Journal of Environmental Protection and Ecology 15, No 4, 1650–1659 (2014) Marine ecology
POLLUTANT BIOACCUMULATION IN THE LONG-SNOUTED SEAHORSE AT THE ROMANIAN COAST M.-I. NENCIUa*, V. COATUa, A. OROSa, D. ROSIORUa, D. TIGANUSa, N. ROSOIUb NIRDEP – ‘Grigore Antipa’ National Institute for Marine Research and Development, 300 Mamaia Blvd., 900 581 Constanta, Romania E-mail:
[email protected];
[email protected] b Department of Biochemistry, Faculty of Medicine, Constanta, ‘Ovidius’ University Constanta, Academy of Romanian Scientists, 54 Splaiul Independentei Street, 050 094 Bucharest, Romania a
Abstract. In recent years, pollution has become one of the major threats aquatic species are facing, due to anthropogenic discharges into the marine environment. Many species are threatened by these influences, while other are insufficiently studied. Hippocampus guttulatus (C u v i e r, 1829) is the seahorse species inhabiting the Black Sea coastal areas, being rated by the International Union for Conservation of Nature (IUCN) as data deficient, and the actual extent to which pollutant discharges influence the long-snouted seahorses at the Romanian Black Sea coast is unknown. Under these circumstances, the aim of this paper is to assess the pollutant bioaccumulation in the body of seahorses. In addition, some biochemical parameters (dry/wet weight, moisture, crude protein, crude lipid, ash) were analysed, with the view to establishing potential correlations between such parameters and xenobiotic intake. The analysed contamination compounds were organochlorine pesticides, polycyclic aromatic hydrocarbons (PAHs) and heavy metals (Cu, Cd, Pb, Ni and Cr), the main indicators used for assessing marine environment pollution. Keywords: H. guttulatus, pollutants, bioaccumulation, biochemical parameters, polycyclic aromatic hydrocarbons, organochlorine pesticides, heavy metals.
AIMS AND BACKGROUND Given the fact that one of the major threats on seahorses around the World Ocean is pollution1, this paper aims at analysing the pollutant bioaccumulation in the body of the long-snouted seahorse – Hippocampus guttulatus (C u v i e r, 1829) from the Romanian Black Sea coast. While it is known that mussels or the rapa whelk, for instance, have a remarkable capacity to accumulate xenobiotics2–6, there are few data on how the body of seahorses absorbs xenobiotics, especially in the Black Sea area. The seahorse species in the Black Sea (H. guttulatus), like all seahorse species, inhabits shallow coastal areas, where they can find a proper habitat in seagrass *
For correspondence.
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meadows and rocky areas covered in macrophyte algae. However, this closeness to the coast makes them extremely vulnerable to anthropogenic discharges7. As such, 3 of the most significant contaminants of the marine environment (organochlorine pesticides, polycyclic aromatic hydrocarbons and heavy metals) were investigated in seahorse tissue collected along the southern Romanian Black Sea coast. EXPERIMENTAL The samples were collected by divers in August-September 2013 from three shallow (0–20 m) sampling stations located along the southern part of the Romanian coast, due to the fact that here seahorses find the propitious habitat (a rocky and rough seabed, which is covered by macrophyte algae – Cystoseira barbata, as well as few of the seagrass meadows in Romania – Zostera noltei8). The three stations were (from south to north) Mangalia, Saturn-Venus and Cazino-Constanta (Fig. 1). 16 Hippocampus guttulatus individuals (9 males and 7 females) were collected from each sampling location, with a total wet weight of 26.33 g (Mangalia), 28.53 g (Saturn-Venus) and 25.83 g (Cazino-Constanta), respectively. Subsequently, the biological material was frozen and later subjected to specific analysis methods. For the proximate biochemical composition of the samples, standard methods were used. Moisture and dry weight were determined by drying in an oven at 105oC for 24 h. Ash was determined by burning in an oven at 550oC for 6 h. The protein
Fig. 1. Sampling stations along the southern Black Sea coast
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content (% dry weight) of H. guttulatus was analysed by the Lowry method. The lipid content was determined by the ether extraction with the Soxhlet apparatus. Concerning the organochlorine pesticides (OCPs), the frozen tissues were freezedried and homogenised. About 3 g of the dried tissue were used for analysis. Internal standard 2,4,5-trichlorobenzene was added to the samples for quantifying the overall recovery of the analytical procedures. The extraction of OCPs from biota samples was done with 30 ml acetone/hexane (1:1, v:v), in microwave extraction system Start E Milestone for 30 min at 120oC. Further processing of the samples followed the steps: concentration of the extracts to rotoevaporator, clean-up on florisil, respectively, alumina/silica column and concentration of the samples using the Kuderna-Denish concentrator and nitrogen flow. The analytical determination of the OCP content was made by the gas-chromatographic method with a Perkin Elmer gas chromatograph CLARUS 500, equipped with electron capture detector. The analysed compounds were HCB, Lindane, Heptachlor, Aldrin, Dieldrin, Endrin, p,p′-DDE (dichlorodiphenyldichloroethylene), p,p′-DDD (dichlorodiphenyldichloroethane) and p,p′-DDT (dichlorodiphenyltrichloroethane). For PAH analysis9, the frozen tissues were freeze-dried and homogenised. About 2 g of the dried tissue were used for analysis. Internal standard 9,10 dihydroanthracene was added to the samples for quantifying the overall recovery of the analytical procedures. Samples were Soxhlet extracted for 8 h with 250 ml of methanol. The extracts were then saponified by adding 20 ml of 0.7 M KOH and 30 ml of water and refluxing for 2 h. The resulting mixture was transferred into a separating funnel and extracted 3 times with hexane – once with 90 ml, twice with 50 ml. The extracts were concentrated by rotary evaporation down to 15 ml, and then further concentrated to about 5 ml under a gentle flow of clean nitrogen. Finally, the extract was cleaned up and fractionated by passing it through a silica/alumina column. Elution was performed using 20 ml of hexane to yield the first fraction (containing the aliphatic hydrocarbons), then 30 ml of hexane:methylene chloride (90:10) and followed by 20 ml of hexane:methylene chloride (50:50). These two eluents containing the aromatic hydrocarbons (PAHs) were combined for analysis. The fraction containing PAHs was evaporated under a weak flow of nitrogen to 1 ml and it was subjected to qualitative and quantitative analysis on GC/MS Perkin Elmer Clarus 500. The analysed compounds were: naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3-cd] pyrene, benzo[ghi]perylene, dibenzo[a,h]anthracene. As far as heavy metals are concerned, the sampling, preservation, preliminary processing and analysis methodologies were consistent with the reference methods recommended in the study of marine pollution10. Before the determination of heavy metals, biological samples were freeze-dried, homogenised, weighed and subjected to digestion with 5 ml nitric acid (HNO3 65%, Suprapur Merck) in 1652
sealed Teflon vessels (60 ml Savillex) on a hotplate at 120°C. After completion of digestion, samples were brought to 100 ml volume with deionised water (18.2 MΩ cm, Millipore). In the solutions obtained after the digestion of samples, metals (copper, cadmium, lead, nickel and chromium) were analysed using graphite furnace-atomic absorption spectrometers, type ATI-UNICAM 939Z and SOLAAR M6 Dual Thermo Electron-UNICAM. The accuracy and precision of the analytical method were checked with certified reference materials (CRM). RESULTS AND DISCUSSION The analysis of the proximate biochemical composition of H. guttulatus (dry weight, moisture, ash, crude protein, crude lipid) was performed (WW = wet weight; DW = dry weight). Dry weight (%) ranged from 26.64±0.50 (Mangalia) to 30.69±0.27 (Cazino-Constanta). Moisture (WW%) ranged from 69.31±0.27(Cazino-Constanta) to 73.36±0.5 (Mangalia). Ash (DW%) ranged from 17.90±0.95 (Saturn-Venus) to 19.38±0.62 (Mangalia). Protein content (DW%) ranged from 70.82±5.15 (Cazino-Constanta) to 74.07±3.55 (Mangalia). Lipid content (DW%) ranged from 1.17±0.13 (Mangalia) to 1.28±0.21 (Cazino-Constanta). Dry weight and lipid content recorded the minimum values in seahorses collected in Mangalia and maximum values at Cazino-Constanta. Moisture and protein content registered minimum values in seahorses collected at Cazino-Constanta and maximum values at Mangalia (Fig. 2a, b). The analysis of the organochlorine pesticide (OCPs) levels in seahorse tissue revealed certain differences between the sampling stations and between the analysed pesticides themselves. The maximum value recorded was for aldrin in Mangalia (0.208173 μg/g WW), while lindane was almost below the detection limit (0.0004 μg/g WW) in all three sampling locations. The values recorded for hexa chlorobenzene (HCB) were quite low in all three sampling sites (mean 0.016189 μg/g WW) and for heptachlor the value recorded in Cazino-Constanta (0.083625 μg/g WW) was significantly higher compared to the other two stations (0.0003 μg/g WW). p,p′-DDT values in Cazino-Constanta (0.051445 μg/g WW) were also significantly higher than those recorded in Saturn-Venus (0.026322 μg/g WW) and Mangalia (0.012507 μg/g WW). For the remaining pesticides analysed (dieldrin – mean 0.068848 μg/g WW, endrin – mean 0.072671 μg/g WW, p,p′-DDE – mean 0.082492 μg/g WW, p.p′-DDD – 0.084484 μg/g WW), the values recorded were quite similar, with slight differences between stations (Fig. 3).
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Fig. 2. Dry weight-moisture ratio (a) and protein, lipid and ash content (%DW) (b) in Hippocampus guttulatus tissue
Fig. 3. Organochlorine pesticide contamination (μg/g WW) of Hippocampus guttulatus on compounds
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The total organochlorine pesticide content in seahorse whole tissue ranged between 0.43502 μg/g WW in Saturn-Venus and 0.55348 μg/g WW in Cazino Constanta (mean 0.50475), thus pointing out an overall higher contamination with organochlorine pesticides in the station close to the Constanta port (Fig. 4).
Fig. 4. Total organochlorine pesticide contamination of Hippocampus guttulatus
This overview of the OCP values in seahorse tissue revealed that, despite its low lipid content compared to other marine organisms (mussels, for instance), seahorses still bioaccumulate organochlorine compounds, which are usually absorbed by fatty tissues. However, the extent to which their metabolism and enzymatic activity is affected by OCPs is yet to be investigated during further research. Similarly to organochlorine pesticides, polycyclic aromatic hydrocarbons (PAHs) were also identified in the seahorse tissue, with differences between the 16 analysed compounds (Fig. 5).
Fig. 5. Polycyclic aromatic hydrocarbon contamination (ng/g WW) of Hippocampus guttulatus on compounds
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The highest level was recorded by fluorene both in Saturn-Venus and CazinoConstanta (265.7 ng/g WW and 267.3 ng/g WW, respectively), while naphthalene, anthracene, benzo[k]fluoranthene values were below the detection limit. Fluorene is a low mollecular weight PAH, with 2–3-aromatic rings, characteristic for oil discharghes and oil product spills11. This high fluorene value recorded in seahorse tissue in all stations may indicate this type of pollution of water in the sampling areas, resulting in a rapid intake of this PAH by the seahorse body. Three of the polycyclic aromatic compounds, namely acenaphthene, fluorene and benzo[a]anthracene recorded higher values (means 100.0 ng/g WW, 226.4 ng/g WW and 93.5 ng/g WW, respectively), while the other remaining PAHs did not show an alarming contamination. However, the total polycyclic aromatic hydrocarbon content in seahorse tissue samples showed a higher degree of contamination in the Cazino-Constanta Station (587.8 ng/g WW), decreasing southwards to 439.5 ng/g WW in Saturn-Venus and 322.5 ng/g WW in Mangalia (Fig. 6).
Fig. 6. Total polycyclic aromatic hydrocarbon contamination (ng/g WW) of Hippocampus guttulatus
No data were available to compare the levels of organochlorine pesticides and polycyclic aromatic hydrocarbons in seahorses in the Black Sea to seahorse species in other marine areas worldwide, to see whether these are normal or exceptional values. It may be possible that these high values are the results of some interference compounds, as pigments still had remained in the extract, after the clean-up step. Consequently, the analyses must be repeated, using the same sampling stations. For heavy metals, however, some information for comparison with other seahorse species were available12. As such, the heavy metal content in H. guttulatus tissue analysed was similar and comparable for copper, cadmium and lead, as follows: Cu – 5.56 μg/g DW in H. guttulatus and 6.03 μg/g in Hipppocampus kuda (B l e e k e r, 1852), Cd – 1.51 μg/g in H. guttulatus and 1.54 μg/g DW in Hippocampus spinosissimus (R o u l e, 1916), Pb – 2.50 μg/g DW in H. guttulatus 1656
and 2.07 μg/g DW in H. histrix (K a u p, 1856). These comparable values confirm the accuracy of the analysis perfomed on the Black Sea samples and indicate that seahorses along the Romanian Black Sea coast do bioaccumulate heavy metals. The highest value recorded in the samples was for copper in Saturn-Mangalia (5.56 μg/g DW), while the lowest contamination level was for cadmium in the Cazino-Constanta Station (0.02 μg/g DW) (mean 2.66 μg/g DW). Lead contamination was also significant, with a mean value reaching 1.88 μg/g DW. Cadmium, nickel and chrome recorded similar mean values, ranging from 0.54 μg/g DW (Cr) to 0.82 μg/g DW (Ni) (Fig. 7).
Fig. 7. Heavy metal contamination (μg/g DW) in the species Hippocampus guttulatus
Due to the high copper and lead levels in Saturn-Venus, this station resulted in being the most contaminated with heavy metals (total 11.19 μg/g DW), followed by Mangalia (6.10 μg/g DW) and Cazino-Constanta (2.39 μg/g DW). This high level of copper in H. guttulatus is probably a result of copper contamination of the surrounding water, which may be caused by vessel traffic (it is used as antifouling agent on hulls) (Fig. 8).
Fig. 8. Total heavy metal contamination (μg/g DW) of Hippocampus guttulatus
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CONCLUSIONS The overall conclusion of this first research attempt on the bioaccumulation level in the Black Sea Hippocampus guttulatus confirms the hypothesis that seahorses, like most of marine species, absorb the xenobiotics reaching the aquatic environment. In summary, the organochlorine pesticide values recorded in seahorse tissue revealed that, despite their low lipid content compared to other marine organisms, seahorses still bioaccumulate organochlorine compounds, which are usually absorbed by fatty tissues. Higher overall levels of OCPs were recorded in the Cazino-Constanta Station. Concerning the total polycyclic aromatic hydrocarbon content in seahorse tissue, samples also showed a higher degree of contamination in the Cazino-Constanta Station, decreasing southwards (probably caused by the vicinity of the Constanta Port). For heavy metals, however, the highest values (for copper) were recorded in one of the southern stations, Saturn-Venus, likely to be caused by vessel traffic in the area. All these compounds are highly toxic, even in low concentrations, especially if they accumulate in the metabolically active sites, and they could significantly influence the survival of a sensitive species such as the long-snouted seahorse. It is, thus, essential to perform future research aimed at analysing the extent to which OCPs, PAHs and heavy metals influence the metabolic and enzymatic activity of H. guttulatus. REFERENCES 1. L. WOODALL: Hippocampus guttulatus. In: IUCN 2013. IUCN Red List of Threatened Species. Version 2013.2. www.iucnredlist.org; 2012. 2. M. BRATU, L. TOFAN, V. COATU, M. CRASMARU: Comparative Study of Catalase Activity from the Mid-gland of Three Mollusk Species from the Black Sea. J Environ Prot Ecol, 5 (2), 341 (2004). 3. D. MIRCEA, N. ROSOIU, A. OROS, D. TIGANUS: Some Eco-biochemical Aspects of Mollusk Shells from Romanian Black Sea Coast. J Environ Prot Ecol, 6 (4), 838 (2005). 4. A. OROS, M.-T. GOMOIU: A Review of Metal Bioaccumulation Levels in the Romanian Black Sea Biota during the Last Decade – A Requirement for Implementing Marine Strategy Framework Directive (Descriptors 8 and 9). J Environ Prot Ecol, 13 (3A), 1730 (2012). 5. A. OROS, I. PECHEANU, R. MIHNEA: Some Aspects Concerning Trace Metals Bioaccumulation in Mytilus galloprovincialis along the Romanian Black Sea Coastal Area. J Environ Prot Ecol, 4 (4), 850 (2003). 6. D. ROSIORU, V. COATU, A. OROS, D. VASILIU, D. TIGANUS: Marine Environment Quality of the Growth and Exploitation of the Main Mollusks from the Romanian Black Sea. J Environ Prot Ecol, 13 (3A), 1799 (2012). 7. M. I. NENCIU (ZAHARIA), D. ROSIORU, V. COATU, A. OROS, N. ROSOIU: Characterization of the Environmental Conditions of the Long-snouted Seahorse Habitat at the Romanian Coast. J Environ Prot Ecol, 14 (4), 1695 (2013). 8. O. MARIN, V. ABAZA, D. SAVA: Phytobenthos – A Key Biological Element in Shallow Marine Waters. Recherches Marines, (43), 197 (2013).
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9. IAEA-MEL: Training Manual on the Measurement of Organochlorine and Petroleum Hydrocarbons in Environmental Samples. Marine Environmental Studies Laboratory, 1995. 10. IAEA-MEL: Standard Operating Procedures for Trace Metals Analyses. 1999. 11. NIMRD Research Team: Report on the State of the Marine and Coastal Environment in 2012 Recherches Marines, (43), 5 (2013). 12. QIANG LIN, LIN JUNDA, LU JUNYI, LI BINGJI: Biochemical Composition of Six Seahorse Species, Hippocampus sp., from the Chinese Coast. J World Aquacult Soc, 39 (2), 225 (2008). Received 3 October 2014 Revised 20 November 2014
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