Acute Toxicity to Peptone Concentrations in the Polychaete Perinereis aibuhitensis under Laboratory Culture

  • cc icon

    Organic pollution causes eutrophication and dystrophication, which occur when excessive amounts of organic matter enters seawater. Eutrophication can contaminate sediment and harm aquaculture. Polychaeta species have been shown to restore eutrophic sediment. In this study, we used peptone to simulate a eutrophic environment and detect the levels at which eutrophication became toxic to the polychaete Perinereis aibuhitensis. Peptone concentrations were 0, 100, 200, and 500 mg/L. The median lethal concentrations were 950.35 mg/L at 48 h, 340.34 mg/L at 72 h, and 120.22 mg/L at 96 h, which are much higher than those of other aquatic species. Polychaeta species are highly tolerant of eutrophication. During the 15-day long-term experiment, sediment loss on ignition, as well as seawater total organic carbon and total nitrogen all decreased significantly (P<0.05). However, NH4+ concentration increased with time. Perinereis aibuhitensis slowed the increment of NH4+ but could not prevent its increase. Our results indicate that this polychaete is helpful in the recovery of seawater and sediment from eutrophication.


    Entrophication , LC50 , LOI , Peptone , TOC , TN , NH4+ , Perinereis aibuhitensis

  • Introduction

    The human population has increased drastically during the last several decades, with industry and agriculture developing rapidly. Environmental problems caused by rapid development are now coming to the forefront. In coastal areas, substantial waste is released into the marine environment. These anthropogenic inputs can cause eutrophication of seawater and sediment (Lenhart et al., 2010). Nutrient enrichment due to anthropogenic activities appears to be the main cause of eutrophication in coastal areas (Cloern, 2001). Seawater eutrophication can increase biomass production (Cadee and Hegeman, 2002) and lead to changes in species composition (Philippart et al., 2000). In combination with adverse meteorological and hydrographical conditions, eutrophication is an important factor in oxygen depletion and subsequent mortality of benthic communities (Meyer-Reil and Koster, 2000).

    Peptone, an enzyme that digests animal protein, is commonly used as a source of organic nitrogen in microbiological and cell culture media, as well as for preparing eutrophic seawater (Matsushige et al., 1990).

    The polychaete Perinereis aibuhitensis is a segmented worm, a keystone invertebrate that lives in the mud of the in-tertidal zone. This polychaete has been used as an indicator of heavy metals, agricultural pesticides, and petrol pollution in estuarine ecosystems (Eisler and Hennekey, 1977; Scaps et al., 1997; Durou et al., 2007).

    However, relatively few studies have investigated environmental recovery in eutrophic areas. We evaluated the toxicity of peptone-synthesized eutrophic seawater to P. aibuhitensis and the effect of peptone on the ability of the polychaetes to improve eutrophic sediments and seawater, including sediment loss on ignition (LOI) and seawater total organic carbon (TOC) and total nitrogen (TN). The aim of this study was to determine whether polychaetes can contribute to the restoration of eutrophic environments.

    Materials and Methods

      >  Animal and sediment collection

    The polychaete Perinereis aibuhitensis, which was used as the test animal, was collected in Mokpo, Jeonnam, Korea. After transport to the lab, the polychaetes were acclimated to test conditions for 96 h before being exposed to synthetic eutrophic seawater. Sediments without infaunal organisms were purchased from the biotech company (Thrivecn Biological Technology CO., Ltd., Yangzhou, China). The pH-7 sediment had a particle diameter <1 mm and was composed of 10% sand, 70% silt, and 20% clay. The polychaetes could burrow into the sediment and survive without exhibiting abnormal appearances or behaviors.

      >  Acute toxicity and degradation experiment

    Peptone (Sigma-Aldrich, St. Louis, MO, USA) dissolved in seawater was used as an organic pollutant to create synthetic eutrophic seawater. The primary tests for acute toxicity were conducted using concentrations of 100 and 1,000 mg/L for 1 day. None of the animals in the 100-mg/L group died, whereas all of those in the 1,000-mg/L group died. Based on these results, we prepared a series of peptone test concentrations of 0, 100, 200, and 500 mg/L for the formal acute toxicity bioassays. In the long-term experiment, a concentration of 100 mg/L resulted in no deaths over 48 h.

    For the experiment, 30 polychaetes (body length 16.0±1.3 mm; body weight 0.4±0.1 g) were placed in a 10 L tank with 5 cm sediment on the bottom. The volume of circulating seawater was 30 L. Average water quality conditions during the experiment were 22℃ , pH 7.5, and salinity 30.6 practical salinity unit. Air pumps and air stones provided water and sediment oxygenation and turbulence. Each experimental group was replicated three times. During the acute toxicity bioassays, the polychaetes were examined every 24 h, and dead animals were removed. For the 15-day long-term experiment, seawater and sediment samples were collected at 0, 2, 5, 7, 9, and 15 days and stored at -20℃ for further analysis.

      >  Chemical analysis

    Sediment subsamples were first analyzed to determine the LOI concentration at 0, 2, 5, 7, and 15 days, according to the Korean standard method for sediments and seawater. Sediment samples were dried and ground to a fine powder smaller than 230 mesh using a mortar and pestle. A known weight of sample was placed in an aluminum crucible, which was then ignited in a muffler furnace at 550℃ for 2 h. The sample was cooled in a dessicator and weighed. LOI was calculated as the difference between the initial and final sample weights, divided by the initial sample weight times 100%.

    TOC and TN of seawater samples were analyzed at 0, 2, 5, 7, 9 and 15 days using a TOC/TN analyzer (TOC-V/TNM-1; Shimazu, Kyoto, Japan).

    Seawater NH4+ was detected using the o-phenylphenol [(1, 1´-biphenyl)-2-ol] (OPP) method of Yamaguchi and Machida (1968), as modified by Kanda (1995). The method is based on the reaction of indophenols with o-phenylphenol [(1, 1´-biphenyl)-2-ol] (the Berthelot reaction) as an alternative to phenol. The extracted indophenol was used to recover ammonium nitrogen to determine the 15-N isotope ratio and, thus, the concentration of NH4+ in seawater using a Europa Scientific ANCA-MS system (Europa Scientific Ltd., Crewe, UK).

      >  Statistical analysis

    Statistical comparisons of mean cumulative mortalities were examined using an ANOVA test at α=0.05. Lethal concentrations of peptone, defined as those at which the test organisms suffered 50%mortality (LC50), and their 95%confidence limits were estimated at 24 h and 48 h using a Probit analysis (Finney, 1971). The safe concentration was calculated as LC50X0.1.

    A one-way ANOVA, followed by Duncan’s multiple comparison procedure, was used to determine significant differences in LOI in sediment and in TOC, TN, and NH4+ concentrations in seawater at different elapsed times.


      >  Acute peptone toxicity tests

    Table 1 summarizes our results. No polychaetes died at the earliest time point. At 48 h, mortalities in different concentrations were still low, with mortality at 300 mg/L being 13.3%, which differed significantly from mortalities at other concentrations (ANOVA, P<0.05). However, after 96 h, mortalities

    increased sharply, with no significant differences (P<0.05) in cumulative mortalities between 200 mg/L and 500 mg/L. Almost all of the polychaetes in the 500 mg/L concentration were dead.

    Probit analysis comparing concentration and cumulative mortality was conducted using SPSS software version 18.0 (IBM, Yorktown Heights, NY, USA). Mortality probability was linearly dependent on log concentration (Table 2). The r2s of the three regression equations indicated that the probability of mortality and log concentration at 96 h were the most linearly dependent.

    Using regression equations, we calculated LC50 and the 95%confidence limit at different times. Table 3 shows the results. At 48 h, 72 h, and 96 h, LC50 was 950.35, 340.34, and 120.22 mg/L, respectively. At 96 h, the 95% confidence limit was statistically significant at 105.64-133.62. The safe concentration was 12.02 mg/L.

      >  Sediment analysis of long-term exposure experiment

    Fig. 1 shows the trends in LOI. Control sediment LOI was elevated significantly (P<0.05), whereas the LOI of the experimental group increased after the addition of the eutrophic seawater. Over time, LOI increased in both control and experimental groups. However, the LOI of the experimental group began to decrease at 5 days. At the end of the experiment, sediment LOI was significantly lower than the initial value.

      >  Seawater analysis of the long-term exposure experiment

    We monitored seawater TOC and TN in both control and experimental groups (Figs. 2 and 3). In both groups, TOC and TN increased significantly (P<0.05) after the addition of eutrophic seawater, and then decreased over time. The decrease

    of TOC and TN in the experimental group was greater than that of the control group.

    The concentration in seawater of NH4+ (Fig. 4) increased with time. At 9 days, a significant difference (P<0.05) became

    apparent in both groups. However, the final NH4+ concentration in the control was significantly higher (P<0.05) than in the experimental group.


      >  Sediment and water improvement

    High levels of peptone, which is a source of organics and nitrogen, can lead to eutrophication (Shikano and Kawabata, 2000). Thus, the addition of synthetic eutrophic seawater to the experimental group resulted in a significant increase in sediment LOI, as well as seawater TOC and TN (Figs. 1-3). However, in the experimental group, LOI and TN increments were lower than those of the control and had decreased below initial levels by the end of the experiment. Our results indicate that polychaetes can accelerate the recovery of sediments and seawater from eutrophication. Polychaetes have been shown to improve sediment conditions in scallop aquaculture (Kang et al., 2010). As a deposit-feeder, polychaetes ingest sediment and digest organic matter in the sediment, before defecating the altered sediment. Some research has indicated that the digestive fluids of deposit-feeders are more effective than water at solubilizing organic materials associated with sediments (Ahrens et al., 2001). In addition, disturbances caused by polychaetes aid the growth of specific bacterial groups, which break down, use, and enrich specific types of organic matter (Cuny et al., 2007). These two mechanisms may explain how polychaetes can reduce organics in seawater and sediment.

      >  Peptone toxicity and fate

    Peptone-rich peptide can be reduced to toxic ammonia and nitride by the activities of microorganisms (Shikano and Kurihara, 1988). Few studies have investigated the toxicity of peptone, in contrast to the plethora of studies on ammonia toxicity. Ammonia is acutely toxic to marine animals. Chronic exposure of fishes to sublethal levels of ammonia causes reduced food intake and slower growth (Pinto et al., 2007). Endocrine system functions appear to be especially affected in marine animals (Spencer et al., 2008). Elevated concentrations of ammonia in the surrounding water may reduce or prevent ammonia excretion by fishes, leading to a buildup of ammonia in the plasma and ultimately leading to death (Brinkman et al., 2009). In cobia, the 96-h LC50 of ammonia has been estimated at 1.13 mg/L (Rodrigues et al., 2007), and that of cutthroat trout is about 0.3-0.8 mg/L (Thurston et al., 1978). During our acute toxicity bioassays, the LC50 of peptone was 120.22 mg/L, and even when 1%of the peptone was reduced, the LC50 ammonia concentration was greater than that of the two species above. The ability of polychaetes to exist in highly eutrophic areas lends support to the idea that polychaeta can improve eutrophic environments.

    The NH4+ concentration in seawater increased with time (Fig. 4), which accords with a previous study showing that peptone-rich peptides can be reduced to ammonia (Shikano and Kurihara, 1988). We found that polychaetes slowed the increase in NH4+ compared to controls. However, the NH4+ concentration still increased with time, reaching 0.5 mg/L, which could impact other species. To control the increase in NH4+, integrated bioremediation should be applied, perhaps using nitrifying bacteria (Yang et al., 1999).

    In summary, we found that, under lab conditions, polychaetes can be used to alleviate eutrophication. Further research should be conducted to determine whether our data are applicable to real environmental conditions.

  • 1. Ahrens MJ, Hertz J, Lamoureux EM, Lopez GR, McElroy AE, Brownawell BJ 2001 The role of digestive surfactants in determining bioavailability of sediment-bound hydrophobic organic contaminants to 2 deposit-feeding polychaetes [Mar Ecol Prog Ser] Vol.212 P.145-157 google doi
  • 2. Brinkman SF, Woodling JD, Vajda AM, Norris DO 2009 Chronic toxicity of ammonia to early life stage rainbow trout [Trans Am Fish Soc] Vol.138 P.433-440 google doi
  • 3. Cadee GC, Hegeman J 2002 Phytoplankton in the Marsdiep at the end of the 20th century: 30 years monitoring biomass primary production and Phaeocystis blooms [J Sea Res] Vol.48 P.97-110 google doi
  • 4. Cloern JE 2001 Our evolving conceptual model of the coastal eutrophication problem [Mar Ecol Prog Ser] Vol.210 P.223-253 google doi
  • 5. Cuny P, Miralles G, Cornet-Barthaux V, Acquaviva M, Stora G, Grossi V, Gilbert F 2007 Influence of bioturbation by the polychaete Nereis diversicolor on the structure of bacterial communities in oil contaminated coastal sediments [Mar Pollut Bull] Vol.54 P.452-459 google doi
  • 6. Durou C, Smith BD, Romeo M, Rainbow PS, Mouneyrac C, Mouloud M, Gnassia-Barelli M, Gillet P, Deutsch B, Amiard-Triquet C 2007 From biomarkers to population responses in Nereis diversicolor: assessment of stress in estuarine ecosystems [Ecotoxicol Environ Saf] Vol.66 P.402-411 google doi
  • 7. Eisler R, Hennekey RJ 1977 Acute toxicities of Cd2+ Cr+6 Hg2+ Ni2+ and Zn2+ to estuarine macrofauna [Arch Environ Contam Toxicol] Vol.6 P.315-323 google
  • 8. Finney DJ 1971 Probit Analysis google
  • 9. Kanda J 1995 Determination of ammonium in seawater based on the indophenol reaction with o-phenylphenol (OPP) [Water Res] Vol.29 P.2746-2750 google doi
  • 10. Kang KH, Zhang ZF, Bao ZM, Zhou B, Kang KH, Kim YH, Webster CD 2010 Effects of clamworm Perinereis aibuhiteusis density on acid volatile sulfide chemical oxygen demand and loss of ignition in benthic sediment and on survival of scallop Chlamys farreri Spat [J World Aquac Soc] Vol.41 P.131-138 google doi
  • 11. Lenhart HJ, Mills DK, Baretta-Bekker H, van Leeuwen SM, van der Molen J, Baretta JW, Blaas M, Desmit X, Kuhn W, Lacroix G, Los HJ, Menesguen A, Neves R, Proctor R, Ruardij P, Skogen MD, Vanhoutte-Brunier A, Villars MT, Wakelin SL 2010 Predicting the consequences of nutrient reduction on the eutrophication status of the North Sea [J Mar Syst] Vol.81 P.148-170 google doi
  • 12. Matsushige K, Inamori Y, Mizuochi M, Hosomi M, Sudo R 1990 The effects of temperature on anaerobic filter treatment for low-strength organic wastewater [Environ Technol] Vol.11 P.899-910 google doi
  • 13. Meyer-Reil LA, Koster M 2000 Eutrophication of marine waters: effects on benthic microbial communities [Mar Pollut Bull] Vol.41 P.255-263 google doi
  • 14. Philippart CJM, Cadee GC, van Raaphorst W, Riegman R 2000 Long-term phytoplankton-nutrient interactions in a shallow coastal sea: algal community structure nutrient budgets and denitrifi cation potential [Limnol Oceanogr] Vol.45 P.131-144 google doi
  • 15. Pinto W, Aragao C, Soares F, Dinis MT, Conceicao LEC 2007 Growth stress response and free amino acid levels in Senegalese sole (Solea senegalensis Kaup 1858) chronically exposed to exogenous ammonia [Aquac Res] Vol.38 P.1198-1204 google doi
  • 16. Rodrigues RV, Schwarz MH, Delbos BC, Sampaio LA 2007 Acute toxicity and sublethal effects of ammonia and nitrite for juvenile cobia Rachycentron canadum [Aquaculture] Vol.271 P.553-557 google doi
  • 17. Scaps P, Demuynck S, Descamps M, Dhainaut A 1997 Effects of organophosphate and carbamate pesticides on acetylcholinesterase and choline acetyltransferase activities of the polychaete Nereis diversicolor [Arch Environ Contam Toxicol] Vol.33 P.203-208 google
  • 18. Shikano S, Kawabata Z 2000 Effect at the ecosystem level of elevated atmospheric CO2in an aquatic microcosm [Hydrobiologia] Vol.436 P.209-216 google doi
  • 19. Shikano S, Kurihara Y 1988 Analysis of factors controlling responses of an aquatic microcosm to organic loading [Hydrobiologia] Vol.169 P.251-257 google doi
  • 20. Spencer P, Pollock R, Dube M 2008 Effects of un-ionized ammonia on histological endocrine and whole organism endpoints in slimy sculpin (Cottus cognatus) [Aquat Toxicol] Vol.90 P.300-309 google doi
  • 21. Thurston RV, Russo RC, Smith CE 1978 Acute toxicity of ammonia and nitrite to cutthroat trout fry [Trans Am Fish Soc] Vol.107 P.361-368 google doi
  • 22. Yamaguchi R, Machida T 1968 Colorimetric determination of ammonia. I. A new spectrophotometric method for the determination of ammonia using o-phenylphenol [Yakugaku Zasshi] Vol.88 P.1383-1387 google
  • 23. Yang L, Chang YF, Chou MS 1999 Feasibility of bioremediation of trichloroethylene contaminated sites by nitrifying bacteria through cometabolism with ammonia [J Hazard Mater] Vol.69 P.111-126 google doi
  • [Table 1.] Toxicity of peptone to polychaeta
    Toxicity of peptone to polychaeta
  • [Table 2.] Regression equation at different times
    Regression equation at different times
  • [Table 3.] The LC50 and safe concentration of peptone for the polychaeta
    The LC50 and safe concentration of peptone for the polychaeta
  • [Fig. 1.] The loss on ignition (LOI) of sediment at different elapsed time.Initial LOI of sediment before treatment. *Significantly different from initial LOI (P<0.05).
    The loss on ignition (LOI) of sediment at different elapsed time.Initial LOI of sediment before treatment.  *Significantly different from initial LOI (P<0.05).
  • [Fig. 2.] The total organic carbon (TOC) of seawater at different elapsed time.Initial TOC of seawater before treatment. *Significantly different from initial TOC (P<0.05).
    The total organic carbon (TOC) of seawater at different elapsed time.Initial TOC of seawater before treatment. *Significantly different from initial TOC (P<0.05).
  • [Fig. 3.] The total nitrogen (TN) of seawater at different elapsed time.Initial TN of the seawater before treatment. *Significantly different from initial TN (P<0.05).
    The total nitrogen (TN) of seawater at different elapsed time.Initial TN of the seawater before treatment. *Significantly different from initial TN (P<0.05).
  • [Fig. 4.] The NH4+ concentration of seawater at different elapsed time.Initial NH4+ concentration of the seawater before treatment. *Significantly different from initial concentration (P<0.05).
    The NH4+ concentration of seawater at different elapsed time.Initial NH4+ concentration of the seawater before treatment. *Significantly different from initial concentration (P<0.05).