Integrated multi-trophic aquaculture: - HZ University of Applied ...

Integrated multi-trophic aquaculture:Bioremediation of particulate sea bass waste by the polychaetes Capitella sp. and Nereisdiversicolor in a Recirculating Aquaculture SystembyVera van BruggenHZ University of Applied SciencesFebruary 2012Internship supervisionM. Callier; IfremerJ. van Houcke; HZ University of Applied SciencesJ. Heringa; HZ University of Applied Sciences

Integrated multi-trophic aquacultureBioremediation of particulate sea bass waste by the polychaetes Capitella sp. and Nereisdiversicolor in a Recirculating Aquaculture SystemBy:Vera van BruggenStudent number: 41417Hogeschool ZeelandFebruary 2012Internship supervisionM. Callier; IfremerJ. van Houcke; HZ University of Applied SciencesJ. Heringa; HZ University of Applied Sciences

Foreword and AcknowledgmentThe internship was part of the RAAK project (the Salty Gold) on land based integrated aquacultureand ECOSYM/IFREMER project on "Fish waste bioremediation".RAAK, ‘the Salty Gold’ is an international project for applied research with goals in understandingand acquiring knowledge on IMTA. The province of Zeeland is a pioneer when it comes to initiativesfor aquaculture in the Netherlands. The RAAK project aims to have cooperation and exchange ofknowledge between small and medium enterprises (SME) from different countries. The centralresearch question of RAAK project is: How can we realize a sustainable, reliable and competitiveproduction in land based marine water systems? Particularly in France, the necessary practicalknowledge is developed on integrated aquaculture in recirculating aquaculture system andintegrated cultivation in lagoons and former saltpans which is relevant to the concepts of extensiveoutdoor aquaculture which is on the increase in Zeeland.ECOSYM is a joint research unit which includes well established research institutions: CNRS, theUniversity of Montpellier II, the public research institute for the development of Southern countries(IRD) and The French Institute for Exploitation of the Sea (Ifremer). ECOSYM focuses its researchactivities on the study of the “effects of local and global changes linked to human activities onlagoons and marine coastal ecosystems”. Ifremer has been involved for 30 years in research onaquaculture, marine biology and aquatic ecosystems. Ifremer addresses all the major scientificfields related with aquaculture, notably fish physiology for growth and reproduction, nutrition,genetics, rearing systems technology, health and welfare management both on fish and shellfishbiological models.During the internship, I have received help and advice from the team in Ifremer Palavas. I wouldlike to thank Myriam Callier, Jean-Paul Blancheton, Cyrille Przybyla, Thibault Geoffroy and LorenaDediu. I would also like to thank Jasper van Houcke, Jouke Heringa and Jorik Creemers.ii

SummaryRAAK, ‘the Salty gold’ is an international project for applied research with goals in understandingand acquiring knowledge on integrated multi-trophic aquaculture (IMTA). The province of Zeeland isa pioneer when it comes to initiatives for aquaculture in the Netherlands. The RAAK project aims tohave cooperation and exchange of knowledge between small and medium enterprises (SME) fromdifferent countries. The central research question of RAAK project is: How can a sustainable,reliable and competitive production in land based marine water systems be realized? Particularly inFrance, the necessary practical knowledge is developed (integrated cultivation in lagoons andformer saltpans) which is relevant to the concepts of extensive outdoor aquaculture which is on theincrease in Zeeland.Research on the treatment of waste water of marine aquaculture began 15 years ago at a researchinstitute based in the south of France (Ifremer). This treatment was based on the use of macroalgae. Now Ifremer is interested in the possibilities of using Nereis diversicolor and Capitella sp.as astepping stone for remediation of inorganic fish waste from the Sea bass farm at location. It iscalled a stepping stone because the waste will not only be treated by Nereis diversicolor andCapitella sp. for the particulate organic fish waste, but it probably will be treated afterwards bymacro algae to decrease the concentration of nutrients. In the end, the water will be re-circulatedinto the fish farm.The possibilities of bioremediation of waste with the use of Nereis diversicolor and Capitella sp.were examined by using different scaled experiments. The initiator was interested in thepossibilities of these polychaetes for remediation of particulate organic fish waste. These wormswould not only be used for degradation of the particles but Capitella sp. and Nereis diversicoloralso have an economic value (feed for fish or fish bait).During an experiment (on mesocosm scale) it was examined what the possibilities are for Nereisdiversicolor and Capitella sp. to remediate particulate waste that comes from a sea bass farm. Thefocus will be on characterization of the particulate fish waste and water quality (nitrogen,phosphorous and organic matter) and biomass of worms (wet weight, dry weight and ash free dryweight). Two experiments were done. One experiment only with Capitella sp. and one experimentwith both worms (Capitella sp. and Nereis diversicolor). Different densities of worms were usedduring the two different experiments. The objective of the first experiment was to evaluate thefeeding capacity of Capitella sp. raised at different densities (0, 100, 1000 and 10.000 individualsper m -2 ) and fed with fish waste. The objective of the second experiment was to compare the fishwaste bioremediation by the two polychaetes Capitella sp. and N. diversicolor, raised individuallyand in combination (0 worms, 300 Nereis diversicolor, 150 Nereis diversicolor and 10.000 Capitellasp., and 20.000 Capitella sp. individuals per m -2 ).The underlying processes (converting waste load into biomass and converting of particulate organicfish waste into dissolved nutrients) are very dependent on the stocking density of worms and wasteload of the fish farm (applicable to both experiments). Observed was that after 6 days of bothexperiments most of the particulate organic fish waste were used by the worms for biomass but alsodissolving the nutrients in the water column. The treatments with the highest number of wormswere breaking down the particulate organic fish waste very fast even a shortage of input wasobserved. This means that a bigger input of particulate organic fish waste is possible for moreremediation. The treatment with both Capitella sp. and Nereis diversicolor was the most idealbecause of the lowest mortal rate and good results on remediation.During the experiments several ideas came up to improve and change the follow-up experiments.One recommendation is to do a research on the ideal Capitella sp. Nereis diversicolor combinationto enhance the reproduction of Capitella sp. but also the habitat of Nereis diversicolor. Also a flowthrough system could be a solution for a better experimental setup. During the experiment ashortage of particulate organic fish waste occurred. With a flow through system this could beprevented, necessary will be to do research on the ideal flow through ratio (l/h) and organic load.iii

Table of Contents1.0 Introduction ............................................................................................................................... 11.1 Context ................................................................................................................................... 11.2 Candidate species and remediation .................................................................................. 21.3 Problem analysis ................................................................................................................ 42.0 Material and Method ................................................................................................................ 52.1 General Methods ................................................................................................................... 52.2 Experiment 1: Effects of Capitella sp. density on waste bioremediation ................ 92.3 Experiment 2: Nereis diversicolor and Capitella sp. experiment ............................. 103.0 Results and Discussion ........................................................................................................... 123.1 Capitella sp. experiment .................................................................................................. 123.1.1 Physical Parameters ....................................................................................................... 123.1.2 Nutrients analysis ........................................................................................................... 123.1.3 Organic matter ................................................................................................................ 153.1.4 Growth of Capitella sp. ................................................................................................. 163.2 Capitella sp. and Nereis diversicolor experiment ........................................................ 173.2.1 Physical Parameters ....................................................................................................... 173.2.2 Nutrient analysis ............................................................................................................. 173.2.3 Organic matter ................................................................................................................ 213.2.4 Growth of Capitella sp. and Nereis diversicolor ...................................................... 224.0 Conclusion ................................................................................................................................ 255.0 Recommendations .................................................................................................................. 26References ........................................................................................................................................... 27Annex .................................................................................................................................................... 29I. Physical parameters Capitella sp. experiment ................................................................. 29II. Physical parameters Capitella sp. and Nereis diversicolor experiment ....................... 30III. Capitella sp. and Nereis diversicolor experiment input and output of worms ........ 31

1.0 Introduction1.1 ContextAquaculture industry is growing worldwide and is already providing over a quarter of the seafoodsupply (FAO, 2010). This figure will probably increase as aquaculture expands and fish catchdecreases because of overfishing and global changes (FAO, 2010). Aquaculture can cause damage tothe environment which is raising questions on how to best meet the food demands but also not puttoo much pressure on the ecosystem. To develop the aquaculture sector in a sustainable way, it iscrucial to have good management practice and control nutrient waste induced by fish farming. Oneof the major environmental concerns is related to the inorganic (mainly ammonium and phosphate)and organic fish farm effluent (faeces and uneaten food) to the environment (Hargrave 2005).Excessive release of the waste water is leading to severe problems (pollution of estuaries) andbecame a major concern for the aquaculture industry. Nowadays a lot of research is done to solvethis problem. The use of land based recirculating aquaculture system (RAS) allows reduction ofaquaculture effluent and the amount of water used, and thus reduces impacts on the environment(Martin et al. 2009). RAS systems combine rearing tanks with sediment traps to collect sedimentedparticulates (faeces and food). A mechanical filter is used to remove suspended particles and a biofiltration for the nitrification and denitrification.Another approach is the development of a modern polyculture, called integrated multi-trophicaquaculture (IMTA). This concept combines different trophic levels in one system, for example, fedaquaculture(Sea bass e.g.) with extractive inorganic aquaculture (example: seaweed) andextractive organic aquaculture (shellfish, sea cucumbers) (Chopin et al. 2006). This principle isbased on the possibility of using waste (fish faeces and uneaten food) as a food source for anotheraquatic culture. To improve RAS system, it is possible to add biological compartments and thereforedevelop a “RAS-Integrated multi-trophic aquaculture”. Research has been done on the optimizationof RAS waste treatment in combining High Rate Algal Ponds. It has been shown that Ulva rigidavalorizes dissolved nutrients (mainly nitrate output from the bio filter (Neori et al. 2004 Metaxa etal. 2006)).Little research has been done on the organic particulate waste in RAS, although almost 60% of thephosphorus not assimilate by fish is lost as organic phosphorus in faeces. The reduction of theparticulate organic fish waste within aquaculture plants may be achieved by the use of livingorganisms. One is the focus on the remediation of particulate organic fish waste by worms. Specificexamples in the literature reveal that some invertebrates are potential remediators of heavymetals, microbial contaminants, hydrocarbons, nutrients and persistent organic pollutants (Beckeret al. 2009). In particular, filter-feeding marine macro invertebrates filter large volumes of watersfor their food requirements and exert high efficiency in retaining small particles including bacteria(Matthew et al. 2011). Many of these macro invertebrates such as oysters, mussels, clams,polychaetes and sponges, are suitable bioremediators of particulate organic fish waste and have theability to generate an economic return following remediation activities. Bioremediation is anattractive option for aquaculture without many negative impacts. In particular, filter-feedersinvertebrates on account of their capability to remove organic compounds through the filtrationprocess, have been recently proposed as potential bioremediators (Zhanga et al. 2011).The presentproject is part of a program carried out at Ifremer Palavas station on the use of polychaetes for theremediation of fish waste (ECOSYM/IFREMER). The initiator is interested in the possibilities of thesepolychaetes for remediation of particulate organic waste. The objectives of the program are 1) toconvert the organic waste into worm biomass for bioremediation, 2) to produce a co-product witheconomic value (polychaetes could be used as feed for fish or as fish bait), 3) to treat waste waterfrom the fish and polychaete cultures via algal ponds.1

The aim of the present project was to test the use of two polychaetes: Capitella sp. and Nereisdiversicolor on the mineralisation of fish waste. Nereis diversicolor and Capitella sp. have beenchosen because they are known to be able to use fish waste as food source and are common specieson the Mediterranean coastal zone. The worms are also used for their capability to enhance wastemineralization. Indeed the various sediment activities of deposit-feeders (feeding and bioturbation)enhance organic matter mineralisation and recycling (Aller et al. 1994). Through bioturbation, theseanimals enhance sediment reworking and oxygen penetration to the sediment depth, therebystimulating mineralization of organic matter (Caron et al. 2003)1.2 Candidate species and remediationNereis diversicolorThe rag worm (Nereis diversicolor) is a marine polychaete in the family Nereididae. These wormsare commonly found in the littoral zone of coastal areas in Western Europe. They live in U-shapedsediment burrow up to 50 cm depth, in sandy mud, gravel and clay (Duroua et al. 2008).Reproduction of N. diversicolor happens once a year at full moon (N. diversicolor is mature at twoyears of age) at the end of April. The worms live at densities varying from 35 to 3700 individuals persquare meter. N. diversicolor is a brackish and euryhaline species which means the species istolerant and can adapt to a wide range of salinities. The worm is also known as an indicator oforganic pollution and is characterized by a high physiological tolerance to extreme variation ofseveral environmental parameters such as temperature and salinity. Nereis diversicolor is able tochange the feeding mode from filter feeding to deposit feeding or active carnivorous feeding onsmall meiobentic organisms (Bischoff et al. 2008). Feeding of N. diversicolor happens mostly aroundthe openings of the burrows by swallowing surface mud. As a predator, N. diversicolor had beensuggested to be an important structuring factor in soft bottom communities (Gillet et al. 2008).Capitella sp.Capitella sp. is a polychaete belonging to the family Capitellidae. These worms are found in manydifferent sediment types from intertidal areas to the deep sea, most of the time in a highabundance. Capitella sp. commonly lives in mucous lined tubes or burrows. Most of their dietconsists of detritus, algae, bacteria, microfauna and meiofauna (http://www.eol.org/pages/153,consulted May 2011) Capitella sp. is an opportunistic deposit feeding polychaete. Capitella sp.commonly inhabits in muddy estuarine sediments. The sizes of adult worms range from 20 to 40mm, and the wet weight ranges from 3 to 12 mg. Capitella sp. has a short life cycle (52 days at20°C (Tsutsumi et al. 2005)). Capitella sp. also has a very short generation time of six days at atemperature of 20° C. Its sex is controlled through a simple system of female heterogamety(Gremare et al. 1988). Deposit feeding polychaetes obtain their nutrients from the sediment.Deposit feeders are also known of their importance as secondary producers in wetland ecosystems.They play a role in the ecological cycle by transferring energy and material from the primaryproducers and detritus to the organisms (fishes and birds) in the higher trophic levels (Hua et al.2003). A bioremediation technique has been developed using artificially mass-cultured colonies ofCapitella sp. known is Capitella sp. favors organically enriched sediment, aiming at acceleratingdecomposition of organic matter and oxidation of reduces substances in the organically enrichedsediment (Tsutsumi et al. 2005).2

RemediationNutrient availability on the sediment particles can influence the growth and the survival Capitellasp. and Nereis diversicolor. Polychaete worms change their ingestion rates (especially when foodavailability is limited) to maintain the homeostasis of metabolism (Bayne and Newell 1983; Calow1975; Phillips 1984; Taghon 1981). Animals may also increase their ingestions rates with the increaseof food concentrations with the purpose to obtain more nutrients. Thus the sediment nutrients caninfluence the feeding behavior of deposit feeders, which in turn has its effects on growth andreproduction. The sediment nutritional values to deposit feeders can be estimated by two methods,chemical analyses and bioassay approaches (Cheng et al. 1993; Page et al. 1992). The combinationof these two methods can determine what the limiting nutrients are to the worms, but also itsavailability in the field.Food availability can influence the nutrient uptake, the feeding and growth responses of depositfeeders can be used as the bioassay approaches to determine the sediment nutritional values. Theingestion rate can represent the response of individual animals to the change of food quality. Thegrowth rate of immature animals, on the other hand, reflects the amount of energy assimilatedafter food ingested.3

1.3 Problem analysisFigure 1: Schematic problem analysis. Sea bass aquaculture produces dissolved and particulate waste.Polychaetes could enhance the mineralization of the organic waste (faeces and uneaten particles) increasingnutrient concentration in the water that could be used by macro algae. Extra oxygen was put into eachmesocosm for the worms.Key question:What are the possibilities of Nereis diversicolor and Capitella sp. to remediate particulate organicfish waste?Sub questions:What is the rate of removal of organic particulate fish waste by Nereis diversicolor and Capitellasp. in relation to species composition?How much of the organic waste is allocated in respectively worm biomass and dissolved nutrients?To what extend are the underlying processes dependent on stocking densities and waste load?Two experiments have been carried out to answer these questions.4

2.0 Material and MethodIn this research two different experiments were carried out. The objective of the first experiment(Effects of Capitella sp. density on waste bioremediation) was to evaluate the feeding capacity ofCapitella sp. raised at different densities (0, 100, 1000 and 10.000 individuals per m 2 ) and fed withfish waste. The objective of the second experiment (Nereis diversicolor and Capitella sp.experiment) was to compare the fish waste bioremediation by the two polychaetes Capitella sp.and N. diversicolor, raised individually and in combination (0, 300 Nereis diversicolor, 150 Nereisdiversicolor and 10.000 Capitella sp., and 20.000 Capitella sp. individuals per m -2 ). As outlined intable 1.Table 1: Number of Capitella sp. and Nereis diversicolor in the second experiment where the nutrient uptakeof different species ratios was tested.N.diversicolor in individuals/m 2 Capitella sp. in individuals/m 2Situation 1 150 0Situation 2 0 10.000Situation 3 300 10.000Situation 4 150 20.000During this second experiment, two types of substrates were tested (natural (fine) sand andmedium-coarse sand), to determine if the habitat could influence the activity of the worms andthus their remediation capacity. Similar methodology was used in both experiments and is describedin the first section (2.1), the sections (2.2) and (2.3) described the methods for the first and thesecond experiment, respectively.2.1 General MethodsBelow, the methods used for both experiments are described.Figure 2: Particulate organic fish waste (left) and sediment trap (right).First, the quantity and quality (% organic matter, %OM) of solid fish waste was determined. Solidwaste was collected from the sediment trap (connected to the fish farm) (Figure 2) with a bucket.5

The content of the bucket was stirred for homogenization and 200 ml subsamples were poured intobeakers (Figure 2). This quantity was then poured into each mesocosm.3 sub-samples samples of 30 ml were taken following the same method and were filtered on predriedand weighed filters and placed in the oven at 80 °C for 24 h. Filters were weighed todetermine the dry weight (DW). Filters were then burnt at 500 °C for 4.5 h and weighed again, todetermine the ash free dry weight (AFDW). Percentage of organic matter was determined on weightloss on ignition: %OM= ((DW-AFDW)/DW)*100%.For both experiments, filters and aluminum cups have been used. All filters and cups were pre-driedat a temperature of 80 °C for 24 h. Cups with filters were weighed with an analytical balance andhandled with tweezers to avoid contact.Physical parametersFigure 3: Odeon multi-parameter liquid analyzerThe physical parameters (pH, O 2 (mg/l), temperature (° Celsius) and salinity (mg/l)) were measuredevery day during the experiments using an Odeon Multi-parameter liquid analyzer: pH, conductivity,ORP, dissolved oxygen) (figure 3). These measurements were done to check the experiment, tomake sure there is only a slight difference of these parameters during the experiment.The material was rinsed with Mili Q water before each measurement. The probe was stirred forapproximately 20 seconds to obtain stable values. The Oxygen electrode consuming oxygen itself, itis important to stir gently the probe to obtain an accurate O 2 value and a stable pH. Salinity wasrecorded when the value was stabilized.Drying filters and aluminum cups:During the experiments organic content measurements were done. As said before the filters andcups were pre dried to be sure no moisture was left behind in the material. The filters andaluminum cups were dried at a temperature of 80 °C for 24 hours. After this the cups with filtersare weighed with an analytical balance. Filters and cups are handled with tweezers to avoid contactwith human tissue.6

Collecting Capitella sp.Figure 4: Left; plastic pipette is shown (used for collecting Capitella sp.). Right schematic example ofCapitella sp. which is carrying eggsThe collection of Capitella sp. was done by getting a subsample of sediment from the open sewer atIFREMER station (all water coming from the sea bass farm runs through this sewer), rinsing thesample with water and putting the residue in a tray. After 10 to 15 minutes, the worms wereseparated from each other because they are naturally clustered together when removed from thesediment. Worms were counted under a binocular and collected with a plastic pipette (Figure 4).Capitella are very small (3 mm to 10 mm long) (Figure 4). Females with eggs were avoided duringthe experiments because of the short lifecycle of the worm (52 days) (Tsutsumi et al. 2005) and tobe sure to have a fixed number of worms per mesocosm. These worms were collected with plasticpipettes, rinsed with filtered seawater and put on a petri dish. Worms were left in filtered seawaterfor 12 hours to empty their guts. This was important to determine their initial weight. In figure 4 aplastic pipette and a schematic representation of a Capitella sp. is shown.7

Collecting Nereis diversicolorFigure 5: Collecting Nereis diversicolor in the fieldNereis diversicolor was collected in the field (Figure 5) with the use of a pvc-pipe, a bucket and asieve. The worms were collected note that these worms do not differ too much from size. In figure6, the collection spot is shown on the map.PalavasCollection spotIfremerFigure 6: the collection spot of Nereis diversicolor.Dry weight and Ash free dry weight of worms:The dry weight of Capitella sp. and Nereis diversicolor was determined at the beginning and end ofan experiment. The average dry weight of the worms at the beginning and the end of theexperiment and biomass gain or loss was determined: W = W1 – W0 where W is difference ofaverage biomass of the worms, W1 the average biomass at T1 and W0 the average biomass at thebeginning of the experiment.At the start of the experiment 3 subsamples of 10 Capitella sp. and 1 Nereis diversicolor (to notwaste too many worms, collecting worms is very time consuming) were collected, left 12 hours infiltered seawater to empty their gut and placed in a pre-weighed cup. Worms in cup were put in anoven at 80 °C for 24 hours and put in an oxicator for an hour to cool down and weight again (dryweight). Finally, the samples were put in a furnace (500 °C for 4 hours) to determine the ash freedry weight (AFDW). This was done with tweezers to avoid contact with human tissue.8

Nutrient analysisAlso nutrient analyses were carried out during the two experiments. The analyses were done byThibault Geoffroy with an auto analyzer. PO 4 3- and NH 4 + can be found in the results. UnfortunatelyNO 3 - and NO 2 - are not concluded in the experiment because of the inconvenience of a brokenanalyzer.2.2 Experiment 1: Effects of Capitella sp. density on waste bioremediationThis experiment was done with Capitella sp. only (no sediment). The choice for the use of only thisworm was made to examine the characteristics of this worm. The reason was see the influence ofbacteria on remediation of particulate organic waste but also the possible cooperation of the wormand the bacteria on remediation. Different densities of worms per m 2 (0, 100, 1000 and 10.000)were tested which means 0, 4, 37 and 372 worms per mesocosm (in this experiment bottles (r= 5.4h= 11, surface 0.037 m2) were used as mesocosms) (adult worms, avoiding females with eggs)(Figure 7: B, C, D).During this experiment no substrate was added. The reason was to examine if Capitella sp. couldmanage without substrate. The possibility was for these worms to function without substrate couldbe important to know, not only for the species itself but also for commercial use (when substrate isnot required it can be cost effective for the economy). If there is no necessity to implementsubstrate these worms will have an advantage in the upcoming industry for the usage of worms. Theexperiment was done from 28-6-2011 till 11-7-2011 (table 2).Figure 7: Setup of the Capitella sp. experiment, the numbers in the mesocosms represents the amount ofCapitella sp. per mesocosmAn increase in abundance of worms during the 13 days of the experiment was expected. At thesedensities, 18 mesocosms mean 3717 worms were used during this experiment. A comparison wasmade between treatment with and without Capitella sp. This gives an idea of particulate organicfish waste degradation without Capitella sp. (only bacterial effect) (figure 7: A).9

The particulate organic fish waste was put in the mesocosms (200 ml of organic matter in solution)(figure 7) to examine if there is an influence variability of remediation and remineralization byCapitella sp. 200 ml sea water was added. At day 0, the sample of organic matter in solution wasanalyzed to know the percentage of organic material in the fish faeces at the start of theexperiment. At day 6 two bottles of each treatment were removed to determine the organic matterleft after six days, also the worms were counted and the weight (WW, DW and AFDW) wasdetermined. In every mesocosm aeration was added.Table 2: Time planning of experiment 1, green box means this measurement was done on the specific day2.3 Experiment 2: Nereis diversicolor and Capitella sp. experimentIn the experiment the capability of the worms Nereis diversicolor and Capitella sp. to remediatewaste coming from the seabass farm was examined (separate and the combination of the twospecies).The experiment was done with two different types of sediment. The reason for that was toinvestigate if there is a significant difference between remediation in the two different types ofsand. Aquarium sand (

24 mesocosms were used, 12 mesocosms with the natural sand type and 12 with the aquarium sand.The experiment was carried out in triplicate. The capability of both worms to break down theparticulate organic matter was monitored. A layer of 8 centimeter of sediment was put in themesocosms. The mesocosms were put in place randomly. In every mesocosm aeration was added.Total number of individuals Nereis diversicolor and Capitella sp. are calculated as follows: Anormal density of rag worms in a rag worm farm is 300 (pubescent) worms per square meter.Derived from this, the number Nereis diversicolor is 0.037 m2 (surface mesocosm) *300 = 11 Nereisdiversicolor per mesocosm when only Nereis diversicolor is stocked in these mesocosms.Table 3: Amount of worms used during the experiment, the experiment was carries out in triplicateSurface Nereis diversicolor Capitella sp. Nereis diversicolor/ Capitella sp.0.037 11 643 5/372From this number of rag worms the wet weight of the rag worms is determined. The same biomasswas applied to mesocosms only containing Capitella sp. and the number of Capitella sp. wasdetermined as well. All parameters (number of individuals and wet weight) are monitored duringthe experiment in order to calculate growth in biomass in (mg) (table 3).After the set-up phase, an acclimation period of two days (first sediment 24 hours and after thatthe worms had 24 hours of time to create their burrows) is used. After this period 200 ml of faeceswere put into the mesocosms and the experiment started. This experiment took 12 days from 11-8-2011 till 22-8-2011.Table 4: Time schedule of experiment 2 (when a box is green the measurement was done)After day 12 all of the mesocosms were cleaned and the worms were filtered out of the sediment.The total number of individuals, wet weight, dry weight and ash free dry weight (AFDW) weredetermined. Also the growth of Nereis diversicolor and Capitella sp. can be calculated. In table 4 atime schedule can be seen which was used during the experiment.11

3.0 Results and Discussion3.1 Capitella sp. experiment3.1.1 Physical ParametersDuring the experiment physical parameters were measured (pH, temperature in °C, oxygen in mg/land salinity in g/l. Temperature varied between 21 °C and 23 °C. The oxygen concentration variesbetween 7.8 mg/l and 9.8 mg/l. pH fluctuates between 7.6 and 8.3. Salinity varies between 33.4g/l and 37.1 g/l. These values did not influence the experimental set up because there were noextreme fluctuations during the experiment. The figure can be seen in Annex I.3.1.2 Nutrients analysisDuring the experiment several chemical measurements were done. The purpose of the experiment isto remediate the particulate organic fish waste. It is expected that a part of the organic matter willbe used by the worms as food source and thus will be converted into biomass and the worm willenhance the mineralization of the organic matter and increase the flux of nutrients into the watercolumn.Phosphate2,5PO 43-concentration (mg/l)21,510,500 2 4 6 8 10 12 14Time (days)0 Capitella sp.4 Capitella sp.37 Capitella sp.372 Capitella sp.Figure 9: Nutrient concentration of PO 4 3- during 12 days in different densities of worms. Observation was doneduring the first experiment in Capitella sp. experiment.During the first days of the experiment, the nutrient concentration of PO 4 3- was lower in thetreatments with high abundance of worms (37 and 372) compared to treatment without Capitellasp. or with low abundance (figure 9). After 6 days, no difference was observed 0, 4 and 372Capitella sp. the treatment of 37 Capitella sp. was lower between the 4 treatments an increase wasobserved until day 9 and at day 10 a decrease of PO 4 3- was observed. To explain this decrease thereare several factors to take into account. An explanation could be fixation of phosphate in thesubstrate; this was not the case because no substrate was used during this experiment. During theexperiment no extra sea water was added to the experiment and there was no flow through of12

water in the system. Also no algae were observed during the experiment to explain the decrease ofphosphate. Note that the samples were not checked for any algae under a microscope.The nutrient flux of PO 4 3- was made to see the actual increase of PO 43-per 3 days over theexperimental time of 13 days (shown in figure 10). What can be seen is the difference in flux at daysix between the treatments of 0 Capitella sp. together with 4 Capitella sp and 37 Capitella sp.together with 372 Capitella sp. observed was a severe bigger increase for the last two treatments.Which means more dissolved PO 4 3- is available in the water.0,500Nutrient flux of PO3-4 i in mg/l per 3days0,4000,3000,2000,1000,000-0,1000 Capitella sp. 4 Capitella sp. 37 Capitella sp. 372 Capitella sp.Day 3 Flux 1Day 6 Flux 2Day 9 Flux 3Day 12 Flux 4-0,200Figure 10: Nutrient flux of PO 4 3- in mg/l per 3 days, observation was done during the Capitella sp. withoutsediment experiment.13

AmmoniumAs NH 4+is an important factor for Capitella sp. shown in the graph (figure 11) a decrease of NH 4+wasobserved. After six days an increase of NH 4+occurs for the experiment with 37 and 372 Capitella sp.An explanation for the increase is the dying organisms found in the mesocosms. Thereby a higherconcentration of NH 4+from day 9 until the last day of the experiment occurred. The overalldecrease of 0 and 4 Capitella sp. could be explained by the fact that nitrification and denitrificationhas taken place in the system. Unfortunately this cannot be proved by actual results because of themissing results of nitrate and nitrite.8NH 4+ concentration (mg/l)76543210 Capitella sp.4 Capitella sp.37 Capitella sp.372 Capitella sp.00 2 4 6 8 10 12 14Time (days)Figure 11: An overview of the nutrient flow of NH 4+during 13 days. Observation was done during the Capitellasp. experiment.2,500Nutrient flux of NH+4 in mg/l per 3days2,0001,5001,0000,5000,000-0,500-1,0000 Capitella sp. 4 Capitella sp. 37 Capitella sp. 372 Capitella sp.Day 3 Flux 1Day 6 Flux 2Day 9 Flux 3Day 12 Flux 4-1,500Figure 12: Nutrient flux of NH 4+in mg/l per 3 days, observation was done during the Capitella sp. experiment.14

3.1.3 Organic matterAt the start of the experiment 200 ml of particulate organic fish waste was put into the mesocosms.Before this step the input of organic matter was determined as described in the method. Theorganic matter at the start of the experiment was 2.91 grams. In figure 13 a bar graph is shown atday six and day twelve. What can be seen is there has been a decrease of organic matter after 6days for all treatments. The decrease after six days for the treatment of 37 Capitella sp. and 372Capitella sp. is the strongest. After 12 days a strong increase occurs also for these two treatments.This could be because of the density of the worms compared to the available particulate organicfish waste. What can be seen is an overall decrease of particulate organic fish waste.0,14Total organic material (g)0,120,10,080,060,040,02T6T1200 Capitella sp. 4 Capitella sp. 37 Capitella sp. 372 Capitella sp.Figure 13: Organic content particulate organic fish waste. At T6 a severe amount of organic waste is brokendown. At T13 the remediation stays quite the same. T0 is 2.9115

3.1.4 Growth of Capitella sp.At the start of the experiment the average weight of one individual Capitella sp. was determined asdescribed in the method section 2.1. The average ash free dry weight (AFDW) of 1 Capitella sp. is0,000479 g. In figure 14 an increase can be seen for the treatment of 4 Capitella sp. For the othertwo treatments the biomass in AFDW stays more or less the same over time. It must be said that forthe treatment with 372 Capitella sp. dead organisms were observed in the mesocosms. Thetreatment with 37 Capitella sp. and 4 Capitella sp. were observed with small eggs which the wormsare carrying.0,0060AFDW of Capitella sp. Organiccontent in grams0,00500,00400,00300,00200,0010T0T6T120,00004 Capitella sp. 37 Capitella sp. 372 Capitella sp.Figure 14: Growth of Capitella sp. shown as ash free dry weight expressed per 1 organism (in grams)16

3.2 Capitella sp. and Nereis diversicolor experimentThe experiment was carried out with two different types of worms; Capitella sp. and Nereisdiversicolor raised individually and in combination in two types of sand: a fine sediment, (

In figure 15 the flux of PO 43-can be seen. The flux can be used as a comparison to the nutrientgraph, this to understand both graphs better. What can be seen is in the first 5 days of theexperiment there has been an overall increase of phosphate (day 5 flux 1). At Day 11 flux 2 (the next6 days) a greater increase can be seen observed.Nutrient flux of PO3-4 in mg/l per 5days0,3000,2500,2000,1500,1000,0500,000-0,0500 worms 11 Nereis d 643 Capitella sp. 5 Nereis d./ 372Capitella sp.Natural sandDay 5 Flux 1Day 11 Flux 2Figure 16: Nutrient flux of PO 43-in mg/l per 3 days, observation was done during the Capitella sp. and Nereisdiversicolor with sediment experiment2,500PO 43-concentration in mg/l2,0001,5001,0000,500Medium sand0 worms11 Nereis d.643 Capitella sp.0,0000 2 4 6 8 10 12 14Time (days)5 Nereis d./ 372Capitella sp.Figure 17: An overview of the nutrient flow of PO 4 3- during 12 days. Observation was done during theCapitella sp. and Nereis diversicolor with sediment experiment under the medium sand conditions18

In figure 18 the nutrient flux of PO 4 3- is shown of the medium sand treatment. What can be seen isan overall increase of PO4 3- , but also a stronger increase at the first 5 days of the experiment.Nutrient flux of PO3- 4 in mg/l perday0,3000,2500,2000,1500,1000,0500,0000 worms 11 Nereis d 643 Capitella sp. 5 Nereis d./ 372Capitella sp.Medium sandDay 5 Flux 1day 11 Flux 2Figure 18: Nutrient flux of PO 43-in mg/l during the 5 th and 12 th day, observation was done during the Capitellasp. and Nereis diversicolor with sediment experimentNH 4+is an important factor for Capitella sp. and Nereis diversicolor but also for Ulva lactuca as anelement for growth. Shown in figure 19 and figure 21 is an increase of NH 4 + . After five days and astrong increase of NH 4 + occurs for all treatments except for the control. A cause could be that therehas been a lack of particulate organic fish waste which resulted in dead organisms (lack ofparticulate organic fish waste) this is also an explanation why the treatment with 0 worms has asevere lower value. Which means that these densities of worms could not cope with the amount ofparticulate organic fish waste which was offered another reason could be that the worms had anextra influence on the breakdown of the particulate organic fish waste. In figure 20 and 22 thenutrient fluxes NH 4+is shown in the natural sand and the medium sand. This to give a clearerperspective to see what happens with the nutrient fluctuation over time. The treatment with 0worms shows a decrease of NH 4 + which could mean nitrification and denitrification processes takeplace. Unfortunately these values do not exist because of a broken auto analyzer for nitrate andnitrite.NH4+ concentration in mg/l9,0008,0007,0006,0005,0004,0003,0002,0001,0000,000-1,0000 2 4 6 8 10 12 14Time (days)Natural sand0 worms11 Nereis d.643 Capitella sp.5 Nereis d./ 372Capitella sp.19

Figure 19: Nutrient concentration over time of NH 4+during the Capitella sp. and Nereis diversicolor sedimentexperiment (natural sand treatment).Nutrient flux of NH+ 4 in mg/l per 5days1,0000,8000,6000,4000,2000,000-0,2000 worms 11 Nereis d 643 Capitella sp. 5 Nereis d./ 372Capitella sp.Natural sandAverage Flux 1Average Flux 2Figure 20: Nutrient flux of NH 4+in mg/l during the 5 th and 12 th day, observation was done during the Capitellasp. and Nereis diversicolor with sediment experiment (natural sand treatment).NH4+ concentration in mg/l25,00020,00015,00010,0005,0000,000-5,0000 2 4 6 8 10 12 14Time (days)Medium sand0 worms11 Nereis d.643 Capitella sp.5 Nereis d./ 372Capitella sp.Figure 21: An overview of the nutrient flow of NH 4+during the Capitella sp. and Nereis diversicolor sedimentexperiment (medium sand treatment).20

Nutrient flux of NH+ 4 in mg/l per 5days2,5002,0001,5001,0000,5000,0000 worms 11 Nereis d 643 Capitella sp. 5 Nereis d./ 372Capitella sp.Medium sandAverage Flux 1Average Flux 2Figure 22: Nutrient flux of NH 4+in mg/l during the 5 th and 12 th day, observation was done during the Capitellasp. and Nereis diversicolor with sediment experiment (medium sand treatment).At the end of the experiment, dead organisms were found. This occurred predominantly in theNereis diversicolor treatment. This could either be caused by the input of feacal material (lack offood). Another reason could be that an aeration error occurred at day 11. This means that there hasbeen a lack of oxygen in the mesocosms. This was not a problem for the nutrient samples (thesewere already collected)3.2.3 Organic matterAt the start of the experiment 200 ml of particulate organic fish waste was put into the mesocosms.As in the Capitella sp. experiment without sediment the organic matter input was determined(described in Chapter Method). This start value of organic matter is 2.97 grams. In figure 23 andfigure 24 a bar graph is shown at day twelve (T12). The start value was taken out of these bars tosee the differences between the different treatments. What can be seen for the Natural sandtreatment is that the experiment with Nereis diversicolor is the least effective in reducing organicmaterial. This could be as said before caused by the dead organisms which were found in thistreatment.Total organic material (g)0,140,120,10,080,060,040,02Natural sandT1200 worms 11 Nereis d. 643 Capitella sp. 5 Nereis d./ 372Capitella sp.21

Figure 23: An overview of the organic content particulate organic fish waste. At T12 a severe amount of organicwaste is broken down (natural sand treatment) T0 was 2.97 grams.Remediation of organic matter ingrams0,140,120,10,080,060,040,0200 worms 11 Nereis d. 643 Capitella sp. 5 Nereis d./ 372Capitella sp.Medium sandT12Figure 24: An overview of the organic content particulate organic fish waste. At T12 a severe amount of organicwaste is broken down (medium sand treatment)3.2.4 Growth of Capitella sp. and Nereis diversicolorAt the start of the experiment the average wet weight of one individual Capitella sp. and anindividual Nereis diversicolor was determined and described in the method. The average wet weightof 1 Capitella sp. is 0.00311 g. The average wet weight of one Nereis diversicolor is 0.19g. In figure25 and figure 26the wet weight at the start and end are shown for the Capitella sp. treatments. Infigure 26 and figure 28 the wet weight of Nereis diversicolor is shown of the start and end. Chosenwas to display the wet weight of one organism. It was not possible to display the AFDW figure. Notall the material was burned in the furnace (a part of the dried worms would go to a lab to check onnutrients inside the worm)Biomass in wet weight (g) ofCapitella sp.0,0070,0060,0050,0040,0030,0020,0018E-18-0,001WW 1 Capitella sp. (643 Capitellasp.)WW 1 Capitella sp. (5 Nereis d./ 372Capitella sp.)Natural sandT0T12Figure 25: Growth of Capitella sp. shown as wet weight, expressed per 1 organism (in grams). (Natural sandtreatment)22

In figure 25 an almost stable figure can be seen. The worms have been stable in wet weight duringthe experiment as well for the 643 Capitella sp. as the 5 Nereis diversicolor and 372 Capitella sp.treatment.Biomass in wet weight (g) of Nereisdiversicolor0,250,20,150,10,050WW 1 Nereis d. (11 Nereid d.) WW 1 Nereis d. (5 Nereis d./ 372Capitella sp.)Natural sandT0T12Figure 26: Growth of Nereis diversicolor shown as wet weight, expressed per 1 organism (in grams). (Naturalsand treatment)0,007Biomass in wet weight (g) ofCapitella sp.0,0060,0050,0040,0030,0020,001Medium sandT0T120WW 1 Capitella sp. (643 Capitella sp.) WW 1 Capitella sp. (5 Nereis d./ 372Capitella sp.Figure 27: Growth of Capitella sp. shown as wet weight, expressed per 1 organism (in grams) (Medium sandtreatment)23

Biomass in wet weight (g) of Nereisdiversicolor0,250,20,150,10,050WW 1 Nereis d. (11 Nereid d.)Medium sandT0T12WW 1 Nereis d. (5 Nereis d./ 372 Capitella sp.Figure 28: Growth of Nereis diversicolor shown as wet weight, expressed per 1 organism (in grams). (Mediumsand treatment)24

4.0 ConclusionKey question:What are the possibilities of Nereis diversicolor and Capitella sp. to remediate particulate organicfish waste?There are possibilities to remediate particulate organic fish waste with Capitella sp. and Nereisdiversicolor. The combinations of both worms are giving the best overall results. This is based onthe results of wet weight, the increase of wet weight of Nereis diversicolor and Capitella sp. in thecombination treatment together with the survival rate was the most positive (Annex III).. After sixdays of the first experiment the removal of the particulate organic fish waste was the highest(observed in Capitella sp. experiment).Sub questions:What is the rate of removal of organic particulate fish waste by Nereis diversicolor and Capitellasp. in relation to species composition?The start value of the input of organic matter during the Capitella sp. experiment was 2.91grams.After 6 days the value was decreased. 0 Capitella sp.:0.082 grams, 4 Capitella sp.: 0.081 grams, 37Capitella sp.: 0.049 grams and 372 Capitella sp.: 0.078. The treatment of 37 Capitella sp. was themost effective.The start value of organic matter during the Capitella sp. and Nereis diversicolor experiment was2.97 grams. After 12 days the value decreased for the different treatments. First the natural sandtreatment will be described. 0 worms: 0.081 grams, 11 Nereis diversicolor: 0.13 grams, 643Capitella sp.: 0.079 grams and 5 Nereis diversicolor/Capitella sp. 0.081 grams. Medium sand: 0worms: 0.078 grams, 11 Nereis diversicolor:0.068 grams, 643 Capitella sp.: 0.075 grams and 5Nereis diversicolor/Capitella sp.: 0.076 grams.Together with the results of the survival rate during the experiments can be concluded that thecombination of Capitella sp. and Nereis diversicolor was the most effective. Also visual observationhad an important role for the conclusion.How much of the organic waste is allocated in respectively worm biomass and dissolved nutrients?In the Capitella sp. experiment an increase of biomass occurred for assuming that these worms didnot have a shortage of food supply. The overall increase of biomass in % organic matter was0.0040-0.0005 = 0.0035 grams. Also a part of the organic waste was converted to dissolvednutrients. PO 43-and NH 4 + are treated during the results. Also NO 2 and NH 2 were examined and can befound in Annex II. The NO 3 + would be examined, the auto analyzer was broken therefore it could notbe included.During Capitella sp. and Nereis diversicolor experiment there was the same problem as for theCapitella sp. experiment (a broken auto analyzer) therefore it was not possible to do part of thedesired nutrient analysis. During this experiment a decrease occurred of biomass of the worms.There is a clear increase of nutrients seen (Chapter 3.2 Capitella sp. and Nereis diversicolor andAnnex III).25

To what extend are the underlying processes dependent on stocking densities and waste load?The underlying processes (converting waste load into biomass and converting of particulate organicfish waste into dissolved nutrients) are very dependent on the stocking density and waste load(applicable to both experiments). Observed was that after 6 days most of the particulate organicfish wastes (which were suitable for the worm to use) were used by the worms for biomass but alsodissolving the nutrients in the water column. Also was seen that the stocking density is an importantfactor for the remediation of particulate organic fish waste. The treatments with the majority ofworms were breaking down the particulate organic fish waste very fast even a shortage of input wasobserved (this was also a visual observation).5.0 RecommendationsDuring the experiments and by the results several ideas came up to improve and change the followupexperiments. A lot of observations were done. In this chapter an overview of recommendationscan be seen.During the Capitella sp. and Nereis diversicolor experiment a decrease of worms occurredespecially in the Capitella sp. (642 worms) and the Nereis diversicolor treatment. The density ofworms in the mesocosms was calculated based on worms in a rag worm farm. What could berecommended is that there should be research done on the ideal combination of these two speciesof worms or the ideal Capitella sp. / Nereis diversicolor ratio. The ideal combination of worms isnecessary to improve the reproduction of Capitella sp. but also the habitat of Nereis diversicolor.Thought is of a flow through system. During the experiments a shortage of input of particulateorganic fish waste occurred for some treatments. When there is a flow through system there cannotbe a problem with the input of particulate organic fish waste. Seen during past experiments is thatafter 6 days most of the organic content which can be used by the worms to remediate theparticulate organic fish waste is used. A constant flow of water together with particulate organicfish waste as the input of the follow up experiment could be a solution, this could be constructed atthe bottom (above the substrate) of the experimental set up. The output of the flow through systemcould be in the water column, so the nutrient rich water can flow out of the experimental setup andused as a food supply for Ulva rigida. What should be examined and researched is what the idealvelocity would be of the mix of seawater and particulate organic fish waste and the output (nutrientrich water in the water column). The worms need time to remediate the waste but this could bevery interesting for the future of remediation of waste by Nereis diversicolor and Capitella sp.Another option is to apply an airlift system. An airlift means that the air is directed (with a pump) inthe mesocosm by a pipe directly through the sediment. This could mean that the food could becomeeasier available for the worms because of the movement of the faeces. Also the aeration could havea positive effect on converting the particulate organic fish waste into dissolved nutrients in thewater column. Also this recommendation should be well researched.26

ReferencesAller RC, 1994; Bioturbation and remineralization of sedimentary organic-matter - effects of redoxoscillation. Chemical Geology, Volume 114, Issues 3–4, 1 June 1994, Pages 331-345Bayne, B.L and R.C. Newell, 1983; Physiological energetics of marine molluscs. In The Mollusca,Vol. 1st (1983), 407--515Bischoff Adrian A. , Patrick Fink, Uwe Waller, 2008; The fatty acid composition of Nereisdiversicolor cultured in an integrated recirculated system: Possible implications for aquaculture.Aquaculture, Volume 296, Issues 3–4, 16 November 2009, Pages 271-276Calow P. 1975; The feeding strategies of two freshwater gastropods, Ancylus jluviatilis M11. andPlanorbis contortus Linn. (Pulmonata) in terms of ingestion rates and absorption efficiencies.Oecologia. 20:33-49.Caron A., G. Desrosiers, P.J.W. Olive, C. Retiere, C. Nozais, 2003; Comparison of diet and feedingactivity of two polychaetes, Nephtys caeca (Fabricius) and Nereis virens (Sars), in an estuarineintertidal environment in Quebec, Canada. Journal of Experimental Marine Biology and Ecology,Volume 304, Issue 2, 30 June 2004, Pages 225–242Cheng S.Y., 1993; Urea excretion by Penaeus japonicas Bate exposed to different concentrations ofambient ammonia J. Mar. ecol. Prog. Ser., vol. 153: 197-202Chopin T, Robinson S, MacDonald B, Haya Kand others (2006) Integrated multi-trophic aquaculture:Seaweeds and beyond... The need of an interdisciplinary approach to develop sustainableaquaculture. Journal of Phycology 42, 11Cohen I., A. Neori, 1991; Ulva Lactuca Biofilters for marine fishpond effluents. I. Ammonia yotakekinetics and nitrogen content, Botanica Marina 34:475-482Duroua Cyril, Catherine Mouneyraca, Claude Amiard-Triquet ,2008, Environmental qualityassessment in estuarine ecosystems: Use of biometric measurements and fecundity of the ragwormNereis diversicolor (Polychaeta, Nereididae), ;42(8-9):2157-65. Epub 2007 Nov 28FAO, 2010 The State of World Fisheries and Aquaculture 2010P. Gillet, M. Mouloud, C. Durou, B. Deutsch, 2007; Response of Nereis diversicolor population(Polychaeta, Nereididae) to the pollution impact e Authie and Seine estuaries (France). Estuarine,Coastal and Shelf Science, Volume 76, Issue 2, 20 January 2008, Pages 201-210A. Gremare, A. G. Marsh and K. R. Tenore, 1988; Short-term reproductive responses of Capitella sp.I (Annelida : Polychaeta) fed on different diets. Journal of Experimental Marine Biology andEcology, Volume 123, Issue 2, 22 November 1988, Pages 147-162Hargrave, B. T., Ed. (2005). Environmental effects of marine finfish aquaculture. The handbook ofenvironmental chemistry. Water pollution, Part M., Springer-Verlag, Berlin. Published online 19 July2005.27

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AnnexI. Physical parameters Capitella sp. experimentTemperature24,0023,00A (0 Capitella)B (4 Capitella)22,00C (37 Capitella)21,00D (373 Capitella)20,0019,0018,000 2 4 6 8 10 12 1445,00Salinity (mg/l)40,0035,0030,0025,000 2 4 6 8 10 12 1410,00Oxygen (mg/l)9,008,007,006,000 2 4 6 8 10 12 149,008,00pH7,006,000 2 4 6 8 10 12 14Time (days)Figure 29: Variation of temperature, salinity, Oxygen and pH during the first Capitella sp. experiment

II. Physical parameters Capitella sp. and Nereis diversicolor experimentFigure 30: Variation of temperature, salinity, Oxygen and pH during the second experiment30

III. Capitella sp. and Nereis diversicolor experiment input and output ofwormsTable 5: Input and output of worms during the Nereis diversicolor and Capitella sp. experiment. Alsothe offspring of Capitella is registered, to see if reproduction is possible during the experiment.Sand type NaturalinputinputfoundFoundBalansBalansCapitellaNereisd.Capitellasp.Nereisd.Capitellasp.Nereisd.Capitellasp.withoffspring0 worms (1) 0 0 0 0 0 00 worms (2) 0 0 0 0 0 00 worms (3) 0 0 0 0 0 011 Nereis d. (1) 11 0 6 0 -5 011 (Nereis d. (2) 11 0 7 0 -4 011 Nereis d. (3) 11 0 6 0 -5 0643 Capitella sp. (1) 0 643 0 203 0 -440 x643 Capitella sp. (2) 0 643 0 197 0 -446 x643 Capitella sp. (3) 0 643 0 216 0 -4275 Nereis d./ 372 Capitellasp (1)5 Nereis d./ 372 Capitellasp (2)5 Nereis d./ 372 Capitellasp (3)5 372 5 103 0 -269 x5 372 4 152 -1 -220 x5 372 5 132 0 -240 x31

Table 6: Input and output of worms during the Nereis diversicolor and Capitella sp. experiment. Alsothe offspring of Capitella is registered, to see if reproduction is possible during the experiment.Sand type MediuminputNereisd.inputCapitellasp.foundNereisd.FoundCapitella sp.BalansNereis d.BalansCapitellasp.Capitellawithoffspring0 worms (1) 0 0 0 0 0 00 worms (2) 0 0 0 0 0 00 worms (3) 0 0 0 0 0 011 Nereis d. (1) 11 0 2 0 -9 011 (Nereis d. (2) 11 0 8 0 -3 011 Nereis d. (3) 11 0 7 0 -4 0643 Capitella sp. (1) 0 643 0 294 0 -349 x643 Capitella sp. (2) 0 643 0 359 0 -284643 Capitella sp. (3) 0 643 0 322 0 -3215 Nereis d./ 372 Capitellasp (1)5 Nereis d./ 372 Capitellasp (2)5 Nereis d./ 372 Capitellasp (3)5 372 5 240 0 -132 x5 372 4 266 -1 -106 x5 372 5 227 0 -145 x