Indicators of Diminished Organic Matter Degradation Potential of Polychaete Burrows in Philippine Mariculture Areas

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1 Organic THE PHILIPPINE Matter Degradation AGRICULTURAL Potential of Polychaete SCIENTIST Burrows S.M.S. ISSN Santander et al. Vol. 91 No. 3, September 2008 Indicators of Diminished Organic Matter Degradation Potential of Polychaete Burrows in Philippine Mariculture Areas Sheila Mae S. Santander 1,2,*, Maria Lourdes San Diego-McGlone 1 and Wolfgang Reichardt 1 1 Marine Science Institute, University of the Philippines, Diliman, Quezon City, 1101 Philippines 2 Southeast Asian Fisheries Development Center/Aquaculture Department, Tigbauan, Iloilo, 5021 Philippines * Author for correspondence; sheila.santander@yahoo.com; telefax: Sediments from underneath fish cages and in mesocosm tanks were examined to establish indicators of diminished organic matter degradation potential of polychaete burrows from increased mariculture activities in Bolinao-Anda, Pangasinan, Philippines. Results showed that simple sediment characteristics may be used as sediment quality indicators to describe the contribution of burrows to biodegradable processes in sediments with extremely high deposition of organic particles. The indicators of diminished organic matter degradation potential of polychaete burrows are low redox potential (-113 to -150 mv for F. Spionidae and F. Eunicidae) at the 1 cm surface layer, absence or decreased size of burrow openings, presence of H 2 S, disappearance of apparent Redox Potential Discontinuity Layer (arpd), formation of black sediment, and presence of Beggiatoa (sulfide oxidizing bacteria) and gas bubbles. Key Words: burrows, mariculture, organic matter recycling, polychaetes, sediment quality indicators INTRODUCTION In coastal areas, the oxygen-containing layer of the sediment is typically only a few millimeters thick (Revsbech et al. 1980; Revsbech and Jorgensen 1986; Holmer et al. 2005). The infauna extends this oxygenated layer into burrows by means of bioturbation processes such as irrigation, ventilation and mixing of the sediments (Reise 2002). Upon deposition of organic matter (OM), oxygen is consumed first by aerobic respiration, and then bioavailable OM is further remineralized via fermentation and different processes of anaerobic respiration. Once SO 4-2 has become the predominant terminal electron acceptor via sulfate reduction, the sediment starts to show the Fe-sulfide black color (Lyle 1983; Hall et al. 1990; Karakassis et al. 1998) and emit the rotten egg smell of H 2 S. Sediments below fish cages are usually anoxic, have a low redox potential (Hargrave et al. 1993), high organic material content (Hall et al. 1990), impoverished or depleted in benthic fauna (Brown et al. 1987; Ye et al. 1991; Holmer and Kristensen 1992; Lu and Wu 1998), and contain mats of the sulfide-oxidizing bacteria Beggiatoa (Ross 1989; Hall et al. 1990; Holmer and Kristensen 1992; Karakassis et al. 1998). Hargrave et al. (2008) showed that ph, redox potentials and dissolved sulfides can be used as indicators of the biogeochemical changes related to organic matter enrichment in marine sediments. The indicators of anaerobic sediments are known but changes in organically enriched sediments containing burrows and their qualitative indicators to serve as a monitoring tool for fish farmers has yet to be documented and developed. This study compares sediment cores in an area with and without organic matter deposition from fish farming. It aims to identify indicators of diminished organic matter degradation capacity of the burrows with the influence of mariculture feed input. MATERIALS AND METHODS The study was conducted in 2006 and composed of 1) a sediment mesocosm experiment designed to examine induced effects of organic particle inputs; and 2) a comparative observational or field study between milkfish cage and no-cage areas of Bolinao-Anda waters, Pangasinan located at the northwestern Philippines to represent difference of organic matter input. Sediment Preparation Sediments for mesocosm cores were collected in March 2006 from a neritic no-cage area using an Ekman grab (4L) and sieved through a 0.3-mm mesh steel sieve to remove The Philippine Agricultural Scientist Vol. 91 No. 3 (September 2008) 295

2 the fauna. The sieved sediments were allowed to settle for 5 7 d in a plastic crate kept in a covered concrete tank before they were cored using 5.0-cm internal diameter and 30-cm height plexiglass cylinders. The cores were then equilibrated to allow development of burrows for 3 5 d in an aerated darkened flow-through tank with ambient coastal water temperatures. Treatments Presence of polychaete and fish feed level were the two factors considered that may affect the organic matter degradation potential of the burrows. Polychaete treatments were small-polychaete ( mm width, cm length), large-polychaete ( mm width, cm length) and no-polychaete. Each polychaete treatment was applied to 8 replicate cores. Small polychaetes were collected from the no-cage area (Fig. 1), while the large polychaete was from the intertidal area (16 o N, 119 o E) where fishcages are not present. A polychaete survey was done in the cage and no-cage areas but no large polychaetes were found. The small polychaetes belonged to Family Spionidae (Fauchald 1977) Prionospio sp. (Blake and Kudenov 1978). The large polychaete was identified under Family Eunicidae (Marphysa sp.). Six small and one large Fig. 1. Map of sampling areas located in Bolinao-Anda waters, Pangasinan, northwest Philippines. Sampling was conducted in March, April, June and September Cage and no-cage samples were collected at 16º N, 119º E and 16º N, 119º E, respectively. Inset is the Philippine map. polychaete were added to the respective treatment cores. The number added was based on polychaete density at the source site. The density of the large polychaete treated cores was determined from three 0.5 x 0.5 x 0.3 m sediments collected using a spade (Holme and McIntyre 1984; Radashevsky et al. 2006) while small polychaete density was determined using 20 pcs (5.0 cm i.d. and 30 cm height) plexiglass cores collected through SCUBA with 1 m distance between cores in the no-cage area. First observation was done 3 5 d after the addition of the polychaetes to determine the organic matter degradation potential of burrows in the absence of feed input. Indicators such as bioturbation depth (burrow depth), qualitative color profile, presence/absence of apparent redox potential discontinuity (arpd) layer, number of burrow holes seen at the surface and size of burrows were estimated using a ruler and observed using a magnifying glass. H 2 S and redox potentials were determined. H 2 S was detected in two ways, through the sense of smell (Gallizia et al. 2004) and using a silver stick (Frederiksen et al. 2006) where its presence was indicated by the formation of silver sulfide, or the silver stick turning black. The silver stick was left in the sediment column for 2 h. The silver stick only measures 10 cm of the sediment column. Redox potential was measured using a redox Pt probe (Cyberscan ph/mv/ºc) at the top 1 cm layer and in the burrow linings of the large polychaete burrows. Small polychaete burrows were too small to be measured directly by the probe. Redox potential was corrected with +200mV. arpd layer was measured as the shift between oxidized brownish layer to reduced black sediment (Rosenberg et al. 2002). This boundary indicated the shift from ferric (Fe +3 ) to ferrous (Fe +2 ) ions (Karakassis et al. 1998). Sediment profiles were photographed using a digital camera to describe the details of changes in the mesocosm and field sediment cores. The relatively high bioturbation depth, light brown color, large number of burrow holes seen at the surface, wide size of burrows and absence of H 2 S/smell (Gallizia et al. 2004) were indicators used to determine the presence of organic matter degradation potential of burrows. The sediment core was then subsampled for total protein concentration (Lowry et al. 1951) as measure of organic matter degradation. Subsampling was done first by siphoning out the overlying water from the cylinders. Subsamples were then taken using a sawed-off syringe from the top most sediment layer (2 cm) for the nopolychaete treated sediments, and using a Pasteur pipette or sawed-off syringe (depends upon the depth) at the burrow/linings of the large and small polychaete treated sediments. Subsampling of fauna-treated sediments was targeted in the burrow linings for a more direct examination of the bioturbation activity and to avoid large differences in the replicates. The sediment sample was then transferred from the syringe to the petri dish for homogenization. 296 The Philippine Agricultural Scientist Vol. 91 No. 3 (September 2008)

3 Fish feed level treatments were low-feed (163 mg per cylinder) and high-feed (491 mg per cylinder). Treatments were based on 50% of the average sedimentation rates in the no-cage site (167 g m -2 d -1 ) for low feed and the cage areas (500 g m -2 d -1 ) for high feed treatment. These were measured using sediment traps (2.0 cm i.d.) deployed for 3 consecutive 5-d periods in February To avoid resuspension, the traps were deployed at mid-depth. Feed was added to the cores after 3 5 d equilibration from the first visual observation and subsampling. The 8 replicates of each polychaete treatment were divided into two. Feed level treatments were added in the corresponding 4 replicate cylinders. Commercial milkfish feed pellets (not less than 28% crude protein based on manufacturer s analysis) were weighed and soaked in seawater for approximately 10 min and mashed using stainless spatula before they were added to the plexiglass cylinders to simulate particle size sinking at the bottom. The feeds were observed to float in the surface so cylinders were then covered with nets to avoid loss of feeds. Second observation was done 48 h from feed addition, the time when the worm started to die based on the initial experiment. The same parameters and methods were used as in the first observation and subsampling. The sediments were sieved to examine survivorship of the polychaetes. No feed treated sediments which were sediments with large or small polychaetes but without feed input were also conducted using the same methods. Collection of Field Samples Sediments from the field were taken from cage and no-cage sites in the months of March, April, June and September 2006 (Fig. 1). The cage site had 171 fish cages (Sagip Lingayen Gulf Project, 2006, per com). The no-cage site was ~2 km away from the cage site. The depth of both sites was m. Four (4) sediment core samples were collected in plexiglass cylinders (5.0 cm i.d. and 30 cm height) at 1 m distance from each other starting at the site coordinates using SCUBA. Redox and H 2 S were measured immediately. Equilibration was done in order to allow development of burrows which were disturbed from collection in an aerated darkened flow-through tank with ambient coastal water temperatures. The same parameters and methods used in the mesocosm observation and subsampling were done. Statistical Analysis Feed input may decrease the burrow density and redox potential therefore correlation of total protein concentration to burrow density and redox potential was determined using the Pearson Product Moment Correlation using STATISTICA (Steel et al. 1997). RESULTS AND DISCUSSION Indicators of Organic Matter Degradation Potential of Polychaete Burrows In the mesocosm experiment before feed was added, the color of the sediments with large, small and no polychaetes was light brown from the surface up to 0.5, 0.05 and 0.25 cm arpd layers, respectively (Fig. 2). This was followed by light gray to gray until the bottom of the cores. The sediments with the no-polychaete treatment contained no burrows and a redox potential of 137 ± 10 mv. The small polychaete burrows were approximately 60 in number and were of a pen point size (~0.025 cm) with redox potential of 179 ± 6 mv. In cores with large polychaetes, there were 2 12 burrow holes with cm size opening and significantly higher redox potential (249±16 mv) among other treat- Fig. 2. Sediment profile images of mesocosm sediments with large-polychaete (row 1), small-polychaete (row 2), and no-polychaete (row 3) before-feed addition (column 1), with low-feed input (column 2), and high-feed input (column 3) treatments. BO = Burrow opening OL = Oxidized line/layer UBL= Unoxidized burrow line RBL= Reduced burrow line CBO = Closed burrow opening arpd = apparent Redox Potential Discontinuity Layer RL = Reduced Line CW = Clear sediment-water interface TW = Turbid sediment- water interface FP = Feed Particles Calibration marks in cm. The Philippine Agricultural Scientist Vol. 91 No. 3 (September 2008) 297

4 ments (p<0.05). In the 30 cm length cores, the depth of the large visible polychaete burrows reached 4 cm to the bottom, while the small polychaete burrows reached 2 20 cm. Large polychaete burrow lines were brownish and showed an arpd below ~ 0.5 cm of the oxidized layer, while the small polychaete burrow lines were grayish or unoxidized (Fig. 2). The results suggest that large polychaete brownish burrows have the highest potential for aerobic respiration without the influence of feed input. Redox Potential (mv) and Percentage (%) Survival of Polychaetes L-NF L-LF L-HF S-NF S-LF S-HF Polychaete and Feed treatments SURVIVAL REDOX Fig. 3. Percentage (%) survival and redox potential (mv) of sediments treated with large polychaetes (N = 4) with no feed input (L-NF), with low feed input (L-LF), and with high feed input (L-HF); and of sediments treated with small polychaetes (N = 4) with no-feed input (S-NF), with low feed input (S-LF), and with high feed input (S- HF). Changes in the Indicators of Organic Matter Degradation Potential of Polychaete Burrows with Feed Input After feed was added, the burrow openings were closed due to deposition of feeds; overlying water became turbid in all treatments, and more turbid in the no-polychaete cores (Fig. 2). These observations were confirmed by the inverse correlation of burrow density with protein concentration (R = -0.07). There was no remarkable difference in indicators of organic matter degradation with low and high feed input, except for the presence of more feed particles in the surface of the sediments in the upper layer of the high feed input cores in all treatments (Fig. 2). The large and small polychaetes were observed to burrow deeper into the sediment column after addition of feeds. The creation of deeper burrows may be a mechanism for increased irrigation to help remedy the more reduced conditions (Heilskov et al. 2006). The surface arpd disappeared with brown layers turning black in all treatments after 48 h (Fig. 2). The reduced surface sediment layer was scattered by the large polychaete burrows creating a root-like channel of reduction zone. The arpd in the burrow linings of the large polychaetes were maintained in some cores but turned black for others in both low and high feed treatments. The reduction or disappearance of the arpd layer in the sediments after feed addition suggests consumption of oxygen and production of sulfide. Sediments with small polychaetes became highly reducing at -113±3 mv with low feed, and at -130 ± 5 mv with high feed. Redox potential in the sediments with large polychaetes also became highly reducing at -149 ± 10 mv with low feed input, and at -145 ± 11 mv with high feed input. For both cases there may be decreased irrigation capacity of the fauna, hence, making the sediments more reducing (Nilsson and Rosenberg 1994). This is demonstrated as well by the significantly inverse correlation of total protein concentration with redox potential (p < 0.01) which may mean that the deposition of feeds in the sediments may have clogged the burrow openings. Redox potential decreases under moderate oxygen levels (~1.0 mg L -1 ) and in severe hypoxia (~0.5 m.l -1 ) (Nilsson and Rosenberg 1994). Presence of reactive sulfide was detected in the sediments with large polychaetes from 0 4 cm and 8 9 cm (low feed) and cm (high feed). In the sediments with small polychaetes, sulfide presence was detected at 0 10 cm (low feed) and 0 7 cm (high feed), while in the sediments with no-polychaetes, it was present from cm (low feed) and 0 4 cm (high feed). Based on smell, H 2 S was only present in the small and no-polychaete treatments. The discontinuity of sulfide presence along the 10-cm layer in cores with large polychaetes when feed was added suggests particulate transport of feed material into the burrows. Size of the fauna may have influenced this activity as also shown in Nickell et al. (2003) where mixing intensity and depths were highest with the largest macrofauna mean size. Faunal Survival vs. Redox Potential Sulfide build-up creates a toxic environment for aerobic organisms (Duplisea 1998). In no feed sediments, survival was 87.5% for the large polychaetes with Eh 203±mV and 100% for the small polychaetes with Eh 173±mV. The polychaetes in the feed treated sediments, however, started to die when the redox potential decreased to -113 to -150 mv (Fig. 3). At this Eh range, the large polychaetes had an average survival rate of 50% with low feed input and no survival with high feed input. The survival of the small polychaetes with high feed was 12.5%, and with low feed it was 8.5%. With no feeds added, survival was 87.5% for the large polychaetes and 100% for the small polychaetes; redox potential values were also positive (Fig. 3). Faunal mortality especially in the sediments with small polychaetes at this Eh range may be due to the feed particles degrading, a stage when H 2 S accumulates and reaches a toxic 298 The Philippine Agricultural Scientist Vol. 91 No. 3 (September 2008)

5 Fig. 4. Sediment profile images of field survey cage and no-cage cores. Field survey cores contained (A) Beggiatoa (Beg) and (B) Gas bubbles (GB). No-cage cores showed distinct (C) arpd (blue line) at 1 5 cm with small burrows. level (Holmer et al. 2001). The strong negative correlation of redox potential with faunal survival in mesocosm cores indicates the inability of the sediments to recycle organic matter because of loss of fauna due to hypoxic conditions. In addition, this condition may have caused the Eunicids to move away from the sediment surface where they were observed to have died at the bottom of the sediment core in their attempt to move away from H 2 S. The opportunistic polychaetes Spionids, however, moved out from the sediment and were observed to die at the surface. Indicators of Diminished Organic Matter Degradation Potential of Sediments in Mariculture Areas Sediments from below the fish cages were black in color, had no burrows, contained gas bubbles and were covered with 25 mm thick Beggiatoa mats (Fig. 4). Cores from the no-cage control site showed a light brown top layer and gray color beneath. The arpd layer was at 0.05 cm in March and April, absent in June and at 5.0 cm in September. Burrows in the no-cage area were approximately 60 in number and ~ cm in size, which appeared as pen points in the surface sediment. Cage sediments ~ 2 km from the no-cage area were void of fauna. Sediments were most reducing at the cage site (-160 ± 14 mv), while the no-cage site showed positive redox potential (62 ± 86 mv) except for June. Protein concentration was significantly inversely correlated with burrow density (p < ) and with redox potential (p < 0.01). These results are consistent with the mesocosm experiment whereby fish feed input changed the features of the bioturbation structures, i.e., burrows. Anoxic conditions tend to make the sediments unsuitable for macrofaunal organisms, especially infaunal deposit feeders (Karakassis et al. 1998). However, such conditions are favorable to the microaerophilic Beggiatoa spp. which require sulfide from the lower depths of the sediments and traces of oxygen from the upper depths (Duplisea 1998). The presence of Beggiatoa sulfide-oxidizing bacteria, which appears as a white mat on the sediment surface and gas bubbles which could be N 2, CO 2, methane or H 2 S, are the most defined indicators of extreme fish feed input in the cage site. Beggiatoa was also present on sediment surface of small polychaete high feed treatment. The same indicators were observed in Cephalonia Bay, Greece under the fish cage site (Karakassis et al. 1998). Other studies have also reported the presence of Beggiatoa (Ross 1989; Hall et al. 1990; Holmer and Kristensen 1992) in organic rich sediments around cage farms (Nilsson and Rosenberg 1994). Beggiatoa mats in this study were observed to be denser than those reported by Karakassis et al. (1998). The presence of Beggiatoa and gas bubbles in the cage site is inversely linked with faunal presence but may be directly related to high organic matter input. This means that once the Beggiatoa grows, the large fauna and, consequently, the sediment s bioturbation-degradation potential would start to diminish. Altogether, the indicators of diminished organic matter degradation potential of bioturbation structures in the sediments are as follows: (1) Eh of -113 to -150 at the 1 cm surface layer which means bioturbation of small (Family Spionidae) and large polychaetes (Family Eunicidae) have ceased or decreased due to mortality; (2) absence or decreased burrow openings that indicates diminished irrigation capacity and subsequent covering of burrow holes due to deposition of fish feeds; (3) disappearance of the arpd and presence of black sediment that indicates sulfate reduction; (4) presence of reactive sulfides that means a toxic environment for the fauna; and (5) presence of Beggiatoa mat at the surface and gas bubbles that indicate anaerobic processes occurring in the sediments. At the fish farmer s monitoring level, these indicators could be used to distinguish good quality from poor quality sediments (Table 1). The Philippine Agricultural Scientist Vol. 91 No. 3 (September 2008) 299

6 Table 1. Summary of the recommended indicators of good and poor quality sediment in mariculture areas. Indicators Good Quality Poor Quality Color brown surface layer black surface layer H 2 S absent in sediment- present in sedimentwater interface water interface Presence/ present/ > 1 mm absent/ < 0.25 mm Size of burrow openings Redox positive at sediment- negative (-113 to -150 water interface mv) or burrows Beggiatoa absent present Gas bubbles absent present REFERENCES CITED BLAKE JA, KUDENOV JD The Spionidae (Polychaeta) from southeastern Australia and adjacent areas with a revision of the genera. Mem Nat Mus Victoria 39: BROWN JR, GOWEN RJ, McLUSKY DS The effect of salmon farming on the benthos of a Scottish sea loch. J Exp Mar Biol Ecol 109: DUPLISEA DE Feedbacks between benthic carbon mineralization and community structure: a simulation model analysis. Ecol Model 110: FAUCHALD K The Polychaete Worms Definitions and Keys to the Orders, Families and Genera. Natural History Museum of Los Angeles County. Science Series 28. FREDERIKSEN M, HOLMER M, BORUM J, KENNEDY H Temporal and spatial variation of sulfide invasion in eelgrass (Zostera marina) as reflected by its sulphur isotopic composition. Limnol Oceanogr 51(5): GALLIZIA I, VEZZULLI L, FABIANO M Oxygen supply for biostimulation of enzymatic activity in organic-rich marine ecosystems. Soil Biol Biochem 36: HALL POJ, ANDERSON LG, HOLBY O, KOLBERG S, SAMUELSON MO Chemical fluxes and mass balances in a marine fish cage farm. I. Carbon. Mar Ecol Prog Ser 61: HARGRAVE BT, DUPLISEA DE, PFEIFFER E, WILDISH DJ Seasonal changes in benthic fluxes of dissolved oxygen and ammonium associated with marine cultured Atlantic Salmon. Mar Ecol Prog Ser 96: HARGRAVE BT, M HOLMER, CP NEWCOMBE Towards a classification of organic enrichment in marine sediment based on biogeochemical indicators. Mar Pollut Bull 56: HEILSKOV AC, ALPERIN M, HOLMER M Benthic fauna bio-irrigation effects on nutrient regeneration in fish farm sediments. J Exp Biol 339: HOLME NA, MCINTYRE AD Methods for the Study of Marine Benthos. 2 nd ed.. Oxford, London, Edinburgh: Blackwell Scientific Publications. HOLMER M, KRISTENSEN E Impact of marine fish cage farming on metabolism and sulfate reduction of underlying sediments. Mar Ecol Prog Ser 80: HOLMER M, LASSUS P, STEWART JE, WILDISH DJ ICES Symposium on Environmental Effects of Mariculture. J Mar Sci 58: HOLMER M, WILDISH D, HARGRAVE B Organic Enrichment from Marine Finfish Aquaculture and Effects on Sediment Biogeochemical Processes. Hdb Env Chem 5, Part M: Springer-Verlag Berlin Heidelberg. KARAKASSIS I, TSAPAKIS M, HATZIYANNI E Seasonal variability in sediment profiles beneath fish farm cages in the Mediterranean. Mar Ecol Prog Ser 162: LOWRY OH, ROSEBROUGH NJ, FARR A, RANDALL R Protein measurement with the Folin phenol reagent. J Biol Chem 193: LU L, WU RSS Recolonization and succession of marine macrobenthos in organic-enriched sediment deposited from fish farms. Environ Pollut 101: LYLE M The brown-green color transition in marine sediments: A marker of the Fe(III)-Fe(II) redox boundary. Limnol Oceanogr 28 (5): NICKELL LA, BLACK KD, HUGHES DJ, OVERNELL J, BRAND T, NICKELL TD, BREUER E, HARVEY SM Bioturbation, sediment fluxes and benthic community structure around a salmon cage farm in Loch, Creran, Scotland. J Exp Mar Biol Ecol :221. NILSSON HC, ROSENBERG R Hypoxic response of two marine benthic communities. Mar Ecol Prog Ser 115: RADASHEVSKY VI, DIAZ M, BERTRAN C Morphology and biology of Prionospio patagonica (Annelida: Spionidae) from Chile. J Mar Biol Ass U.K. 86: REISE K Sediment mediated species interactions in coastal waters. J Sea Res 48: REVSBECH NP, JORGENSEN BB Microelectrodes: The use in microbial ecology. Adv Microb Ecol 9: REVSBECH NP, JORGENSEN J, BLACKBURN TH Distribution of oxygen in marine sediments measured with microelectrodes. Limnol Oceanogr 25: ROSENBERG R, AGRENIUS S, HELLMAN B, NILSSON HC, NORLING K Recovery of marine benthic habitats and fauna in a Swedish fjord following improved oxygen conditions. Mar Ecol Prog Ser 234: ROSS A Marine fish farming - Scotland s pride or problem. Ecos 10: STEEL R, TORRIE J, DICKEY D Principles and Procedures of Statistics, A Biometrical Approach. 3 rd ed. USA: The McGraw-Hill Companies, Inc. YE LX, RITZ DA, FENTON GE, LEWIS ME Tracing the influence on sediments of organic waste from a salmonid farm using stable isotope analysis. J Exp Mar Biol Ecol 145: The Philippine Agricultural Scientist Vol. 91 No. 3 (September 2008)