfall is
about 2065–2460 mm with maximum rainfall in December and January
(Marfai and King, 2008). The climate generally is hot with temperatures
between 24°C and 30°C, and an average annual temperature of 28.4°C. Two
canalized rivers from Mount Ungaran run through the city, one on the east
side (East Canal) and one through the west side (West Canal).
The Semarang coastal area was originally fringed with mangrove forests, but
is now highly developed for multi-use purposes, including major industrial
facilities, fisheries, and a harbor. The land use pattern and physical
environment in Semarang are uncontrolled and changing rapidly neglecting
the environmental carrying capacity both in the upland and in the lowland
area. The average population density of Semarang has reached 1,010 people
per square kilometer. Agriculture is intensifying as well as industrial
activities. The number of industrial enterprises has reached 4,678 units in
Central Java (Central Java Province Statistical Central Bureau, 2009). A map
of Semarang is shown in Figure 1. A map of Semarang - Indonesia is shown
52
in Figure 1. The map of Indonesia was created using the Ocean Data View
packages, http://odv.awi.de (Schlitzer, 2008), and the map of Semarang was
manually re-drawn from a map provided by Google Maps,
http://maps.google.com.
Figure 1. Semarang, Central Java – Indonesia. The numbers indicate the aquaculture ponds sampled for this study. The sampling sites include: 1. Beringin River 6. Siangker River-1 2. Kyai Gilang River-1 7. Siangker River-2 3. Kyai Gilang River-2 8. West Canal 4. Tapak River-1 9. East Canal-1 5. Tapak River-2 10. East Canal-2
53
Sampling schedule
The sampling locations, marked with “black dots” in Figure 1, are
aquaculture areas along the Semarang coast line. The sampling sites fall
under the influence of different rivers and canals, with different levels of
human interference and a variable degree of exposure to metal
contamination. Surface sediment (5-10 cm) samples were collected using a
plastic grab sampler at ten locations during the late rainy season, February
through March 2009. The water level in the ponds varied from 50 to 150 cm
at the time of sampling. In each pond three samples were taken, each
consisting of about 2 kg of wet sediment. The wet material was stored in 5-L
plastic bags, sieved in the laboratory and the fraction passing through a
diameter size of 5 mm was dried at 105oC for 48 hours. Before extraction,
the dried material was grounded using a porcelain mortar and again sieved
through a plastic sieve with diameter size of 0.5 mm. The fine dried
sediments were stored in a plastic bag under dry conditions until chemical
analysis.
Metal analysis
Dried homogenized sediment samples of 100 mg were digested in 2.0 ml of a
mixture of HNO3 (Sigma-Aldrich, 65%) and HCl (Riedel-de-Haën, 37%) in
a ratio of 4:1, in closed teflon pots placed in an oven with a constant
temperature of 140oC for 7 hours. The digests were diluted with milli-Q
water to a volume of 10 ml and analyzed for seven different metals (Cd, Cr,
Cu, Fe, Ni, Pb and Zn) by flame atomic absorption spectrophotometer
(Perkin-Elmer AAnalyst 100). Quality control of the analyses was
maintained by digesting certified reference material ISE 989. The recoveries
of the different metals in the reference material were 103-108% of the
certified reference values.
54
Sediment characterization
To determine pH, 5.0 g dry weight sediment was shaken with 25 mL 0.01 M
CaCl2 for 2 hours at 200 rpm. The pH was then measured using a Consort
P907 pH meter. Organic matter content was determined as Loss on Ignition
(%LoI) upon burning samples for 6 hours at 500 oC in an oven. The Cation
Exchange Capacity (CEC) was determined using the silver thiourea method
(Dohrmann, 2006) with some modifications. Principally, all positive ions
bound to negatively charged colloid surfaces, mineral and organic, are
replaced by the strong affinity monovalent silver thiourea complex (AgTU)
cation. The dried sediment was constantly mixed with a 0.01 M AgTU
solution in Milli-Q water under end-over-end shaking for 4 hours, followed
by centrifugation (MSE Falcon 6/300, UK) for 15 minutes at 3000 rpm. The
supernatants of samples and blanks were diluted 100 times and the
concentration of silver was measured by flame atomic absorption
spectrophotometry (Perkin-Elmer AAnalyst 100). Quality control of the
analysis was maintained by determining CEC of the LUFA 2.2 standard soil
(Speyer, Germany). The CEC measured in Lufa 2.2 soil was 70-77% of the
certified value.
Statistical analysis
All computations were done using the statistical software package SPSS
16.00. Comparisons between metal concentrations in the sediments from
different sites were done by analysis of variance (one way ANOVA)
followed by Tukey’s post-hoc test (p < 0.05). The possible association
between different metals was pair-wise assessed by determining Pearson
correlation.
55
Results and discussion
Physicochemical characteristics of sediments
The pH, organic matter content and CEC of the ten sampling sites are shown
in Table 1. All sediments were slightly basic with pH-CaCl2 ranging from
7.4 to 8.1. Such characteristics are typical, as normal pH of surface water
(including seawater) is neutral to basic. In addition, the alkaline pH may be
partly due to lime application. In ponds constructed in mangrove areas,
oxidation of pyrite causes release of acid sulfate, and liming is applied to
neutralize the acid (Gräslund and Bengtsson, 2001). All sediments contained
relatively high organic matter contents (> 4%), which are considered normal
as the sampling sites were located in estuarine areas with prior mangrove
vegetation. Sediments had fairly high CEC levels, which were similar to
values of soils along the Semarang coast line. CEC often is correlated with
organic matter, clay and silt contents (Asadu et al., 1997).
Metal concentrations
Metal concentrations found in the aquaculture pond sediments are presented
in Table 2 and Figure 2. Concentrations of cadmium in all samples were
below the detection limit (0.03 μg/g). Zinc, nickel and iron concentrations
varied among locations but were generally low. Widianarko et al . (2000a)
proposed local background concentrations of Cu, Pb and Zn for Semarang
coastal area of 40.7 µg/g, 25.6 µg/g and 132 µg/g, respectively. Only copper
levels in all samples exceeded the local background concentration. In three
sampling sites located close to Beringin River, East Canal-1 and Siangker
River, copper concentrations were 1.7 to 1.8 times the local background
concentration, suggesting pollution due to anthropogenic activities. Lead
concentrations in sediments taken from East Canal-1 (112 ± 14.7 µg/g) and
56
Tabl
e 1.
Sam
plin
g lo
catio
ns a
nd p
hysi
coch
emic
al c
hara
cter
istic
s of
sed
imen
ts c
olle
cted
from
ten
aqua
cultu
re p
onds
alo
ng th
e Se
mar
ang
coas
tal
line
in In
done
sia.
Loc
atio
n
GP
S pH
– C
aCl 2
L
oI (
%)
CE
C (
cmol
/kg)
d.w
. B
erin
gin
Riv
er
06o 57
.405
’S ;
110o 19
.214
’E
8.1
6.1±
0.08
30
.9±0
.47
East
Can
al-1
06
o 57.2
24’S
; 11
0o 27.0
60’E
7.
4 11
.2±0
.05
29.6
±0.9
4 Ea
st C
anal
-2
06o 57
.366
’S ;
110o 27
.194
’E
7.8
8.2±
0.06
28
.9±1
.86
Gila
ng R
iver
-1
06o 57
.065
’S ;
110o 18
.765
’E
7.8
9.7±
0.04
31
.1±0
.21
Gila
ng R
iver
-2
06o 56
.901
’S ;
110o 18
.758
’E
7.9
7.7±
0.14
31
.6±0
.09
Sian
gker
Riv
er-1
06
o 58.0
60’S
; 11
0o 23.1
42’E
7.
8 7.
2±0.
06
29.5
±0.7
5 Si
angk
er R
iver
-2
06o 58
.083
’S ;
110o 22
.912
’E
7.9
8.5±
0.06
30
.4±0
.70
Tapa
k R
iver
-1
06o 58
.036
’S ;
110o 21
.126
’E
7.6
8.8±
0.06
30
.7±0
.86
Tapa
k R
iver
-2
06o 58
.045
’S ;
110o 21
.168
’E
7.9
7.8±
0.06
30
.9±0
.77
Wes
t Can
al
06o 57
.016
’S ;
110o 24
.300
’E
7.8
4.7±
0.13
30
.9±0
.36
• G
PS =
loca
tion
indi
cate
d w
ith G
loba
l Pos
ition
ing
Syst
em
• Lo
I = L
oss o
n Ig
nitio
n (=
org
anic
mat
ter c
onte
nt)
• C
EC =
Cat
ion
Exch
ange
Cap
acity
•
d.w
. is d
ry w
eigh
t •
Nam
e of
loca
tion
refe
rs to
the
clos
est r
iver
or c
anal
•
All
valu
es fo
r LoI
and
CEC
are
mea
n ±
stan
dard
dev
iatio
n (n
=3).
Sam
ple
size
was
3, e
ach
with
2 re
plic
atio
ns o
f lab
orat
ory
anal
ysis
.
57
BR
EC
1 E
C2
GR
1 G
R2
SR
1 S
R2
TR
1 T
R2
W
CB
R E
C1
EC
2 G
R1
GR
2S
R1
SR
2T
R1
TR
2W
C
BR
EC
1 E
C2
GR
1 G
R2
SR
1 S
R2
TR
1 T
R2
WC
BR
E
C1
EC
2 G
R1
GR
2 S
R1
SR
2 T
R1
TR
2 W
CB
R E
C1
EC
2 G
R1
GR
2 S
R1
SR
2 T
R1
R2
WC
Cu (µg/g DW)80 70 60 50Cu (µg/g DW)
120
100 80 60 40 20 0
Pb (µg/g DW)
70
60
50
40
30
Fe (mg/g DW)
22
20
18
16
14
12
Ni (µg/g DW)
35
30
25
20
15Cr (µg/g DW)
Fi
gure
2. B
ox-W
hisk
er p
lots
show
ing
the
varia
tion
in c
oppe
r, le
ad, c
hrom
ium
, nic
kel a
nd ir
on c
once
ntra
tions
in µ
g/g
dry
wei
ght (
mea
n±SE
, n=6
) in
sedi
men
t fro
m te
n di
ffer
ent a
quac
ultu
re p
onds
alo
ng th
e Se
mar
ang
coas
t lin
e. S
ee T
able
1 a
nd F
igur
e 1
for f
urth
er in
form
atio
n on
the
sam
plin
g si
tes,
and
Tabl
e 2
for m
ean
met
al c
once
ntra
tions
. BR
= B
erin
gin
Riv
er; E
C =
Eas
t Can
al; S
R =
Sia
ngke
r Riv
er; G
R =
Kya
i Gila
ng R
iver
; TR
=
Tapa
k R
iver
; WC
= W
est C
anal
.
58
Tabe
l 2. M
etal
con
cent
ratio
ns fo
und
in se
dim
ents
col
lect
ed fr
om te
n aq
uacu
lture
pon
ds a
long
the
Sem
aran
g (I
ndon
esia
) coa
st li
ne in
the
perio
d of
Fe
brua
ry –
Mar
ch 2
009.
Loc
atio
n
Cu
(µg/
g d.
w.)
Pb
(µg/
g d.
w.)
Cr
(µg/
g d.
w.)
Ni
(µg/
g d.
w.)
Fe
(mg/
g d.
w.)
Zn
(µg/
g d.
w.)
Ber
ingi
n R
iver
72
.6±3
.2b,
c 20
.1±0
.1a
21.0
±0.7
a,b
15.7
±0.1
a,b
64.0
±3.3
c 14
8±9.
7b,c
East
Can
al-1
72
.3±1
1.7b,
c 112
±14.
7c
31.0
±1.0
e 19
.7±0
.4d
44.2
±4.6
a 19
4±24
.9d
East
Can
al-2
60
.8±1
3.4a,
b,c
81.3
±30.
2b 28
.0±4
.2d,
e 19
.1±1
.4d
45.0
±2.8
a 10
7±5.
7a,b
Gila
ng R
iver
-1
51.4
±0.9
a 19
.4±0
.4a
26.0
±0.3
b,c,
d,e
15.5
±0.3
a,b
50.7
±5.9
a,b
92.5
±1.8
a
Gila
ng R
iver
-2
64.5
±1.3
a,b,
c 21
.8±0
.4a
22.5
±0.8
b,c
16.3
±0.2
b,c
57.2
±2.9
b,c
104±
0.9a,
b
Sian
gker
Riv
er-1
73
.7±5
.1c
14.4
±0.5
a 17
.2±1
.5a
13.2
±0.8
a 47
.3±0
.9a
121±
21.9
a,b,
c
Sian
gker
Riv
er-2
52
.7±5
.5a,
b 19
.2±2
.7a
25.4
±1.9
b,c,
d 17
.1±0
.9b
49.2
±0.9
a,b
110±
32.2
a,b
Tapa
k R
iver
-1
57.2
±10.
5a,b,
c 22
.5±1
.4a
24.7
±2.6
b,c,
d 18
.5±1
.5c,
d 50
.4±3
.1a,
b 16
2±6.
0c,d
Tapa
k R
iver
-2
47.3
±0.3
a 21
.2±1
.2a
27.5
±0.6
c,d,
e 19
.0±1
.0c,
d 51
.0±2
.5a,
b 12
5±5.
3a,b,
c
Wes
t Can
al
49.4
±1.0
a 18
.7±1
.4a
21.2
±2.4
a,b
15.7
±1.6
a,b
52.5
±0.7
a,b
108±
3.7a,
b
• N
ame
of lo
catio
n re
fers
to th
e cl
oses
t riv
er o
r can
al; s
ee T
able
1 a
nd F
igur
e 1.
•
d.w
. is d
ry w
eigh
t •
All
valu
es a
re m
ean
± st
anda
rd d
evia
tion
(n=3
). Sa
mpl
e si
ze w
as 3
, eac
h w
ith 2
repl
icat
ions
of l
abor
ator
y an
alys
is.
• D
iffer
ent s
uper
scrip
ts sh
ow si
gnifi
cant
diff
eren
ces b
etw
een
loca
tions
(p<0
.01)
, bas
ed o
n Tu
key’
s pos
t-hoc
test
. •
The
valu
es p
rinte
d in
bol
d ex
ceed
the
Sedi
men
t Qua
lity
Gui
delin
es (s
ee T
able
4)
59
East Canal-2 (81.3 ± 30.2 µg/g) were significantly higher than at the other
locations (Tukey’s post-hoc test; p < 0.05). Lead most likely originated from
automobile exhausts since both sites are located less than 50 meters away
from the intercity highway (Jalan Arteri Utara) and around 2 km from the
Semarang harbour. Pearson correlations between metal concentrations found
in the ten sampling sites are shown in Table 3. When metals are found in
high levels at the same site, this may suggest a similar source of pollution.
Chromium and nickel concentrations were significantly correlated (p<0.01).
In geochemistry research, the Cr/Ni ratio is commonly used as a pre-
indication of volcanic rock and stone (Garver et al., 1996). The existence of
chromium together with nickel found at two locations (East Canal-1 and East
Canal-2) is indicative of natural erosion from Mount Ungaran. The
significant correlations between lead, nickel and chromium concentrations,
however, may also suggest an anthropogenic source of nickel and chromium
pollution. These metals often are used together in electroplating industries
(United State Patent, 1995). The significant correlation between copper and
zinc (p < 0.05) might indicate the anthropogenic origin of these metals from
their use in fungicides (complex of copper and zinc with sulphuric acid; Nan,
1995).
Possible sources of copper in pond sediments
Metal contamination of aquaculture ponds can be unintentional and
sometimes even unavoidable due to water quality problems (Gräslund and
Bengtsson, 2001). Boyd (1990) cited by Yang et al. (2007) stated that copper
pollution often results from copper sulphate. Copper sulphate is the most
commonly used algicide, normally applied in the aquatic farming of fish,
mollusks, crustaceans and aquatic plants. Copper sulphate application is the
only algal control method for shrimp ponds recommended by FAO
60
(GESAMP, 1997). CuSO4.5H2O is known as an effective, relatively
inexpensive algicide, which is also useful as an anti-parasitic agent (Watson
and Yanong, 2006). Considering that waters along the north coast of Central
Java normally are turbid and eutrophic (Takarina et al ., 2004), fish farmers
in Semarang coastal ponds may be expected to use the copper-based
algicides to control water quality and to combat parasites.
Copper is an essential mineral for animals and plants, so it is commonly used
for fortification in the food and feed manufacturing industry. Copper
contaminated waste water may be produced by food factories in Semarang -
including among others for the production of noodles, biscuits and bakery
products, dairy products, candy and frying oil. Also these processes may
contribute to the increasing anthropogenic copper pollution of rivers and
canals. Copper is also known as antifouling agent for boats (Warnken et al.,
2004) and often used as wood-preservative (Freeman and McIntyre, 2008) in
plywood and furniture. Semarang harbour (Tanjung Mas) is a trade port
where many ferries and boats often lie at anchor for several days. Given that
the local government did not ban copper-based antifouling yet, relatively
high copper concentrations may be found in the port area. Furthermore, a
large plywood factory is operating in Semarang together with many small-
scale furniture producers, which also use copper-based wood preservatives.
These activities may also contribute the increasing anthropogenic copper
levels. Application of copper-based dyes in Semarang textile and porcelain
industries can be another source of anthropogenic copper pollution. In
developing countries, including Indonesia, 70% of the industrial waste is
discharged untreated into surface water bodies (WWAP, 2000). As any
effluent discharged into the environment eventually finds its way to a river,
pond or sea, aquatic animals may be most vulnerable to the toxic effects
61
Tabe
l 3. P
ears
on c
orre
latio
ns b
etw
een
met
al c
once
ntra
tions
foun
d in
ten
aqua
cultu
re p
onds
alo
ng th
e Se
mar
ang
coas
t lin
e.
Lo
g_Pb
(μ
g/g
d.w
.) Lo
g_C
u
(μg/
g d.
w.)
Log_
Fe
(mg/
g d.
w.)
Log_
Zn
(μg/
g d.
w.)
Log_
Ni
(μg/
g d.
w.)
Log_
Cr
(μg/
g d.
w.)
Log_
Pb
(μg/
g d.
w.)
1.00
0
Log_
Cu
(μ
g/g
d.w
.) 0.
277
1.00
0
Log_
Fe
(mg/
g d.
w.)
-0.4
86**
0.
073
1.00
0
Log_
Zn
(μg/
g d.
w.)
0.39
2* 0.
478**
-0
.101
1.
000
Log_
Ni
(μg/
g d.
w.)
0.65
3**
-0.2
34
-0.2
96
0.37
5* 1.
000
Log_
Cr
(μg/
g d.
w.)
0.64
0**
-0.3
87*
-0.3
95*
0.16
8 0.
867**
1.
000
For
this
ana
lysi
s, da
ta w
ere
log-
trans
form
ed
d.w
. is d
ry w
eigh
t **
. Cor
rela
tion
is si
gnifi
cant
at t
he 0
.01
leve
l (2-
taile
d).
*. C
orre
latio
n is
sign
ifica
nt a
t the
0.0
5 le
vel (
2-ta
iled)
.
62
Tabl
e 4.
Cu
and
Pb c
once
ntra
tions
of S
emar
ang
coas
tal p
ond
sedi
men
ts c
ompa
red
to d
iffer
ent S
edim
ent Q
ualit
y G
uide
lines
Sour
ce o
f dat
a C
u
(µg/
g) d
.w.
Pb
(µg/
g) d
.w.
Ref
eren
ce
Se
mar
ang
pond
sedi
men
ts
Sedi
men
t Qua
lity
Gui
delin
e (S
QG
): Lo
w
Hig
h
47
.3 –
73.
7
34a ; 3
6b ; 65c
190b ; 2
70a,
c
18
.7 –
112
50a ; 7
5c ; 85b
21
8c ; 220
a ; 530
b
The
pres
ent s
tudy
a.
AN
ZEC
C-I
SQG
for A
ustra
lia a
nd
New
Zea
land
(Sim
pson
et a
l., 2
006)
b.
SQ
O fo
r the
Net
herla
nds
(Cro
mm
entu
ijn e
t al.,
200
0)
c. I
SQV
for H
ong
Kon
g (C
hapm
an e
t al
., 19
99)
d.
w. i
s dry
wei
ght
AN
ZEC
C-S
QG
, Aus
tralia
n an
d N
ew Z
eala
nd E
nviro
nmen
t Con
serv
atio
n C
ounc
il –
Inte
rim S
edim
ent Q
ualit
y G
uide
line
SQO
, Sed
imen
t Qua
lity
Obj
ectiv
e IS
QV
, Int
erim
Sed
imen
t Qua
lity
Val
ue
63
of pollutants. Considering that some aquatic animals are consumed and
traded as food, pollution in the aquaculture ponds may eventually also pose a
risk to human health.
Seafood quality and safety risk
Metal concentrations found in sediment are not necessarily available for
organisms. Physical and chemical characteristics of the sediments, like
organic matter content, chelating agents, humic substances, ligands, pH and
metal interactions may all influence the bioavailability of metals (Simpson et
al., 2004; Luoma & Rainbow, 2008). Metal bioavailability can affect living
organisms via bioaccumulation processes.
Elevating awareness on seafood safety in the global society is quite logical as
seafood consumption per capita has increased remarkably over the last two
decades (Einarsson & Emerson, 2009). Evidence of copper-contaminated
seafood products sold in the local markets has been reported by several
authors from various countries (Maher, 1985; Han, et al ., 1994; Hashmi et
al., 2002; Canli and Atli, 2003; Pourang et al ., 2005; Mishra et al ., 2007;
Sivaperumal et al ., 2007). The European Copper Institute proposed a legal
limit of copper in foodstuff of 5 μg/g wet basis (Van Lysebetten et al., 2010).
The World Health Organization (WHO) and the Food and Agricultural
Administration (FAA) suggest the intake of copper should not exceed 12
mg/day for adult males and 10 mg/day for adult females. Some clinical
features of copper toxicity include among others, fatigue, depression,
headaches, cold extremities, lack of concentration and poor memory (Nolan,
1983).
64
Although there is evidence that copper may accumulate in seafood products,
it still is uncertain whether the increased concentrations found in Semarang
coastal pond sediments indeed pose a risk for seafood quality and safety. The
fact that also other metals, like Cr and Ni, show increased sediment
concentrations asks for further research to assess the risk for seafood
consumers and therefore for human health.
Conclusions
• Compared to the SQG values for copper and lead, Semarang coastal
ponds sediments are categorized as mid-range contaminated. In addition
to copper and lead, also nickel and chromium at some sites show
elevated sediment concentrations.
• Metal pollution in Semarang coastal pond sediments can at least partly be
attributed to anthropogenic input.
• The pond sediments along the Semarang coast line are slightly basic,
relatively high in organic matter contents, and have fairly high CEC
values. As a consequence, metal bioavailability may be reduced.
• Nevertheless, copper and lead contamination in Semarang coastal ponds
may threaten local aquaculture practices and may pose a risk for the
quality and safety of seafood products.
65
Chapter 3
Copper toxicokinetics in marbled crayfish
(Procambarus sp.): implications for
food quality and safety
66
Abstract
Metal pollution in coastal areas may threaten seafood quality through metal
bioaccumulation in edible organs. Although copper is an essential trace metal,
it is a common pollutant in sediments. The aim of this study was to investigate
the effect of copper exposure concentration on copper bioaccumulation in
marbled crayfish (Procambarus sp.) by determining uptake and elimination
kinetics. Crayfish were exposed to average sub-lethal copper concentrations of
0.031 and 0.38 mg Cu/L for 14 days and transferred to copper-free water for
another 14 days. At different time points during the uptake and elimination
phases copper concentrations were measured in five organs (exoskeleton, gills,
muscle, ovaries and hepatopancreas). At 0.031 mg Cu/L, copper levels in the
crayfish organs were not significantly increased compared to the control
animals, suggesting effective regulation. Exposure to 0.38 mg Cu/L did lead to
slightly, but not significantly increased copper levels in muscles and ovaries,
while the gills and exoskeleton, which are in direct contact with the water,
showed significantly increased copper concentrations. In these four organs,
copper showed fast uptake kinetics with equilibrium being reached within 10
days of exposure. Copper accumulation was highest in the hepatopancreas;
uptake in this storage organ steadily increased with time and did not reach
equilibrium within the 14-day exposure period. The maximum copper
concentration reached in edible parts (muscle) of the crayfish did not exceed
the Australian National Health and Medical Research Council recommended
standard value for edible crustaceans, suggesting that the risk for food quality
and safety is limited at the exposure concentration of 0.38 mg Cu/L.
67
Introduction
One of the major groups of environmental pollutants are metals. Industrial and
municipal discharges, including metal processing and acid mine drainage
(Powell, 1988), smelter emissions (Nriagu and Rao, 1987), wastewater
discharge by manufacturing industries (Camusso and Tartari, 1991; Oyewo
and Don-Pedro, 2003) and domestic sludge disposal (Thongra-ar et al., 2008)
can lead to elevated metal concentrations in the environment. Among the metal
pollutants, copper is commonly found in the Earth's crust and water bodies
(Nriagu, 1979). But elevated copper concentrations in surface waters are
mostly caused by human activities. According to Graedel et al . (2004) the
average anthropogenic copper discharge to surface waters was estimated at
499 ± 550 Gg per year. As a consequence aquatic animals might habitually be
exposed to copper in various concentrations. Bossuyt and Janssen (2003)
stated that environmentally relevant copper concentrations in freshwater
bodies are 0.5 – 100 μg Cu/L. Among the aquatic organisms, crustaceans are
important and possibly the most susceptible group. For example, Daphniidae,
as a representative of crustaceans are known to be quite sensitive to pollutants
(including metals) in aquatic ecosystems (Zhou et al ., 2008). From an
ecological and food quality point of view, research on larger crustacean
species is also highly relevant. In the freshwater food web, larger crustaceans
take a position as predators, which may enhance the effect of bioaccumulation
along the food chain. The advantage of using larger species as animal model in
toxicological research is the possibility to determine the internal distribution of
the compound of interest.
Copper is an essential trace metal for all living organisms but is also highly
toxic when the concentration exceeds species-specific thresholds (Peňa et al .,
68
1999; US-EPA, 2007). In earlier studies, 15 μg Cu/L caused 50% mortality of
rainbow trout juveniles after 35 days of exposure (McKim et al ., 1978).
Further, Bini and Chelazzi (2006) reported that high internal copper
concentrations caused red swamp crayfish (Procambarus clarkii ) to loose
muscular control and eventually die. For an essential metal such as copper, the
range between essential and toxic concentration levels can be rather narrow
(Mastin and Rodgers, 2000).
Understanding of the distribution of metals in certain tissues of organisms is
vital, especially to identify specific organs particularly selective and sensitive
to metal bioaccumulation (Szefer et al., 1990). There are several mechanisms
that might be relevant for the uptake of metals in animals, including passive
diffusion, facilitated transport, active transport and endocytosis (Simkiss and
Taylor, 1995 in Veltman et al., 2008). In the case of an essential metal such as
copper, the uptake is normally via membrane transport proteins (Veltman et
al., 2008). But metal uptake does not always result in metal bioaccumulation.
Bioaccumulation is a complex mechanism including uptake and elimination
processes. Luoma and Rainbow (2005) mentioned that the bioaccumulation
kinetics of metal in animals significantly varies, depending on the metal, with
species and ecosystem investigated.
The parthenogenetic marbled crayfish (Procambarus sp.) is a freshwater
crustacean with unknown geographic origin and taxonomic identity (Scholtz et
al., 2003) that has been reported as an invasive species in Europe and
Madagaskar (Marzano et al ., 2009; Jimenez and Faulkes, 2010). Studies on
marbled crayfish so far have focused on morphological and molecular analysis
(Schiewek et al ., 2007), life stage (Seitz et al ., 2005) and reproductive
components (Vogt et al ., 2004), development of the central nervous system
69
(Vilpoux et al ., 2006), production of genetically uniform offspring (Martin et
al., 2007) and also embryonic development of the histaminergic system
(Rieger and Harszch, 2008). This makes it a suitable species also for toxicity
tests. However, currently no research on metal kinetics in marbled crayfish has
been reported. As a model-species of edible crustaceans and with regard to
food quality and safety assessment, organ-specific accumulation data,
especially for the muscles of crayfish, are highly required.
The objectives of the present study were (1) to investigate the effect of
exposure concentration on copper bioaccumulation in marbled crayfish organs
by determining copper uptake and elimination kinetics, and (2) to evaluate the
possible consequences of copper bioaccumulation in edible parts of marbled
crayfish from a food quality and safety point of view.
Materials and methods
Animals
Marbled crayfish (Procambarus sp.; Marmokrebs; Malacostraca, Decapoda,
Astacida), used as a model of edible crustacean, were provided by Alterra, part
of Wageningen University and Research Centre - the Netherlands. The animals
measured 3.5-7.2 cm and weighed 1.3-7.4 g (fresh weight). The animals were
acclimated in continuously aerated filtered copper-free water (pH 7.3 ± 0.1;
hardness 2–4 mmol CaCO3/L) for one week prior to exposure, under constant
laboratory conditions (temperature 20 ± 1 ºC; day/night cycle of 12 hours light
and 12 hours dark). Copper-free tap water was obtained from a special piping
system without copper linings available at VU University. Animals were fed
commercial crayfish food pellets three times a week.
70
Copper exposure
The copper concentrations used in this study were based on Bini and Chelazzi
(2006) who showed that a concentration of 0.05 mg Cu/L caused no effects
while at 0.5 mg Cu/L a (non-lethal) reduction was observed in both heart and
gill chamber ventilation rates of red swamp crayfish (Procambarus clarkii ).
Test concentrations of 0.05 mg Cu/L and 0.5 mg Cu/L were prepared by
dissolving CuSO4.5H2O (Merck, p.a.) in copper-free water. The test animals
were randomly divided in three groups and exposed individually in 800 mL
glass jars at 20 ± 1 ºC. The first two groups, each having 31 animals, were
exposed to the two different levels of copper (0.05 and 0.5 mg Cu/L). The
third group, containing 7 animals, was the control and was kept in copper-free
water. The animals were incubated in a climate-controlled room under the
same conditions as during acclimatization. They were fed three times a week
with commercial crayfish food pellets, shortly before renewing the exposure
solution. The wet weight of all test animals was measured at the start of the
test and upon sampling. The exposure took 14 days after which all remaining
animals were transferred to copper-free water. At different time points during
the copper uptake (1, 2, 4, 8 and 14 days) and elimination phases (15, 16, 18,
22 and 28 days) three animals from each treatment, in the inter-moult stage,
were sampled and killed by cutting off the head using a lancet. Control
animals were sampled only after 14 and 28 days. The animals were dissected
to collect the gills, hepatopancreas, ovaries, muscle and exoskeleton. The
organs were placed in plastic tubes with lid, frozen in liquid nitrogen,
weighted and kept in a freezer (-20 ºC) until analysis.
Copper concentration in animal organs and exposure solutions
All tissue samples were freeze-dried for 48 hours and then powdered using a
pestle and mortar. The dried homogenized samples were digested by two
71
different methods, depending on the amount of sample available. For samples
< 50 mg, a micro-digestion was done using 600 µl of a mixture of HNO3 (JT.
Baker, Ultrex II ultra pure) and HClO4 (JT. Baker, Ultrex ultra pure) in the
ratio of 7:1 in small glass tubes placed in a heater block with a gradual
temperature increase (85 ºC for 60 min; 130 ºC for 60 min and 160 ºC for 60
min). Samples weighing > 50 mg were digested in 2.0 ml of a mixture of
HNO3 (Sigma-Aldrich, 65%) and HCl (Riedel-de-Haën, 37%) in a ratio of 4:1
in closed teflon pots placed in an oven with a constant temperature of 140 ºC
for 7 hours. The digests in the glass tubes and teflon pots were diluted with 0.1
M HNO3 to volumes of 3.0 and 10 ml, respectively. For animals exposed to
0.5 mg Cu/L, organs were analyzed using flame atomic absorption
spectrophotometry (AAS, Perkin-Elmer AAnalyst 100), while the organs of
control and 0.05 mg Cu/L exposed animals were measured by graphite furnace
AAS (Perkin-Elmer 5100). Also copper concentrations in the exposure
solution were measured by flame or graphite furnace AAS. Quality control of
the analysis was maintained by digesting certified reference material Dolt-2
(CNRC, Ontario, Canada). The recoveries of copper in the reference material
averaged 57% (small glass tube digestion) and 108% (teflon pots digestion) of
the certified reference value, respectively. Considering the large deviations for
the micro-digestion method, all data were corrected for copper recovery in the
certified reference material.
Data analysis
The copper uptake and elimination kinetics in each crayfish organ were
analyzed using a one-compartment first-order kinetics model described by
Janssen et al . (1991). The model considers each organ as an independent
compartment with specific uptake and elimination rates. From the first 14-day
72
copper exposure period (the uptake phase), the copper concentration in the
organs was described as:
Qt = C0 + Cw(k1/k2)(1 – e- k2t)
After 14 days, when the animals were transferred to copper-free water (the
elimination phase), the copper concentration in the organs was described as:
Qt = C0 + Cw(k1/k2)(1 – e- k2t) – Cw(k1/k2) (1 – e –k2(t-tc))
where Qt = copper concentration in crayfish organ (μg Cu/g dry weight) at
given time; C0 = copper concentration in the crayfish organ at t = 0 (μg Cu/g
dry weight), Cw = copper concentration in the water during the uptake phase
(mg Cu/L), k1 = uptake rate (L water/kg organ dry weight per day), k2=
elimination rate (per day), t = time (day), tc = time at which animals were
transferred to copper-free water (day 14). Both equations were used
simultaneously to estimate the parameters C0, k1 and k2 using all data from the
uptake and elimination phases for each copper treatment level. The model was
applied to each organ separately, so it is assumed that all organs take up
copper from a central compartment.
To calculate Cu kinetics in the whole crayfish, these equations were also
applied to average total body concentrations estimated from the weighted sum
of the concentrations in the different organs. In the latter calculation only, the
average animal growth rate was taken into account to check to what extent
elimination rate (k2) might be affected by growth dilution. The effect of copper
concentration in the exposure solution and exposure time on the copper
73
concentration in each organ was analyzed using 2-way analysis of variance
(ANOVA). All calculations and statistical analyses were performed with the
statistics software SPSS, version 17.0 (SPSS Inc. Chicago, Illinois, USA).
Results
Of the 69 animals used, 64 (92.8 %) survived until the last day of the
experiment. The fresh weight of the animals at the start of the experiment was
comparable between treatments, with an average ± SD of 3.55 ± 0.018 g
(n=69). The average weight of the test animals increased with time reaching
3.97 ± 1.60 (mean ± SD; n=39) g after 14 days but did not significantly differ
from the starting weight (ANOVA, p > 0.05). Animal weights also did not
differ between copper exposure levels (ANOVA, p > 0.05). The growth rate
was estimated to be 0.006 and 0.007 g dry weight /day for the 0.05 and 0.5 mg
Cu/L exposure groups, respectively.
The average copper concentrations in the exposure solutions were 0.042 ±
0.004 mg Cu/L and 0.446 ± 0.018 mg Cu/L, respectively. Upon renewal of the
water after 2-3 days, the copper concentrations were considerably lower with
0.020 ± 0.007 mg Cu/L and 0.316 ± 0.057 mg Cu/L, respectively. The
decreased concentration was taken into account to calculate the average
exposure level. The average copper concentrations over the entire exposure
period used to calculate the uptake and elimination kinetics were 0.031 mg
Cu/L and 0.38 mg Cu/L, respectively.
74
Exo
skel
eton
Gill
s
Hep
atop
ancr
eas
Mus
cle
Who
le a
nim
al
Figu
re 1
. Upt
ake
and
elim
inat
ion
of c
oppe
r in
mar
bled
cra
yfis
h (P
roca
mba
rus
sp.)
exos
kele
ton,
gill
s, he
pato
panc
reas
, mus
cle
and
the
who
le
body
. Cra
yfis
h w
ere
expo
sed
for 1
4 da
ys (u
ptak
e) to
an
aver
age
mea
sure
d co
ncen
tratio
n of
0.3
8 m
g C
u/L,
afte
r whi
ch th
ey w
ere
trans
ferr
ed to
co
pper
-fre
e w
ater
(elim
inat
ion)
. Eac
h da
ta p
oint
repr
esen
ts a
mea
sure
men
t of a
n in
divi
dual
ani
mal
. Lin
es re
pres
ent t
he fi
t of a
one
-com
partm
ent
first
-ord
er k
inet
ics m
odel
to th
e da
ta.
75
Copper concentrations in the marbled crayfish and in their exoskeleton, gills,
muscles, and hepatopancreas exposed to an average measured concentration of
0.38 mg Cu/L are shown in Figure 1. Except for the ovaries, a good fit of the
one-compartment model was obtained. In the ovaries, copper concentrations
showed quite some variation and did not show a consistent change with copper
exposure level and time. Copper levels in the ovaries ranged between 12 and
534 mg/kg dry weight. Copper kinetics in the whole animals was not affected
by crayfish growth, suggesting there was no significant effect of growth
dilution. For that reason no further attempts were made to extend the model
with animal growth rate when assessing copper kinetics in the different tissues.
Estimated uptake and elimination rates at an average measured concentration
of 0.38 mg Cu/L are summarized in Table 1. This table also includes
bioconcentration factors (BCF) calculated as the ratio of the uptake and
elimination rate constants. BCF was highest for the hepatopancreas and lowest
for the muscles and exoskeleton. Average BCF for copper accumulation in the
whole animal was approximately 1214 L/kg. At an average measured
concentration of 0.031 mg Cu/L, copper concentrations did not significantly
increase with time, nor did they differ from the concentrations measured in the
control animals (data not shown). As a consequence, the one-compartment
model could not be fit to the data.
Table 2 shows the results of the two-way ANOVA applied to the copper
concentrations in the different crayfish organs. Copper concentrations in the
exoskeleton, the gills and the hepatopancreas of animals exposed to the
average measured concentration of 0.38 mg Cu/L were significantly higher
than levels in the animals exposed to the average measured value of 0.031 mg
Cu/L (ANOVA, p < 0.05). The effect of exposure concentration on copper
concentrations in the ovaries and muscles was not significant (ANOVA, p >
76
Tabl
e 1.
Cop
per u
ptak
e an
d el
imin
atio
n ra
tes
(k1
and
k 2) a
nd in
itial
con
cent
ratio
ns (C
0) w
ith c
orre
spon
ding
sta
ndar
d er
ror i
n di
ffer
ent o
rgan
s of
th
e m
arbl
ed c
rayf
ish
(Pro
cam
baru
s sp
.) ex
pose
d to
an
aver
age
mea
sure
d co
ncen
tratio
n of
0.3
8 m
g C
u/L
estim
ated
usi
ng a
one
-com
partm
ent f
irst-
orde
r kin
etic
s mod
el. A
lso
give
n ar
e bi
ocon
cent
ratio
n fa
ctor
s (B
CF)
cal
cula
ted
as th
e ra
tio o
f k1 an
d k 2
val
ues.
Cra
yfis
h
k 1
(L w
ater
/kg
orga
n d.
w./d
ay)
k 2
(d
ay-1
)
C0
(mg
Cu/
kg o
rgan
d.w
.)
BC
F
(L/k
g)
Ani
mal
41
.3 ±
17.
6 0.
034
± 0.
033
184
± 31
.9
1214
Ex
oske
leto
n 20
.1 ±
10.
1 0.
253
± 0.
120
50.4
± 6
.01
79.4
G
ills
257
± 13
9 0.
269
± 0.
136
490
± 78
.4
955
Mus
cle
13.5
± 7
.25
0.22
3 ±
0.11
4 24
.1 ±
4.8
8 60
.5
Hep
atop
ancr
eas
223
± 11
4 0.
008
± 0.
032
797
± 27
4 27
875
d.w
. is d
ry w
eigh
t Ta
ble
2. T
wo-
way
AN
OV
A m
easu
res
for
the
effe
ct o
f co
pper
exp
osur
e co
ncen
tratio
n an
d tim
e on
the
copp
er a
ccum
ulat
ion
leve
ls in
diff
eren
t or
gans
of m
arbl
ed c
rayf
ish
(Pro
cam
baru
s sp
.). T
he a
nim
als w
ere
expo
sed
to a
vera
ge m
easu
red
conc
entra
tion
of 0
.031
mg
Cu/
L or
0.3
8 m
g C
u/L,
re
spec
tivel
y fo
r 14
days
, afte
r whi
ch th
ey w
ere
trans
ferr
ed to
cop
per f
ree
wat
er u
ntil
day
28.
Dep
ende
nt v
aria
ble
Cop
per
expo
sure
T
ime
Inte
ract
ion
Cu
in e
xosk
elet
on
<0.0
01*
0.16
6 0.
108
Cu
in g
ills
0.04
5*
0.02
2*
0.37
8 C
u in
ova
ries
0.21
3 0.
238
0.29
8 C
u in
mus
cle
0.19
9 0.
285
0.61
2 C
u in
hep
atop
ancr
eas
<0.0
01*
0.00
6*
0.10
8 *S
igni
fican
t at 0
.05
77
0.05). Time had a significant effect on copper concentrations in the
hepatopancreas and the gills, but not in the exoskeleton, the muscle or the
ovaries. The interaction between copper exposure and time was not significant
for any of the organs.
Discussion
Copper concentrations in the marbled crayfish organs at the highest exposure
concentration could well be described by the one-compartment first-order
kinetics model. Copper exposure to aquatic animals showed a large variety of
sub lethal effects, which seem to be species-specific (Vasloo et al., 2002; van
Heerden et al ., 2004; Li et a l., 2007). Osunde et a l. (2004) showed that
survival of the juvenile freshwater prawn Macrobrachium rosenbergii was
significantly reduced compared to the control when exposed to 0.2 and 0.4 mg
Cu/L. In addition, exposure to 0.6 mg Cu/L showed a dramatic decrease of the
survival. Bini and Chelazzi (2006), exposing the crayfish Procambarus clarkii
to different copper concentrations in the water, found that 0.5 mg Cu/L caused
a (non-lethal) reduction in both heart and gill chamber ventilation rates. In this
study, marbled crayfish exposed for 14 days to average measured copper
concentrations of 0.38 mg Cu/L did not show any significant effects on
survival or growth. The nominal copper concentrations of 0.05 mg Cu/L and
0.5 mg Cu/L chosen for this study therefore did not have sublethal effects on
the marbled crayfish. Copper was present in all crayfish organs, also without
exposure to Cu-treated water (see the C0 values in Table 1). Copper is an
essential metal in most organisms, but plays an important role especially in
crustaceans. In these organisms, copper binds oxygen in hemocyanin, the
respiratory protein in the blood (hemolymph) (Anderson et al ., 1997). Uglow
78
(1969) in Donker et al . (1990) reported that hemocyanin constitutes
approximately 90% of the haemolymph protein. Each hemocyanin molecule
contains two copper atoms (Senkbeil and Wriston, 1981). Copper is also
required for normal biological functioning of many proteins, including
enzymes necessary for the moulting in blue crabs (Engel, 1987) and for the
immune response in grass shrimp juveniles (Lee and Shiau, 2002).
Animals exposed to an average measured concentration of 0.031 mg Cu/L
showed no clear uptake suggesting that marbled crayfish are able to regulate
copper in their bodies to a fairly constant level under low copper exposure.
Animals exposed to an average measured concentration of 0.38 mg Cu/L
showed increased uptake of copper, especially in the hepatopancreas. In this
organ, copper levels showed a continuous increase with time until transfer to
copper-free water. After the transfer, elimination of copper from the
hepatopancreas occurred very slowly. The hepatopancreas has an important
role in the copper homeostasis of several species (Donker et al., 1990; Lyon et
al., 1983; Chaves-Crooker et al., 2003) and contains metal-binding proteins, so
metals can be stored in this organ. It regulates the copper level in the crayfish
body to avoid toxicity but also deficiencies. Copper kinetics in the whole body
of marbled crayfish is very similar to that in the hepatopancreas, suggesting
the hepatopancreas is the organ that regulates copper level in crayfish. The
exoskeleton is a bio-mineral composite that serves as structural support
(Sugawara et al ., 2006), so it will mainly take up copper from the water by
adsorption. Gills, the respiratory organs of crayfish showed to be sensitive to
changing copper concentrations in the water. In crayfish, gills are the primary
entry points of copper in the body (Bryan, 1968 in Zia and Alikhan, 1979).
Through respiration, copper in the water binds to hemocyanin (the oxygen-
carrier protein in the hemolymph) and further circulates to all organs in the
79
crustacean body. Copper kinetics in the muscle and ovaries therefore result
from the hemocyanin circulating via the hemolymph. The copper accumulation
pattern in the marbled crayfish found in this study, with hepatopancreas > gills
> exoskeleton > muscle, is similar to the pattern reported for another crayfish
species Astacus leptodactylus (Guner, 2007) as well as for the freshwater
prawn Macrobachium rosenbergii (Li et al., 2005).
The bioconcentration factor (BCF) is a quantitative measure that indicates the
potential toxicological impact of substances (Feijtel et al., 1997). To produce
adverse effects, metals must first bioaccumulate at a specific site (McGeer et
al., 2003). But for an essential metal such as copper, the homeostatic control
and active regulation in animals might affect the bioaccumulation process, thus
also the BCF. Rainbow and White (1989) comparing three taxa of crustaceans
(decapod, amphipod and barnacle), showed active regulation of the copper
body concentration only in Palaemon elegans - a decapod. The BCF in
marbled crayfish of this study was 1214, lower than in Daphnia magna
exposed to 7.5 μg Cu/L (Winner, 1985) as well as in the Antarctic gammarid
Paramorsa walkeri after exposure to 30 μg Cu/L (Duquesne et al ., 2000),
which were ~ 2000 and 2080 respectively. It is suggested that the BCF may be
lower in bigger animals, because their relative surface is smaller, while BCF
also is known to decrease with increasing exposure concentration (McGeer et
al., 2003; DeForest et a l., 2007). No other BCF values for copper uptake in
larger crustacean species were available, however, hampering proper
conclusions about the possible relationship of the BCF with e.g. water
exposure concentration or body size.
The advantage of using marbled crayfish as test animal in this study is due to
the ease of culturing and production of high numbers of genetically identical
80
offspring (Martin et al . 2007). The marbled crayfish is useful in epigenetics
and epigenomics (Vogt, 2008) but may also be suitable as a (eco)toxicological
test species. This study supports the usefulness of this species for
(eco)toxicological tests.
Copper is an essential element also for humans, but excess copper in food-
intake may have negative effects on human health. Clinical features of copper
toxicity in humans include, among others, fatigue, depression, headaches, cold
extremities, lack of concentration and poor memory (Nolan, 1983). The
Australian National Health and Medical Research Council (ANHMRC)
recommends a maximum copper level in crustaceans of 10 µg/g wet weight
(Maher, 1985). Considering that marbled crayfish contains on average
approximately 72% water, the maximum copper concentrations measured in
marbled crayfish muscles (meat) in this study of 40 µg/g dry weight (~ 10 µg/g
wet weight) does not exceed the recommended level. Exposure of crayfish to a
measured concentration of 0.38 mg Cu/L therefore does not pose a threat to
food quality and safety.
Conclusions
• Exposure to sub-lethal copper levels up to 0.38 mg Cu/L for 14 days did
not affect survival and body weight development of marbled crayfish.
• At the lowest exposure level, copper was not significantly accumulated in
the crayfish, suggesting an effective regulation of body concentrations.
• At 0.38 mg Cu/L, copper was rapidly accumulated in most crayfish organs,
but also rapidly eliminated. Copper was mainly stored in the
hepatopancreas and uptake and elimination kinetics in this organ were
much slower.
81
Chapter 4
Effect of copper exposure on histamine concentrations
in the marbled crayfish (Procambarus sp.)
82
Abstract
Crustaceans can store excess copper in the hepatopancreas, an organ playing a
role in digestive activity as well as in neurosecretory control. Here, we studied
the effect of copper exposure on the level of histamine, an indicator of food
spoilage in edible crustaceans. Histamine is also a neuromodulator in the
intestinal nervous system of crustaceans, and a human allergen. Marbled
crayfish (Procambarus sp.) were exposed to average measured values 0.031
mg Cu/L and 0.38 mg Cu/L, respectively for 14 days and then transferred to
copper-free water for another 14 days. Concentrations of copper and histamine
in the hepatopancreas and muscle were evaluated at different time points.
Histamine levels were significantly higher in hepatopancreas and muscle
tissues at the highest exposure level, but only after transfer of the animals to
copper-free water. The increased histamine concentration following copper
exposure may be explained from a (delayed) stress response, and from up-
regulated histidine synthesis induced by copper, followed by decarboxylation
to histamine. Histamine concentrations in the crayfish muscle – the edible part
– did not exceed the United State Food and Drug Administration
recommended limit for seafood safety.
83
Introduction
Metal bioaccumulation in aquatic animals is an important phenomenon in
ecotoxicology and well documented for many years (Spehar et a l., 1978;
Calabrese et al ., 1984; Khan et al ., 1989; Baden et al ., 1999; Canivet et al .,
2001; Luoma and Rainbow, 2005; Veltman et al., 2008; Martins et al., 2011).
Among the metal pollutants in water bodies and sediments, copper is
interesting, because it is both essential and toxic depending on its available
concentration. Especially in crustaceans, copper binds oxygen in hemocyanin,
the respiratory protein in the crustacean blood (hemolymph) (Anderson et al.,
1997). In aquatic crustaceans exposure to excess copper causes physiological
disturbances, due to bioaccumulation. For instance, Bini and Chelazzi (2006)
reported that upon exposure to 0.5 mg Cu/L, Procambarus clarkii showed and
alteration in the cardiac and ventilatory rates. Several researchers reported
variability of the physiological status in crustaceans caused by copper,
including among others reduced osmoregulatory capacity (Haris and Santos,
2000) and changed moulting cycle (Engel and Brouwer, 1993). Furthermore,
physiological effects are preceded by or coincide with changes in biochemistry
of the animals. Li et al. (2007) reported copper effects on the structure of gills
and hepatopancreas in juvenile giant freshwater prawns coinciding with an
impact on the content of metallothionein.
In a previous study (Soedarini et al ., submitted; chapter 3 of this thesis),
marbled crayfish showed accumulation of copper in different tissues at
increased exposure concentrations in the water with excess copper especially
being stored in the hepatopancreas. The pattern of copper bioaccumulation in
crayfish is similar to that in other aquatic crustaceans, such as the penaeid
shrimp Metapenaeus dobsoni (Manisseri and Menon, 1995), the blue crab
84
Callinectes spp. (Sastre et al., 1999) and the freshwater prawn Macrobrachium
rosenbergii (Reddy et al., 2006). In crustaceans, the hepatopancreas is playing
an important role in digestive activity as well as in neurosecretory control.
Several studies have shown that histamine, a biogenic amine, plays a role as
neurotransmitter or neuromodulator in the stomatogastric nervous system of
crustaceans (Pulver et al., 2003; Cebada and Garcia, 2007). Histamine is also
an indicator of food spoilage in shelf-life studies with edible crustaceans.
Spoilage usually coincides with a marked increase of histamine due to growth
of histamine-producing bacteria. Considering that histamine may cause
poisoning with allergy-like symptoms in human, such as nausea, vomiting,
diarrhea, hives, itching, red rash, and hypotension (Taylor et al ., 1989), the
United States Food and Drug Administration (US FDA, 2011) has set a
maximum level of histamine in seafood at 50 mg histamine/kg (fresh weight).
In this study we ask the question whether copper accumulation interacts with
the histamine content of crayfish. Currently there are no studies on effects of
pollution on the histamine level in aquatic crustaceans. This study therefore is
unique in linking ecotoxicology with food safety aspects. We assess histamine
concentrations in marbled crayfish tissues under the influence of different
copper exposure levels.
Materials and methods
Test animals
Marbled crayfish (Procambarus sp.; Malacostraca, Decapoda, Astacida) were
obtained from Alterra, part of Wageningen University and Research Centre,
The Netherlands. Marbled crayfish itself is not a common edible species, but,
85
being a freshwater and parthenogenetic species, it is more easily cultured and
used in experiments than salt water species. The animals used measured 3.5 to
7.2 cm body length and weighed 1.3 to 7.4 g, and were acclimated in an
aquarium filled with aerated, filtered copper-free water (pH 7.3 ± 0.1; CaCO3
hardness 2–4 mmol/L) for one week prior to exposure. The crayfish were
incubated in a climate-controlled room at 20 ± 1 ºC; day/night cycle of 12
hours light and 12 hours dark and fed with commercial crayfish food pellets
(Tetra Wafer Mix) three times a week.
Copper exposure
Aqueous copper concentration of 0.05 mg Cu/L and 0.5 mg Cu/L were
prepared by dissolving CuSO4.5H2O (Merck, p.a.) in copper-free tap water
(pH 7.3 ± 0.1; hardness 2–4 mmol CaCO3/L). Copper-free tap water was
obtained from a special piping system without copper linings available at VU
University. The test animals were randomly divided in three groups and
exposed individually in 800 ml glass jars at 20 ± 1 ºC. The first two groups,
each having 31 animals, were exposed to the two different levels of copper
(0.05 and 0.5 mg Cu/L). The third group, containing seven animals, was the
control and kept in copper-free water. The animals were incubated in a
climate-controlled room under the same conditions as during acclimatization.
They were fed three times a week with commercial crayfish food pellets,
shortly before renewing the exposure solution. The exposure took 14 days
after which all remaining animals were transferred to copper-free water. At
different time points during the copper uptake (1, 2, 4, 8 and 14 days) and
elimination phases (15, 16, 18, 22 and 28 days) three animals from each
treatment, in the inter-moult stage, were sampled and killed by decapitation
using a lancet. Control animals were sampled only at 0 and 28 days. The
animals were dissected to collect the gills, hepatopancreas, ovaries, muscle and
86
exoskeleton. The organs were placed in plastic tubes with lid, frozen in liquid
nitrogen, weighed and kept in a freezer (-20 ºC) until analysis.
Histamine analysis
Histamine was extracted from the wet tissue samples (hepatopancreas and
muscle). Sample at a precise weight between 0.1 – 0.2 g F.W. were placed in
15 ml plastic tubes and 10 ml 0.1 M ethylenediaminetetraacetic acid (EDTA)-
2Na solution (pH 8.0) was added. The samples were boiled for 20 minutes and
then cooled on ice. The samples were subsequently filtered through a
membrane filter (0.2 µm cellulose acetate syringe filter WHAT10462701,
Whatman, UK) (Sato et al ., 2005). To determine the amount of histamine in
the filtrate, the histamine EIA Kit was used (Oxford Biomedical Research),
which is a competitive direct enzyme-linked immunosorbent assay (ELISA) in
a micro-well format that allows users to obtain histamine concentrations in
ng/ml range. A series of standard solutions containing pre-defined amounts of
histamine (0 – 50 ng/ml) was used. Absorbance was read at 650 nm using a
Versamax 340 – 750 nm plate reader (MTX Lab systems, Inc.)
Data analysis
All calculations were run using Excel (Window Microsoft Office 2007). The
effect of the copper concentration in the water and time of exposure on the
histamine concentrations in the crayfish organs were analyzed using two-way
analysis of variance (two-way ANOVA). Effects of copper on histamine
concentrations in the hepatopancreas and the muscle were determined by
Tukey’s test. Statistical analysis were run in the statistics software SPSS,
version 17.0 (SPSS Inc. Chicago, Illinois, USA).
87
Results
Data on measured copper concentrations in the water, crayfish growth and
copper uptake and elimination kinetics have been reported previously
(Soedarini et al., submitted; chapter 3 of this thesis). Average measured copper
concentrations in the water during the first 14-day test period were 0.031 mg
Cu/L and 0.38 mg Cu/L, respectively.
Development with time of the histamine concentrations in the hepatopancreas
and the muscle of marbled crayfish are shown in Figure 1. Results of the
corresponding statistical analysis are presented in Table 1. Overall, histamine
levels in the animals were significantly higher than the control at the highest
exposure level (p < 0.05), but this seemed mainly due to the levels in the
second part of the test when the animals were no longer exposed to copper
(Figure 1). Time did not affect histamine levels in the animals, except for
histamine in the hepatopancreas during the first 14-day test period, which was
significantly affected by time (p=0.040). A closer look to the data shows that
this is mainly caused by the relatively low histamine levels at the start and
after 1 day (Figure 1); this suggests a rapid built-up of histamine in the
hepatopancreas in the beginning of the experiment, unrelated to copper
exposure.
To better visualize effects of copper exposure concentration, histamine
concentrations averaged over all exposure times are shown in Figure 2. Copper
exposure concentrations did not affect histamine concentrations in the muscle
but showed a clear effect on the histamine levels in the hepatopancreas.
Averaged histamine concentrations in the muscle of animals exposed to
copper-free water, 0.031 mg Cu/L and 0.38 mg Cu/L were 1.28 ± 0.31, 1.28 ±
0.38 and 1.62 ± 0.51 mg histamine/kg fresh weight (mean ± SD), respectively.
88
Average histamine concentrations in the hepatopancreas of crayfish exposed to
0.031 mg Cu/L did not differ from the control, with values of 4.67 ± 1.37 and
5.95 ± 2.89 mg histamine/kg fresh weight (mean ± SD), respectively. In
contrast, histamine concentrations in the hepatopancreas of crayfish exposed to
0.38 mg Cu/L were significantly higher, reaching an average value of 9.41 ±
4.72 mg/kg (mean ± SD).
Table 1. Probability (p) values of F-tests in a two-way ANOVA for the effects of exposure time and copper concentration on the histamine concentrations in muscle and hepatopancreas of marbled crayfish (Procambarus sp.), separated for the uptake phase (day 1-14) where the animals were exposed to concentrations of 0.031 and 0.38 Cu mg/L and the elimination phase (day 15-28) where all animals were exposed to copper-free water.
Figure 1. Histamine concentrations in the hepatopancreas and the muscle of marbled crayfish (Procambarus sp.) measured at different exposure times to two different Cu concentrations for 14 days and after transfer to copper-free water for another 14 days. Each data point represents the averaged measured concentration in three test animals.
89
Figure 2. Average histamine concentrations in the hepatopancreas and the muscle tissue of marbled crayfish (Procambarus sp.) exposed for 14 days to different copper concentration levels and transferred to copper-free water for another 14 days. Error bars represent the standard deviations (n = 6, 27 and 29 for copper-free water, 0.05 mg Cu/L and 0.5 mg Cu/L, respectively). Different letters indicate significantly different histamine levels between treatments (ANOVA, Tukey’s post-hoc test).
Figure 3. Histamine concentrations in the hepatopancreas and the muscle of marbled crayfish (Procambarus sp.) expressed as a function of copper concentrations in these tissues. Animals were exposed for 14 days to different copper exposure levels and then transferred to copper-free water for another 14 days. Each data point represents an individual measurement. The “r” represents Pearson correlation coefficient; “p” represents degree of linear relationship between the two variables and “**” indicates strong correlation between copper concentration and histamine concentration in the hepatopancreas of marbled crayfish (SPSS, Pearson correlation test).
90
Figure 3 relates histamine concentrations to the copper levels in the analyzed
tissues. Histamine levels did significantly correlate with copper concentrations
in the hepatopancreas (p < 0.01), but not in the muscle of marbled crayfish
(Procambarus sp).
Discussion
Histamine is a well-known neuromodulator of the intestinal nervous system,
with a local (paracrine) action on motor neurons. In the lobster Homarus
americanus, histamine reduces the firing of neurons associated of the
stomatogastric system (Pulver et al. , 2003). The increase of histamine levels
under the influence of copper, observed in our experiments, may be linked to a
stress response of the animal by which it attempts to slow down pyloric
activity to prevent further uptake of copper. Alternatively, histamine could
arise because of an increase of the concentration of histidine, followed by
decarboxylation to histamine. Histidine has an important role in the copper-
containing, oxygen carrying molecule of crustaceans. Each hemocyanin
molecule contains two copper ions and each copper ion is coordinated by three
histidine residues. A highly up-regulated synthesis of histidine could be part of
a mechanism by which the animal attempts to scavenge and store copper in the
hepatopancreas. Any excess of histidine produced could be metabolized to
histamine. Which of these possibilities is true is difficult to say. They are both
speculative. However, since we observed an increase of histamine
concentrations only in the hepatopacreas, and since this was also the only
organ in which copper was selectively retained, a causal link between
histamine increase and copper accumulation is very likely. Both explanations
may also be valid when trying to explain the reason why histamine levels
91
showed a kind of delayed response, with higher levels arising especially in the
second part of the test when the animals were transferred to copper-free water.
In all cases, histamine concentrations in the muscle were significantly lower
than in the hepatopancreas. Histamine levels in muscle tissue of marbled
crayfish never exceeded 2 mg histamine/kg fresh weight. These concentrations
are lower than the maximum level of histamine in seafood of 50 mg
histamine/kg fresh weight set by the United States Food and Drug
Administration (US FDA, 2011). Ingestion of food containing histamine can
cause histamine poisoning with allergy-like symptoms such as nausea,
vomiting, diarrhea, hives, itching, red rash, and hypotension (Taylor et al .,
1989). But considering that none of the muscle samples in our experiment
exceeded the threshold of histamine set by the US-FDA, exposure of crayfish
to a copper concentration of 0.38 mg Cu/L does not seem to pose a threat to
food quality and safety, despite the fact that there was a significant increase of
histamine in the hepatopancreas.
Conclusions
• Histamine levels in the hepatopancreas but not in the muscle of marbled
crayfish (Procambarus sp.) increased with increasing copper concentrations
and time of exposure.
• Histamine levels in the hepatopancreas significantly correlated with
accumulated copper levels in this organ.
• Possible reasons for the link between increased histamine and copper
accumulation are (1) animal stress, and (2) up-regulated histidine
production associated with copper scavenging.
92
• At an exposure concentration of 0.38 mg Cu/L, histamine concentration in
the crayfish edible tissue (muscle) did not exceed the US-FDA limit level
for fresh seafood.
93
Chapter 5
Interactions between accumulated copper, bacterial
community structure and histamine formation in
stored crayfish meat after copper exposure
94
Abstract
Copper is toxic to microorganisms, but it is not clear whether copper
accumulated from the environment impacts deteriorative processes in stored
seafood, through affecting contaminating microbial communities associated
with histamine production. Crayfish (Procambarus sp.) were exposed to 0.50
mg Cu/L and the effect on microbial community structure in water and stored
meat samples, and on histamine levels were determined and compared to those
of crayfish cultured in copper-free water. Exposure to aqueous copper for 14
days increased the copper concentration by 17% in crustacean meat, but
copper remained within a range acceptable for human consumption. Cluster
analysis of 16S rRNA-based microbial community fingerprints revealed
copper toxicity to the freshwater bacterial community. Histamine in crayfish
was easily measured and an increase with storage was observed over 10 days.
Histamine concentrations in crayfish exposed to copper were significantly
lower and did not increase as rapidly, compared to those in control crayfish.
Thus, exposure to environmental copper appears to reduce histamine
accumulation in crayfish, by affecting the community of environmental (and
possibly intestinal) bacteria, while having only a minor effect on copper
accumulation in the meat itself. The data suggest a relation between bacteria in
the water and bacteria playing a role in deteriorative processes in crayfish
meat.
95
Introduction
World aquaculture has grown tremendously in quantity, but the quality and
safety aspects of aquaculture products (seafood) are still a concern due to the
risk of food-borne illnesses (Feldhusen, 2000). Among seafood, crustaceans
such as shrimp and prawn are important since they are the second most
valuable seafood commodity (FAO, 2006). Although they have high
economical value, crustaceans are considered as perishable food. The
relatively high moisture and free amino acids content (Simpson et al ., 1998)
make crustacean meat quite susceptible to deteriorative bacteria. Ahmed
(1991) stated that microorganisms, histamine and heavy metals are the major
concerns for seafood safety. Here, we investigated the interaction between
these three components.
The microbial status of seafood after catch is closely related to environmental
conditions and microbiological quality of the water (Feldhusen, 2000). During
storage, especially at inadequate temperatures, microbial growth can hardly be
avoided and leads to decay. Further, the presence and activity of histamine-
producing bacteria is often associated with deteriorative processes in seafood
(Kim et al ., 1999). Histamine-producing bacteria possess histidine
decarboxylase, an enzyme which transforms the free amino acid histidine into
histamine (Niven et al ., 1981). Ingestion of food containing histamine can
cause histamine poisoning with allergic-like symptoms such as nausea,
vomiting, diarrhea, hives, itching, red rash, and hypotension (Taylor et al .,
1989). In crustaceans, histamine may not only be produced by bacteria, but is
also naturally present and acts as neurotransmitter or neuromodulator (Cebada
and Garcia, 2007). The United State Food and Drug Administration (US FDA,
96
2011) has set the maximum level of histamine in seafood at 50 mg
histamine/kg (fresh weight).
Crustaceans are generally cultured in ponds in estuarine environments, which
are often exposed, through wastewater discharge, to metals including copper.
Elevated copper concentrations in estuarine environments occur world-wide
(Correa et al., 1999; Kirby et al., 2001; Matthiessen et al., 1999; Schiff et al.,
2007; Thongra-ar et al., 2008). Exposure to copper increases its concentration
in crustaceans, with the accumulation depending on the organ (hepatopancreas
> gills > exoskeleton > muscle; Guner, 2007; Soedarini et al ., submitted;
chapter 3 of this thesis). With regard to food safety assurance, the Australian
National Health and Medical Research Council (ANHMRC) recommends a
maximum copper level of 10 µg/g wet weight in crustaceans (Maher, 1985).
Copper can have an inhibitory effect on the growth of bacteria (Zevenhuizen et
al., 1979; Cabrero et al., 1998). Bacteria do not respond equally to copper, e.g.
Pseudomonas putida and P. syrin gae strains revealed resistance to copper
(Cooksey and Azad, 1992). By its nature, the interaction between metals and
microorganisms is a very complex phenomenon. Some metals are essential to
certain microorganisms and therefore required, whereas others are toxic even
at low concentrations. In a mixture with other metals, copper reduced the
microbial diversity in the digestive tracts of crayfish (Mickéniené and
Šyvokiené, 1999).
It is not yet clear if and how copper present in water will affect deteriorative
processes in stored meat - by affecting the occurrence and growth of
deteriorative bacteria and histamine producing bacteria. Here, we investigated
the interaction between aqueous copper, bacterial community in culture water
and stored meat as well as histamine accumulation in crustacean meat during
97
storage. Marbled crayfish (Procambarus sp.) was chosen as a model of edible
crustacean species due to its genetic uniformity (Martin et al ., 2007) and
availability.
Materials and methods
Source of animals
Marbled crayfish (Procambarus sp.; Malacostraca, Decapoda, Astacida) were
used as a model of freshwater edible crustaceans and were provided by
Alterra, part of Wageningen University and Research Centre, the Netherlands.
The animals measured 3.7-5.8 cm and weighed 1.6-4.7 g.
Copper exposure and sample preparations
Prior to copper exposure, crayfish were acclimated in copper-free tap water
(pH 7.3 ± 0.1; hardness 2–4 mmol CaCO3/L) for one week. Copper-free water
was obtained from a system involving Amsterdam tap water delivered by
plastic rather than metal pipings on the university’s premises. An exposure
solution of 0.5 mg Cu/L was prepared from CuSO4.5H2O (Merck, p.a.)
dissolved in copper-free water. Seventy animals were randomly divided into
two groups and exposed to 0.5 mg Cu/L and copper-free water, respectively,
for 14 days. The exposure took place in ten 3 L plastic aerated-aquaria with
seven animals in each. Throughout the experiment, the animals were incubated
in a climate-controlled room at 20 ± 1ºC with day/night cycle of 12 hours light
and 12 hours dark. They were fed three times a week with commercial crayfish
food pellets (Tetra Wafer Mix, one pellet per crayfish per feeding) three hours
before renewing the exposure solution. To collect microorganisms, 500 mL of
the culture water was vacuum-filtered over 45-mm-diameter, 0.2-μm pore-size
98
filter (Millipore). After 14 days of exposure, all crayfish were killed by
decapitation using a sterile scissor and the headless specimens (edible parts;
crayfish meat and tail) were individually placed in a sterile petridish and stored
in a refrigerator (5 ± 1ºC). At different times of storage (0, 2, 4, 5, 6, 7 and 10
days) five specimens from each treatment were collected. All samples (filter
and specimen) were kept in a freezer (-20 ºC) until analysis.
Copper concentration analysis
All crayfish samples (weighing around 0.25 g fresh weight) were freeze-dried
for 48 hours and digested in 2.0 mL of a mixture of HNO3 (Sigma-Aldrich,
65%) and HCl (Riedel-de-Haën, 37%) in a ratio of 4:1 in a closed teflon pot at
140 ºC for 7 hours. The digests were then diluted with 0.1 M HNO3 to 10 mL
and the copper concentration was analyzed using flame atomic absorption
spectrophotometry (Perkin-Elmer AAnalyst 100). The copper concentrations
in the culture water were directly measured before and after renewing the
water. Quality control of the analysis was maintained by digesting certified
reference material Dolt-2 (CNRC, Ontario, Canada). The recoveries of copper
in the reference material averaged 113% of the certified reference value.
Considering that the deviations were slightly greater than 10%, all data were
corrected for copper recovery in the certified reference material.
Microbial community structure analysis
Microbial DNA isolation
Microbial DNA in the crayfish meat and tail was isolated using PowerFoodTM
Microbial DNA isolation kit (MO BIO Laboratories, Inc., Solana Beach, CA,
USA). A frozen sample of 0.25 g was placed in a bead beater tube, one piece
of glass bead (4.0 mm diameter) and 1000 μL of sterilized PBS buffer were
added. A FastPrep Instrument (MP Biomedicals, Santa Ana, CA) was then
99
used for 40 seconds at speed level of 4 m/s. After 5 minutes of natural
sedimentation, 250 μL of supernatant was collected and centrifuged at 13.000
x g for 3 minutes. The residual liquid was removed and the microbial pellet at
the bottom of the tube was resuspended in 450 μL of PF1 solution from the
PowerFoodTM Microbial DNA isolation kit and further processed following
the manufacturer’s protocol.
The microbial DNA from the Millipore filters containing microorganisms in
the culture water was isolated using FastDNA spin kit for soil (MP
Biomedicals, Solon, OH). The filter was aseptically cut in small pieces and
further processed following the manufacturer’s protocol.
Bacterial DNA amplification
PCR amplification was performed with universal bacterial primers targeting
the 16S rRNA gene, F357GC and R518 (Muyzer et al., 1993). Table 1 shows
the sequence of the primers and the amplification conditions that we used prior
to DGGE. A total volume of 25 μL was used in each PCR reaction, containing
1 μL of 10 μM forward primer; 1 μL of 10 μM reverse primer; 1 μL of 10
mg/mL bovine serum albumin (BSA; New England BioLabs, Leusden, The
Netherlands); 12.5 μL GoTaq Colorless Master Mix 2 x (Promega, Madison,
WI); 8.5 μL DNase-RNasefree water (MP Biomedicals, Solon, OH) and 1 μL
microbial DNA template. Amplified DNA was verified by electrophoresis
using 1.0% agarose in 1 x TAE buffer. The size of the product of PCR
amplification was ~220 bp.
10
0
Tabl
e 1.
Prim
er se
quen
ces a
nd c
ondi
tions
for a
mpl
ifyin
g ba
cter
ial 1
6S rR
NA
gen
es a
nd h
istid
ine
deca
rbox
ylas
e hd
c ge
nes o
f Gra
m-n
egat
ive
and
Gra
m-p
ositi
ve b
acte
ria.
Prim
er
Sequ
ence
(5’
3’)
pr
imer
co
ncen
tratio
n
initi
al
dena
tura
tion
dena
tura
tion
anne
alin
g el
onga
tion
final
el
onga
tion
Ref
eren
ce
F357
GC
R
518
hdc-
f hd
c-r
HD
C-3
H
DC
-4
16S
rRN
A g
enes
C
GC
CC
GC
CG
CG
CG
CG
GC
GG
GC
GG
G
GC
GG
GG
GC
AC
GG
GG
GG
CC
TAC
GG
G
AG
GC
AG
CA
G
ATT
AC
CG
CG
GC
TG
CT
GG
G
ram
-neg
ativ
e hd
c ge
nes
TC
HA
TY
AR
YA
AC
TG
YG
GT
GA
CT
GG
RG
C
CC
AC
AK
CA
TB
AR
WG
GD
GT
RT
GR
CC
G
ram
-pos
itiv
e hd
c ge
nes
GA
TGG
TATW
GTT
TC
KTA
TGA
C
CA
AA
SWC
CD
GC
AT
CTT
C
0.4
μM
0.2
μM
0.2
μM
94
°C; 5
’
94°C
; 5’
94
°C; 5
’
94
°C; 3
0’’
94
°C; 1
’
95°C
; 45’
’
54
°C; 3
0’’
58
°C; 1
’
48°C
; 1’
72
°C; 3
0’’
72
°C; 1
’
72°C
; 1’
72
°C; 5
’
72 °
C; 5
’
72 °
C; 5
’
Muy
zer e
t al.
(199
3)
Taka
hash
i et a
l. (2
003)
C
oton
and
C
oton
(200
5)
101
Denaturing Gradient Gel Electrophoresis (DGGE)
DGGE was performed using the Bio-Rad D Code (Bio-Rad, Hercules, CA,
USA). PCR products were loaded in 8% (wt/vol) polyacrylamide gel with a
denaturing gradient of 30% - 55% (100% denaturant was defined as 7 M urea
and 40% (vol/vol) formamide). The gels were electrophoresed in 1 x TAE
buffer (40 mM Tris, 20 mM acetic acid, 1 mM Na-EDTA; pH 8.0) at 200 V
and 60°C for 4 h. A mixture of 12 different bacterial 16S rRNA gene
fragments was used as the marker. The gels were stained in 1 x TAE buffer
containing 1 μg of ethidium bromide ml-1 and were recorded with a charge-
coupled device camera system. Gel images were converted, normalized and
analyzed with Gel Compar II software package (Applied Maths, Belgium).
Similarity values between fingerprints were calculated using Pearson
correlation, and subjected to UPGMA cluster analysis (Van Verseveld and
Röling, 2004).
Sequencing of DGGE bands
The four most intense DGGE bands were cut out with a sterile scalpel blade
and each diluted in 50 μL water. One microliter of the diluted DNA of each
DGGE band was re-amplified using universal primers F357 and R518 and run
with the PCR program as described above. PCR products that revealed a
correctly sized band on agarose gel were cut out and purified using the Wizard
SV gel and PCR clean up system (Promega, Madison, WI) according to the
manufacturer’s instructions. Sequencing was performed using Big Dye v1.1
chemistry (Applied Biosystems) on an ABI 3100 Genetic Analyzer. To
determine the closest known relatives of the partial 16S rRNA gene sequences
obtained, BLAST searching was performed (Altschult et al., 1990).
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Detection of hdc genes
Table 1 shows primer sequences and amplification conditions used to detect
histidine decarboxylase (hdc) genes. Primers for the amplification of the hdc
gene in Gram-negative bacteria were hdc-f and hdc-r, which amplify a DNA
fragment of 709 bp (Takahashi et al ., 2003). Primers for the amplification of
the hdc gene in Gram-positive bacteria were HDC-3 and HDC-4 which
amplify a DNA fragment of 435 bp (Coton and Coton, 2005). DNA isolated
from Photobacterium damselae subsp. damselae (LMG 7892, BCCM, Gent,
Belgium) and Lactobacillus parabuchnerii (DSM 5987, DSMZ, Germany)
served as positive controls of Gram-negative and Gram-positive histamine-
producing bacteria, respectively.
Histamine analysis
A precise weight (between 0.10 and 0.25 g fresh weight) of crayfish sample
was crushed to homogeneity, 8 x diluted with water, and centrifuged (10,000
g; 5 minutes). Twenty μL of the collected supernatant was added in 180 μL
acylation buffer of the histamarine enzyme Immunoassay kit IM2369
(Immunotech SAS, Marseille, France). The principle analysis is a competitive
direct enzyme-linked immunosorbent assay (ELISA) in a micro-well format. A
series of standard solutions containing pre-defined amounts of histamine (1 –
500 mg/kg) was used. Absorbance was read at 410 nm using a Versamax 340
– 750 nm plate reader (MTX Lab systems, Inc.).
Data analysis
The effect of copper concentration in the exposure solution on the copper
concentration in crayfish meat was analyzed using one-way analysis of
variance (ANOVA). All calculations and statistical analysis were performed
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with the statistics software SPSS, version 17.0 (SPSS Inc. Chicago, Illinois,
USA).
Results
Copper exposure clearly affected the copper concentrations in the crayfish.
Copper concentrations in crayfish meat of the animals exposed to averaged
measured concentration of 0.43 mg/L copper were higher (N= 10; ANOVA, p
< 0.05) than in the control (copper-free water); they averaged 32.2 ± 5.61 μg/g
D.W. and 26.6 ± 5.93 μg/g D.W. (mean ± SD), respectively (Table 2).
Bacterial communities in the culture waters and in crayfish meat were profiled
by cultivation-independent DGGE analysis of amplified 16S rRNA gene
fragments, to reveal the impact of copper exposure during crayfish culturing
and subsequent storage of meat (Figure 1). The fingerprints of the marker of
12 different bacterial 16S rRNA gene fragments of the DGGEs were well
reproducible at > 85% (profiles are not shown).
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Figure 1. Cluster analysis of DGGE profiles of bacteria (30 to 55% denaturant gradient) present in the water and marbled crayfish meat samples after Pearson correlation. The codes next to the profiles designate the copper treatment (+ and – 0.5 mg Cu/L), duration of storage (0 and 10 days) and test replication (a, b and c).
Cluster analysis (Figure 1) revealed clear differences between bacterial
community structure in copper-free water and in aqueous copper (0.5 mg
Cu/L), with a similarity of 33.5% ± 19.1% (mean ± S.D). The DGGE profiles
revealed fewer bands (averaging 5.3) for water samples exposed to copper than
for samples from the control, copper-free water (averaging 13.3). The
similarity between replicates of DGGE fingerprints of microbial communities
in copper-free water was 66.5% ± 8.06%. More variation between replicates
was observed for samples that were exposed to 0.43 mg Cu/L, with a similarity
among replicates of 46.1% ± 36.9%. Bacterial community structures in the
crayfish meats (0-day stored) harvested from both treatments were clearly
different from those in the corresponding culture water. DGGE profiles of
meat from crayfish incubated in copper-free water showed only a single band.
This band was not visible in the DGGE profiles from the incubation water,
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which contained on average 12.3 bands. A similar observation was made for
meat from crayfish exposed to the aqueous copper, containing on average only
3.3 bands compared to 5.3 in the corresponding water samples. During storage
of the meat over a period of 10 days, a few more new bands occurred, both for
crayfish exposed to aqueous copper or copper-free water. The similarity
between 0 day and 10 days stored meat samples fell at 57.5% ± 10.6%
(crayfish exposed to aqueous copper) and 52.2% ± 3.5% (crayfish incubated in
copper-free water). Between the two copper treatments, the similarity of
bacterial community structure in crayfish meat after 10 days stored was 55.5%
± 6.4%. This was slightly lower than the correspondence between the
microbial communities in the meats from the two treatments at the start of the
storage, when the similarity was 60.0% ± 1.4%. Thus, differences in bacterial
community structure in relation to copper exposure were already visible at the
start of meat storage. Also, in both treatments, bacterial community structure
in the crayfish meat and the crayfish tail after ten days of storage showed very
similar profiles of DGGE fingerprints (the similarity fell at 78.5% ± 6.4%; data
not shown). One common band was present in all DGGE profiles from
crayfish meat samples, irrespective of copper treatment or the duration of
storage. This band was removed and sequenced. Based on BLAST searching,
the sequence (162 nucleotides) was 99% identical to Pseudomonas sp.
Histidine decarboxylase encoding genes of neither Gram-negative nor Gram-
positive bacteria could not be detected in any of the water or crayfish meat
samples. However, the histamine concentration in the crayfish meat samples
increased during refrigerated storage, for both control and copper exposed
crayfish (Figure 2). The strongest increase of histamine concentrations was
observed for the meat obtained from crayfish grown in copper-free water.
After five days of storage, histamine concentrations between the two
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treatments were significantly different (t-test, p < 0.05). After 10 days of
storage, crayfish meat from animals exposed to 0.5 mg Cu/L contained less
histamine than the meat of animals incubated in copper-free water, reaching
7.28 ± 2.58 and 21.6 ± 6.63 mg/kg fresh weight, respectively. The histamine
accumulation pattern appeared to correlate with the differences and changes in
bacterial communities over time. Crayfish meat of animals exposed to 0.5 mg
Cu/L after 10 days of storage revealed fewer DGGE bands and contained a
lower histamine concentrations.
Time (days)
His
tam
ine
in c
rayf
ish
mea
t (m
g/k
g f
.w.)
R2 = 0.891
R2 = 0.940
Time (days)
His
tam
ine
in c
rayf
ish
mea
t (m
g/k
g f
.w.)
Time (days)
His
tam
ine
in c
rayf
ish
mea
t (m
g/k
g f
.w.)
R2 = 0.891
R2 = 0.940
Figure 2. Histamine accumulation in crayfish meat samples during refrigerated storage for 10 days, including the trend lines based on polynomial regression (linear for the 0.5 mg Cu/L samples, quadratic for the copper-free samples). Each data point represents the average value of histamine concentration (N = 5) with the standard error bars measured in meat of crayfish exposed to 0.5 mg Cu/L (●) and animals incubated in copper-free water (▲). Coefficients of determination (R2) of the regressions are indicated in the graph. Histamine concentration is expressed in mg histamine / kg sample fresh weight.
107
Discussion
This study contributes knowledge that links animal toxicology and food
microbiology. Copper is known to have anti-microbial activity (Flemming and
Trevors, 1989), but how copper in aqua-culturing will affect the histamine
accumulation in crustacean meat during storage, especially by changing the
microbial community structure, is not clear yet. Copper and copper-containing
compounds are widely used as bactericide (Cooksey, 1990), algicides and
fungicides (Foye, 1977 in Cervantes and Gutierrez-Corona, 1994). On the
other hand, copper in general is an essential element for (micro)organisms
(Cervantes and Gutierrez-Corona, 1994) and not harmful at low concentrations
(1 – 10 μM). Copper especially plays an important role in crustaceans, where it
binds oxygen in hemocyanin, the respiratory protein in the crustacean
haemolymph (Anderson et al., 1997).
We observed that while copper exposure had a minor, acceptable effect on
copper concentrations in crayfish meat, it had a large impact on both the
microbial community structure in the culturing water and stored meat, and in
fact also resulted in decreased histamine production in stored meat. This
suggests that copper contamination may have a positive effect on the shelf life
of crustacean meat.
Copper exposure, copper accumulation and safety limits
A high copper concentrations in culturing water generally lead to elevated
copper concentrations in the organs or tissues of crustaceans (Rainbow and
White, 1989; Paganini and Bianchini, 2009), as also observed in this study
(Table 2). In crustaceans, excess copper is mostly accumulated in the
hepatopancreas and is also present in high concentration in the gills – the
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respiratory organ – but not in the muscle (Guner, 2007; Li et a l., 2008;
Soedarini et al., submitted; chapter 3 of this thesis). The copper concentration
in crayfish meat was increased by 17.4 %, to 32.2 μg/g dry weight, upon
exposure to 0.5 mg/L copper. The Australian National Health and Medical
Research Council recommends a maximum copper level in crustaceans of 10
µg/g wet weight (Maher, 1985). Considering that crayfish meat contains 78%
water (data not shown), the copper concentration measured in this study does
not exceed the recommended value and does not threaten human health.
Interactions between copper, microbial communities and histamine levels.
Despite the fact that copper concentrations in meat remained within food
safety limits, clear effects of copper exposure were observed on microbial
communities and histamine accumulation in stored crayfish meat. The
histamine accumulation pattern appeared to correlate with the differences and
changes in bacterial communities over time. Meat of crayfish exposed to 0.5
mg Cu/L and stored for 10 days revealed fewer microbial species and
contained lower histamine concentrations. We observed a correlation between
microbial communities in water and in meat. Different communities in water
and in meat, as the result of copper exposure were observed. Mickéniené and
Šyvokiené (1999) also found that copper exposure - in a mixture with other
metals - reduced microbial diversity and changed the microbial community
structure in the digestive tracts of crayfish.
The crayfish intestinal tract, running on the dorsal side of the animal (Figure 3)
contains high numbers of bacteria (DePaola et al ., 1994), including putative
deteriorative bacteria. As the edible, and stored, portion (consisting of the
posterior of the animal) usually still contains the intestine, contamination of
meat by intestinal material after harvesting and cutting the crayfish will be a
109
major source for deterioration during storage. In the present study the crayfish
was killed with a sterilized scissor and further treated under aseptic conditions,
therefore the risk of bacterial contamination from other sources during sample
handling was minimal.
Figure 3. Digestive system of crayfish (Barnes, 1987). The edible portion consists of the hind part of the animal (abdomen and part of the thorax), including the intestinal tract. During storage, the bacterial community in the intestines may grow and migrate into the muscle tissue.
It is likely that aquatic microbial communities determine the composition of
the intestinal communities and that deteriorative bacteria during decay derive
from the culture water, via intestinal communities. This can be concluded from
the following four arguments: (1) aqueous copper changed the bacterial
community structure in the culturing-water, (2) copper concentrations in
muscle tissue were only insignificantly affected by copper exposure and thus
likely had a minor direct influence on intestinal communities, (3) yet
communities in muscle tissue were different from communities in the water
and (4) histamine levels in the crayfish meat during storage were different
between the treatments. The copper concentration in the meat itself most likely
110
is not the major determinant of the histamine accumulation pattern in crayfish
meat during storage.
In general, copper toxicity for microbes and its effect on microbial community
structure in aquatic samples depends on a number of factors, among others the
exposure concentration (Timbrell, 1989), the chemical forms of the toxic
substance (Beswick et al ., 1976; Jonas, 1989; Tubbing et al ., 1994), with
especially the free ionic form (Cu2+) being toxic (Domek et al., 1984), and the
difference between microorganisms in the intolerance for copper (Reteuna et
al., 1989). Many species of bacteria show resistance to copper, among others
Pseudomonas syringae pv. tomato (Bender and Cooksey, 1986), Xanthomonas
campestris pv. juglandis (Stall et a l., 1986) and Escherichia coli strains that
carry the pco determinant (Brown et al ., 1992). Depending on the species,
copper resistance genes can be located either on transferable plasmids or on
chromosomal DNA (Cooksey et al., 1990; Brown et al., 1995).
Furthermore, the toxic effect of the cupric ions to aquatic microorganisms is
also influenced by dissolved organic material such as humic substances
(Santos et al., 2008), aerobic – anaerobic conditions (Beswick et al., 1976) and
salinity (Fleming and Trevors, 1989), which may all vary depending on the
geographical region and aquaculture practises. Thus, while a positive effect of
copper exposure on the quality of stored crayfish was observed in this study, it
may well be that under other environmental conditions other microbial
communities might be selected with different effects on histamine
accumulation in stored meat.
Pseudomonas sp. occurred in all crayfish meat samples, from both treatments
and throughout storage. Previously, Pseudomonas strains isolated from water
and sediment revealed copper resistance (Binder and Cooksey, 1987; Cooksey
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and Azad, 1992). Furthermore, Ryser et al. (1984) found that some species of
Pseudomonas (P. fluor escens, P. putida and non-fluorescent Pseudomonas
spp.) showed the ability to produce low concentrations of histamine.
Application of molecular tools to deteriorative processes.
Cultivation-independent DNA-based fingerprinting of bacterial communities,
followed by clustering analysis, is a reproducible, rapid and relatively
unbiased technique to describe the dynamics in bacterial communities and
their responses to experimental treatments. Isolation and analysis of DNA
directly from food samples (without cultivation of microorganisms) has an
advantage over prior cultivation and analysis of the cultivated bacteria.
Amman et al. (1995) mentioned that only a minor fraction of microorganisms
is cultivable under laboratory conditions, even when using enriched or
selective media. By using specific primers, also genes encoding special
functional characteristics can be determined relatively easily. However,
considering that histamine production was observed, but hdc genes were not
detected in our study, likely microbes harbouring hdc genes that deviate from
common hdc genes, were present. Thus, there is the need to attempt to culture
these species or use other culturing-independent approaches, such as
metagenomics, to gain insight in which of their genes are involved in
histamine production, so these genes can be targeted in the future using PCR
amplification approaches. Indeed the primers currently available for detecting
hdc genes are specific for bacteria with an ability to produce high
concentrations of histamine, such as Photobacterium damselae subsp.
damselae or Lactobacillus parabuchnerii - the positive controls of Gram-
negative and Gram-positive histamine-producing bacteria used in this study,
respectively.
112
113
Chapter 6
General discussion
114
Food safety appears to be a greater problem with aquaculture products
compared to marine wild-captured products. The risk of contamination – by
chemical and biological agents – is greater in freshwater as well as in coastal
ecosystems than in open sea (Reilly and Käferstein, 1999). The quality and
safety of aquaculture products in general varies from region to region,
depending on the production methods and management practices (Hopkins et
al., 1993) and also on the environmental quality of the region (Moullac and
Haffner, 2000). Considering water pollution is a global problem
(Schwarzenbach et al ., 2010), a comprehensive study on the interaction
between pollutants, particularly metal and microbial, and their impact on
edible aquatic animals as described in this thesis may contribute knowledge in
eco(toxicology), microbiology and food safety. It may also be useful for policy
makers working on aquaculture development programmes.
In this study, freshwater marbled crayfish (Procambarus sp.) was chosen as a
model of edible crustacean species. Marbled crayfish is accessible to
biological experiment due to ease of culturing and production of large numbers
of genetically identical offspring (Martin et al ., 2007). From an ecological
point of view, crustaceans tend to accumulate more metals than fish (Rainbow,
1992). Crustaceans are also known to produce histamine, a human allergenic
substance (Mietz and Karmas, 1978). In terms of seafood trading, crustaceans
are known as economically valuable seafood and cultured world wide.
This thesis is dealing with the interaction between metals in the aquatic
environment, microbial community structure and histamine production in a
surrogate seafood species. The conceptual model, introduced in the
Introduction, which links the three factors (metals, microorganisms and
seafood) is shown in Figure 1. Metals in the aquatic environment will affect
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the microbial community in the water but also result in metal bioaccumulation.
Both factors will affect the safety status of the seafood. Metal accumulation in
animal organs may also lead to toxic effects on the organisms cultured, with
increased histamine level as one of the toxic effect indicators. Changes in the
microbial community structure due to metal exposure may not only occur in
the water but also in the seafood. As a consequence of these changes, the
population of histamine-producing bacteria in the seafood items may change,
both in size and activity. This may possibly affect seafood shelf-life and
quality, by increasing histamine production. In this thesis, all these factors
have been studied and their effect on food quality and safety will be discussed
here.
Copper was selected as the metal of interest referring to the situation found in
shrimp ponds along the Semarang coast line (Chapter 2). We observed that
cadmium concentrations in all sediment samples were below the detection
limit (0.03 μg/g d.w.), while zinc, nickel and iron concentrations varied among
locations but were generally also low. Sediments taken from two shrimp ponds
located close to the intercity highway had lead concentrations that were
significantly higher than at the other locations. In three sampling sites, copper
concentrations were significantly higher than at the other sites, up to twice the
local background concentration, which is approximately 40 µg/g dry sediment.
In addition to copper and lead, nickel and chromium at some sites also showed
elevated sediment concentrations. According to the Sediment Quality
Guidelines (SQGs) applied in several countries, the Semarang coastal pond
sediments can be classified as mid-range (medium range) contaminated. The
contamination may partly be attributed to anthropogenic inputs. Copper
contamination in coastal areas is occurring
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Figure 1. A model of the interactions between copper and microorganisms in the aquatic environment and their impact on marbled crayfish as a model of edible crustaceans species, with special concern for food quality and safety. Copper bioaccumulation in crayfish meat and histamine formation in the crustacean triggered by copper exposure has only minor effects on seafood safety. Copper exposure changes bacterial community structure both in the culture water and in the crayfish, and also decreases histamine accumulation in the edible tissues during storage.
worldwide, among others in the Atlantic (Chou et al., 2002; Dean et al., 2007),
Aegean (Kucuksezgin et al ., 2010), Northern Adriatic (Munari and Mistri,
2007), Far East (Alagarsamy, 2006) and Southeast Asia (Nicholson and Lam,
2005). In the case of aquaculture ponds, there are at least two major ways for
copper entering the water system, i.e. intentional and accidental. Application
of chemicals intended to clear the water and suppress excessive growth of
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algae likely contributes to copper contamination. Copper is the active
compound of several algicides, e.g. copper sulphate, and algicides containing
copper are still used worldwide (Song et al., 2010; Jacinthe et al., 2010). The
use of algicides in aquaculture practices sometimes is hard to avoid because of
its benefits: (1) to avoid oxygen depletion in the water that leads to sudden
mass mortality in fish and crayfish, especially at night due to excessive growth
of algae stimulated by fertilizer application (Diana et al ., 1991) and (2) to
avoid off-flavour in aquaculture products due to accumulation of blue-green
algae (Gibson, 1972; Tucker, 2000). Copper may also enter aquaculture ponds
by the intake of copper-contaminated water – from rivers or estuaries – used
for maintaining the water level of the ponds. Several sources of anthropogenic
copper in water bodies have been reported, including among others a wide
range of industrial waste (Graedel et al ., 2002; Widmer et al ., 2005),
antifouling paints (Srinivasan and Swain, 2007), and domestic waste water
(Isaac at al., 1997). The higher the population density and the industrial
activity present in a coastal area, the higher the input of anthropogenic copper
in water bodies may be. The fact that tributyltin (TBT) was banned as an
antifouling agent on small boats in 1990, may also lead to an increased use of
copper-containing paints (Helland and Bakke, 2002) and this further increases
the risk of copper contamination in coastal areas. Considering the fact that
Semarang is a densely populated industrial city with an active harbour, there
are many possible sources of copper, however, the main ones are probably
derived from (1) algicides, (2) industrial waste, (3) domestic waste, and (4)
copper-based antifouling agents.
Our study on copper toxicokinetics in marbled crayfish (Procambarus sp.)
showed there was no clear uptake in animals exposed to 0.031 mg Cu/L,
suggesting that animals are able to regulate low copper concentrations in their
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body at a fairly constant level. This is seen in many animals because copper is
an essential trace metal playing an important role in e.g. hemocyanin and
antioxidant enzymes such as superoxide dismutase. However, at higher
exposure levels, the internal copper concentration is not regulated at the same
level, at least not in all organs. Copper concentrations in the exoskeleton, the
gills and the hepatopancreas of marbled crayfish exposed to 0.38 mg Cu/L
were significantly higher than levels in the animals exposed to 0.031 mg Cu/L.
The exoskeleton, gills, muscle and ovaries accumulated copper relatively fast
and reached equilibrium within 10 days of exposure. Copper accumulation was
highest in the hepatopancreas as uptake in this storage organ steadily increased
with time and did not reach equilibrium within the 14-day exposure period
(Chapter 3). The copper accumulation levels in the marbled crayfish found in
this study were hepatopancreas > gills > exoskeleton > muscle. The tendency
of marbled crayfish to accumulate copper especially in hepatopancreas is
typical for crustaceans (Icely and Not, 1980; Ahsanullah et al ., 1981; Bagato
and Alikhan, 1987; Vogt and Quinitio, 1994; Anderson et al ., 1997; Chavez-
Crooker et al., 2003; Guner, 2007). All species that have hemocyanin in their
blood system (haemolymph) store an excess of copper in the hepatopancreas.
In terms of food safety, the maximum copper concentrations measured in the
marbled crayfish muscles (meat) were 40 µg/g dry weight (~10 µg/g wet
weight). This level does not exceed the recommendations set by the Australian
National Health and Medical Research Council (ANHMRC) for seafood,
which is 10 µg/g wet weight (Maher, 1985).
Metal accumulation in marbled crayfish organs affected the concentrations of
histamine, an important indicator of food spoilage used in food safety research.
Histamine levels showed that the higher the copper exposure concentrations,
the higher the histamine levels were in the hepatopancreas. A rapid built-up of
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histamine in the hepatopancreas was started right from the beginning of the
copper exposure. Copper exposure concentrations did not affect histamine
concentrations in the crayfish muscle. Histamine concentrations in the
hepatopancreas of crayfish exposed to 0.38 mg Cu/L approximated 10 mg
histamine/kg fresh weight (Chapter 4). In animal biochemistry, physiology and
neurobiology, histamine is known as one of the major neuromodulators
(besides dopamine) that plays a role in the animal nervous system (Pulver et
al., 2003; Cebada and Garcia, 2007). Histamine is specifically associated with
modulation of muscle action in intestinal tissues. Why histamine would
increase with copper accumulation is unknown. The phenomenon we observed
is interesting but so far has not been reported in the literature. The proposed
mechanisms in explaining this phenomenon are (1) a stress response of the
animal or (2) upregulation of histidine production followed by decarboxylation
reactions. The amino acid histidine is an important chelator of metals. Copper
is bound to histidine residues in hemocyanin. In this hypothesis, copper would
induce the production of histidine, of which part is decarboxylated to produce
histamine. Testing these hypotheses poses an interesting subject for further
research. Histamine concentrations in the muscle in all cases never exceeded 2
mg histamine/kg fresh weight, which is much lower than the maximum level
of histamine in seafood of 50 mg histamine/kg fresh weight set by the United
States Food and Drug Administration (US FDA, 2011) thus the experiment
indicated that copper exposure itself did not affect seafood safety.
Histamine has a negative impact on seafood quality and safety. The level of
histamine is an accepted index of seafood deterioration due to the growth of
histamine-producing bacteria (Russel and Maretic, 1986). In our study, we
observed that 0.50 mg Cu/L significantly changed the bacterial community in
the freshwater. Upon storage, histamine concentrations in meat of crayfish
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exposed to 0.50 mg Cu/L were significantly lower and did not increase as
rapidly compared to those in control crayfish. After 10 days of storage, meat
from crayfish exposed to 0.50 mg Cu/L contained approximately 7.5 mg
histamine/kg fresh weight, significantly less than the meat of animals
incubated in copper-free water, which approximated 22 mg/kg fresh weight.
Considering that copper can slow down histamine accumulation during storage
by changing the bacterial community structure in the culture water, we suggest
that the increased histamine level during storage is derived from the bacteria
present in the water (Chapter 5). Aquatic bacteria will also be present in the
gut, and these might be the source of bacterial growth in the animal after it
died. Consequently, when copper affects the bacterial community in the water,
there will also be an effect on the bacterial community growing in the animal
during storage. In this study aseptic conditions were employed during
processing the crayfish meat, because several studies on histamine in seafood
observed that poor sanitation and mishandling during processing are the cause
of the increased histamine levels (López-Sabater et a l., 1994; Tsai et al .,
2004). Elevated levels of histamine in seafood can have severe implications for
human beings because histamine induces physical responses like seafood
allergy with burning sensation in the mouth and asthma (Bucca et al ., 1991),
besides nausea, vomiting, diarrhea, hives, itching, red rash, and hypotension
(Taylor et al ., 1989). In international seafood trade, the seafood quality
standard criteria set by the United States Food and Drug Administration,
including the criteria for histamine (maximum 50 mg histamine/kg fresh
weight), are used as the guidelines (Ababouch et al., 2005). According to our
study, the histamine concentrations found in the crayfish meat from both
treatments were lower than the standard and not causing concern regarding
seafood safety. But considering that in real aquaculture ponds more diverse
bacterial communities may exist – including histamine-producing bacteria –
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higher concentrations of histamine during storage may be produced potentially
leading to food-safety risks. Also, contamination during handling and
processing of the seafood has to be taken into account since it may also lead to
similar effects on histamine accumulation during storage. Determining which
source of bacterial contaminations – from the aquatic environment or from
seafood handling – will play a major role in histamine accumulation is also an
interesting subject for future research.
According to the copper accumulation pattern and histamine levels in marbled
crayfish in our studies (Chapter 3 and Chapter 4, respectively), high copper
concentrations and high levels of histamine especially occur in the
hepatopancreas. This seems irrelevant to seafood safety considering that the
hepatopancreas is non-edible. But in terms of small size edible crustaceans,
such as Euphausia pacifica Hansen, Euphausia superba Dana and Sergia
lucens Hansen (Kubota and Kobayashi, 1988), where the whole body is
considered as the edible portion, information of copper concentration in each
organ as well as in the whole body and histamine concentration in the
hepatopancreas becomes important. Consuming the whole body of
crustaceans, including the gills and hepatopancreas – organs with high
concentrations of copper – indeed may pose a hazard for food safety. In terms
of food safety assessment, the major consideration is dietary exposure. Two
factors are needed for defining the dietary exposure, i.e. data on food
consumption and data on the concentration of chemicals in food (Nesreddine
and Parent-Massin, 2002). A further step is comparing the estimated dietary
exposure to the toxicological reference value of the chemical of concern.
In the food safety risk assessment, the concept of acceptable daily intake
(ADI) is commonly applied. ADI is the level of daily intake of specific
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substances by humans, on a body weight basis, which would not cause an
appreciable risk. The ADI concept has been developed and established by
FAO/WHO together with the International Program on Chemical Safety
(WHO, 1987). The ADI concept has been developed to deal with food
containing specific substances – that are considered toxic or cause adverse
health effects – under an assumption that exposure levels below the threshold
are safe. The ADI concept allows people to consume certain amounts of “food
with caution” without any worry for their health, as long as the concentrations
are below the threshold and amounts of food taken do not exceed
recommended values. The threshold level is based on the “no observed adverse
effect level” (NOAEL), which in most cases is determined, based in laboratory
toxicity studies with test animals. The ADI concept is therefore derived from
the NOAEL with a safety factor of 100 (Kuiper et al., 2001). The provisional
guidelines determined by the World Health Organization of the United Nations
(WHO, 1998) determined the upper limit of copper intake at 2 -3 mg/kg body
weight per day. Intake copper from any source – food and drink – under those
dosages should not give any adverse health effects, even in case of long-term
exposure. Several foods, such as liver and meat, seafood, nuts, and seeds
(NAS, 1989) and drinking water (US EPA, 1991) are considered as high
copper diet materials. In the human body, copper is essential and thus required
for the proper functioning of many enzyme systems, such as superoxide
dismutase, cytochrome oxidase, tyrosinase, monoamine oxidase and
phenylalanine hydroxylase (Linder and Hazegh-Azam, 1996). But Olivares et
al. (2001) mentioned that acute exposure to high copper concentrations can
cause food-poisoning symptoms including as nausea, vomiting, diarrhea, and
abdominal pain. Furthermore, several studies with mice showed that although
copper is not known for its carcinogenicity (Toyokuni & Sagripanti, 1994) it
has a tendency to be mutagenic (BRL, 1968 in WHO, 1998). When
123
considering our data, in case of a person with 60 kg body weight consuming
100 g of a marbled crayfish exposed to 0.5 mg Cu/L (containing 185 mg Cu/kg
dry weight or ~ 46 mg Cu/kg fresh weight), the exposure will be 0.08 mg/kg
body weight. Referring to the WHO standard limit of copper intake of 2-3
mg/kg body weight per day, consuming the whole body of marbled crayfish
does not pose any risk.
According to our findings, exposure to copper at nominal concentrations up to
0.5 mg/L had a minor effect on histamine levels of live crayfish and did not
lead to unacceptable copper levels in crayfish meat. It did, however, have a
large impact on both the microbial community structure in the culturing water
and in the stored meat. In Figure 1, the arrow that connects histamine levels in
live animals to food safety and the arrow connecting copper accumulation to
food safety are printed in white as these interactions are considered to have
minor effects on food safety. In contrast, the arrow that connects histamine
accumulation in the meat during storage is printed in grey colour as it is
considered to represent a serious impact. Copper exposure decreased histamine
production in stored meat. This suggests that copper contamination may have a
positive effect on the shelf life of crustacean meat. This effect of copper is
indirect, by impacting the microbial community structure. Effect of copper
application during seafood handling on seafood quality and safety poses an
interesting subject for future research. Also, whether the copper toxicity on
microorganisms also occurs with other metals or organic pollutants needs
further research. Comparing the copper behaviour in different species of
crustaceans, especially those living in freshwater and brackish water may also
be an interesting subject of future research.
124
A unique feature of this study is the comprehensive link between
environmental quality and food safety. The effect of copper in the aquatic
environment on edible crustacean model species, both alive (i.e. copper
accumulation and histamine levels) and during storage (i.e. microbial
community structure and histamine accumulation) was studied. Several
scientific approaches were involved in this study: toxicology, microbiology
and food safety. In terms of toxicology, copper uptake and elimination kinetics
in each organ of the marbled crayfish was described (Chapter 3). Also, a new
insight was gained on increasing histamine levels in the crayfish, especially in
the hepatopancreas, triggered by copper (Chapter 4). In both chapters, the
relevancy of the interaction between copper accumulation and histamine levels
for food safety was elucidated. Further copper exposure changed the bacterial
community structures both in culture water and crayfish meat, which
eventually affected the histamine accumulation during storage (Chapter 5).
The (molecular)-microbiology and food safety approaches were
comprehensively utilized for explaining the phenomenon. Our finding on
microbial contamination in the aquatic environment affecting seafood quality
supports the statement of Ahmed (1991) that “the quality of seafood reflects
the quality of the water from which the seafood is taken”. Aquaculture
production under poor hygiene and poor sanitation will result in faster
deterioration of seafood, and also in higher levels of accumulated histamine
during storage. Moderate copper contamination in the aquatic environment
does not pose a risk to food safety in terms of metal bioaccumulation and even
leads to the growth inhibition of the deteriorative bacteria in seafood during
storage.
125
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Acknowledgement
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When I recall, all the things associated with my studies in the department of
Ecological Science, VU University Amsterdam, they were full of the
miraculous works of God. After nearly five years of effort to achieve a
scholarship for pursuing a PhD abroad, on September 2008 I finally “won” a
three year full scholarship provided by the Indonesian government.
Something that sounded “impossible” considering that Indonesia is a
developing country that still is struggling to emerge from a long economical
crisis. Completing a set of experiments and writing the thesis under a very
tight schedule and relatively little time was another proof of the divine
intervention of God. So, my first gratitude goes to Jesus Christ and Mother
Mary for all the miraculous works, for turning the “missions impossible” into
a “reality”, and for all the blessings I received throughout the three years and
three months of my study in The Netherland. Also, I am truly appreciative
of the Indonesian government, especially the Directorate General of Higher
Education for providing a three years full scholarship for PhD study abroad
with regards to improving my academic capabilities.
I would like to express my deep gratitude to my promotors and co-
promotors, Prof. N.M. van Straalen (Nico), Prof. B. Widianarko (Pak Bud),
Dr. C.A.M. van Gestel (Kees) and Dr. W.F.M. Röling (Wilfred) for the trust
and exceptional opportunity considering that my educational background is
in food science rather than in biology. I also express my sincere gratitude for
their wonderful support throughout the years of my study. It was definitely
an extraordinary good luck to have a combination of four magnificent
professors! Nico and Pak Bud, both of you always came up with wise advise
and strategic decisions. Your empathy and caring, from the beginning of my
study, or actually long before any scholarship was available for me, that still
exist until now is just expression of your genuine kindness. Nico, that was a
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great experience to have you picked me up from the Schiphol airport and
drive me by car to the FIC monastery Den Haag (a special housing
arrangement where I stayed for the first three months in The Netherland).
Pak Bud, I will never forget your willingness to guide me finding the shrimp
ponds even when it was under a heavy tropical rain! For Kees, I am really
grateful for the “in depth mentoring” you carried out in many ways,
including a weekly discussion on the book entitle “Metal contamination in
aquatic environments”. Your expertise on ecotoxicology combined with high
accuracy, also your patience and indulgence gave me such “confident and
comfort” on dealing with the “toxicokinetic” subject. The twice Christmas
dinners, at the end 2009 and 2010, you arranged in your house in the
Bilthoven were unforgettable moments! Wilfred, without your thoughtful
guidance, constructive advice and keen interest, I would never have been
able to complete the crucial topic of my thesis. Thank you so much for the
kind offer of the Brock-Biology of microorganism book! I wish that there
will be other opportunities for me to collaborate with each of you in the near
future.
Special thank to Dr. Ad Ragas, Dr. Ansje Löhr, Dr. Ir. Dick Roelofs, Dr.
Michiel Rutgers and Prof. dr. Margot van Eck v.d. Sluijs – van de Bor for
spending valuable time, carefully reading, correcting and giving evaluation
to this dissertation.
I am thankful to Rudo Verweij for his genuine kindness and serious
assistance, not only when dealing with the laboratory analysis, but also
picking the crayfish up from the Alterra in Wageningen. Together with
Janine, you revived my “dormant-talent” for pedalling a bicycle. Also, you
provided a great help when I had to move from the Laan van Kronenburg
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117 to the Bouwmeester 61 in Amstelveen. Rudo, I wish one of these days
you will get a chance to visit UNIKA Semarang again; and for sure, I will
bring you to the best food court in Semarang! I am also gratified to Dr. Ivo
Roessink who provided the crayfish and critical reading of some chapters in
this dissertation. I really appreciate Linda Klaver, a master student from the
department of Health Science VU University, for her dedication and helpful
collaboration on the experiments with crayfish. I also thank Jorke Kamstra
for kind assistance on dealing with the Versamax plate reader.
It was a great experience to get the opportunity to socialize with all member
of the sub department of animal ecology, of course under an international-
warm atmosphere. The professors and staffs (Prof. Jacintha Ellers, Prof.dr.
Herman A. Verhoef, Dr. Dick Roelofs, Dr. Joris Koene, Dr. Gerard Driessen,
Dr. Matty Berg and Dr. Toby Kiers), the former and current post-docs
(Thierry Janssens, Roel Pel, Ben Nota, Tjalf de Broer, André Tavares Dias,
Fernando Monroy and Wei Luo), the lab-technicians (Janine, Riet, Kora and
Florrie), the PhD students, the guests, the master and the undergraduate
students who were all friendly, kind and supportive just makes me simply
“homy”. Also, I really thank the secretary of the department of Ecological
Science, Desiree Hoonhoud and Karin, for their truly kind and hospitality. In
particular, special thanks to Maria Diez Ortiz – the angel of the hearth –,
Daniel Giesen – the disseminator of the youth spirit –, Cécile Le Lan – the
cheerful lady–, Elferra Swart – the big mama –, Valentina Zizzari – the
Italian princes –, also Masoud Ardestani, Pauline Kool, Yumi Nakadera,
Elaine van Ommen Kloeke, Bertanne Visser, Erik Verbruggen, Yifu Pei and
Marta Ferreira for friendship, kindness and caring. Further, I greatly
appreciate the member of the molecular cell physiology group, especially
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Suzana Dirieto, Raquel Vargas, Annachiara, James Weedon, Egia Zaura and
Martin Braster for support.
I would like to express my gratitude to the leadership of UNIKA
Soegijapranata Semarang (Prof. Dr. Ir. Budi Widianarko; Ibu Theresia Dwi
Hastuti, S.E., MSi., Akt.; Dr. Angelina Ika Rahutami, MSi., Benedictus
Danang Setyanto, SH., LLM; also Ferdinandus Hindiarto, S.Psi, MSi) for
their constant support. Also, I thank Lenny Setyowati and Susana Jawi for
assistance on the administrative matters. My sincere thanks also to the
members of Department of Food Technology UNIKA Soegijapranata: Ita
Sulistyawati, Inneke Hantoro, Kristina Ananingsih, Kartika Puspa Dwiana,
Lindayani, Laksmi Hartayani, Probo Yulianto, Retnaningsih, Rika Pratiwi,
and Sumardi, also Roswari, Agus Waskito, Wati, Supriyana and Wartono for
consideration and support. Also I would like to thank my colleagues: Yulita
Titik, Octavianus Digdo and Marcella Elwina for the nice time in the IHS
housing Den Haag. Special thanks for Tyas Susanty and Trihoni Nalesti
Dewi, friends who were always supportive and kind. Last but not least, I
would like to express my gratefulness to Felix Sholeh and pak Sondakh, for
assistance on collecting sediments from the ten shrimp ponds. Mas Sholeh
and pak Sondakh, without your help, I would never have been able to bring
any sediment samples to The Netherlands!
During my stay in the Netherlands, I enjoyed the Christmas 2009 in the FIC
house in Maastricht and for the wonderful occasion, I am really grateful to
Bruder Dr. Martinus Handoko – the former Rector of UNIKA –, Bruder
Anton Hadiwardoyo, Bruder Guido and also Bruder Lou. I am so sorry
Bruders, that during the last one and half year I was not able to visit the FIC
house. I will never forget the nice warm house with special arrangement of
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breakfast and supper! I also thank Alida Ruitinga for providing a cozy room
in her house during the last seven months of my stay. Special thanks to
Erryana Martati for the long term friendship and valuable sharing. Also for
the authentic Indonesian cuisine you always provided everytime I visited
your apartment in Bornsesteeg, Wageningen. I am so lucky to meet several
Indonesian friends as the member of “maksi-ma”: Lia Auliaherliaty, Rully
Adi Nugroho, mas Herman, Dessy Rahmawati, Enade and Romo Sunu
Hardiyanto. Lia, without your help, I would never have a representative map
of Semarang in my dissertation. I really enjoyed our friendship and I thank
you very much for showing me many interesting spots of Amsterdam,
Roermond and Lelystad. Pak Rully, thank you very much for all the smart-
tips and valuable sharing! Romo Sunu, I really thank you for letting me
know a piece of the “Jesuit techniques” on dealing with negative emotions.
Oom Bambang and Tante Ida, Oom Markoni and Tante Titin, I will never
forget the celebration of the New Year 2010, which was full of Indonesian
delicious menu! Furthermore, I express my sincere thanks to Ibu Widji
Mardiningsih in Semarang, for her truly caring and deep hospitality since
1998 until now. For Hartati and Sandra – sisters in the faith – thank you very
much for all the prayers. I missed the monthly devotion in Gua Maria Kerep
Ambarawa for many times!
Finally, I would like to convey my sincere gratitude to my parent, Bapak Ibu
Atmawiyana in Yogyakarta, my brother Adi Santosa and my sister Felicitas
Sumarmi for their everlasting supports, deep understanding, love and prayer.
Last but not least, I would like to thank Michael J. McCann, the American
“partner in crime”, for unconditional affection and support, for sharing the
Irish inspiring prayer as well as showing the well-known pilgrimage sites in
Europe.
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Curriculum vitae
Bernadeta Soedarini was born in Yogyakarta - Central Java, Indonesia on
Sunday, February 4, 1968. Prior to starting her academic career in the
Soegijapranata Catholic University (UNIKA) - Semarang in 1993, she
completed all her formal education in Yogyakarta. In 1998, she earned a
Master degree in Food Science and Technology from the Postgraduate
Program, Gadjah Mada University - Yogyakarta. Further in 2001, she got an
opportunity as a UNESCO fellow at the Osaka University, Japan and earned
a diploma in Advance Microbiology in 2002. Following her UNESCO
fellowship, she got a three years full scholarship from the Directorate
General Higher Education of the Indonesian Government on September
2008; and she started her PhD degree at the Department of Ecological
Science, Vrije Universiteit Amsterdam, the Netherlands. As of now, she is
affiliated as a permanent lecturer in the Department of Food Technology,
Faculty of Agricultural Technology, UNIKA – Semarang, Indonesia.
Amsterdam, October 2011
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