Marine Pollution Bulletin 56 (2008) 1570–1577

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Ballast water as a vector of coral pathogens in the Gulf of Mexico: The case of the Cayo Arcas coral reef M. Leopoldina Aguirre-Macedo a,*, Victor M. Vidal-Martinez a, Jorge A.Herrera-Silveira a, David S. Valdés-Lozano a, Miguel Herrera-Rodríguez b, Miguel A. Olvera-Novoa a a Departamento de Recursos del Mar, Centro de Investigación y de Estudios Avanzados del IPN Unidad Mérida (CINVESTAV-IPN), Carretera Antigua a Progreso Km. 6, C.P. 97310 Mérida, Yucatán, Mexico b Gerencia de Seguridad Industrial y Protección Ambiental – RMNE PEMEX Exploración y Producción, Calle 31 S/N, Edificio Complementario 1, Col. Sta. Isabel, Cd. del Carmen, Campeche, Mexico

a r t i c l e Keywords: Coral reef Tankers as vector Coral reef diseases Microbiology Ballast water Vibrio cholerae 01

i n f o

a b s t r a c t The discharge of nutrients, phytoplankton and pathogenic bacteria through ballast water may threaten the Cayo Arcas reef system. To assess this threat, the quality of ballast water and presence of coral reef pathogenic bacteria in 30 oil tankers loaded at the PEMEX Cayo Arcas crude oil terminal were determined. The water transported in the ships originated from coastal, oceanic or riverine regions. Statistical associations among quality parameters and bacteria were tested using redundancy analysis (RDA). In contrast with coastal or oceanic water, the riverine water had high concentrations of coliforms, including Vibrio cholerae 01 and, Serratia marcescens and Sphingomona spp., which are frequently associated with ‘‘white pox” and ‘‘white plague type II” coral diseases. There were also high nutrient concentrations and low water quality index values (WQI and TRIX). The presence of V. cholerae 01 highlights the need for testing ballast water coming from endemic regions into Mexican ports. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction A main concern of the International Maritime Organization (IMO, 1997; Matheickal and Raaymakers, 2003) is the establishment of invasive species in new environments through the ballast water discharges of commercial sea traffic. The role of ballast water as a vector for introduced species has been extensively examined in the Pacific, Atlantic and Gulf coasts of North America (Ruiz et al., 2000a,b; Joachimsthal et al., 2004; Bai et al., 2005). Another potential risk of the discharge of thousands of tons of ballast water is the associated decrease in the water quality in sensitive environments such as coral reefs (Lipp et al., 2002; Patterson et al., 2002). Large oil tankers (350,000–750,000 oil barrels) discharge riverine and coastal water from ports all over the world at the Mexican Oil Company’s (PEMEX) offshore crude oil loading terminal in Campeche Sound, Gulf of Mexico. This terminal is less than four kilometers from the Cayo Arcas coral reef formation (Jordán-Dahlgren and Rodríguez-Martínez, 2004), and there is reasonable cause for concern about the potential negative effects of ballast water discharge in the area. Both the introduction of invasive species and the reduction in water quality associated with this discharge may impact the adjacent coral reef.

* Corresponding author. Tel.: +52 (999) 942 9450; fax: +52 (999) 9812334. E-mail address: [email protected] (M.L. Aguirre-Macedo). 0025-326X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2008.05.022

Some coral pathogens such as Serratia marcescens and Sphingomonas WP type II have been found in ballast water discharge and riverine waters (Lipp et al., 2002; Patterson et al., 2002; Bythell et al., 2004). Here, we analyze the ballast water of oil tankers arriving at Cayo Arcas by determining microbiological indicators of water quality and testing for the presence of bacteria known to be pathogens of corals (Phormidum corallycticus, S. marcescens, Sphingomonas WP type II, Beggiatoa spp. and Desulfovibrio spp.). We also determine possible statistical associations between water characteristics and the presence of bacteria in the ballast water of these tankers. 2. Material and methods Water samples were obtained from 30 tankers loading oil at mooring buoys of PEMEX’s crude oil terminal near Cayo Arcas (20°130 0000 N and 92°000 0000 W, 20°130 0000 N and 91°550 0000 W, 20°080 0000 N and 92°000 0000 W, and, 20°080 0000 N and 91°550 0000 W) in the Gulf of Mexico between 18 June and 6 July 2005 (Fig. 1). Water and sediment samples were obtained from two reference sites near the Cayo Arcas coral reefs. Physicochemical and biological parameters were recorded for all sampled tankers. We focused specifically on bacterial pathogens (Serratia merecens, Phormidium corallycticum, Sphingomonas WP type II, Begiatoa spp. and Desulfovibrio spp.) that are known to be associated with coral diseases.

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Fig. 1. Geographical location of the Cayo Arcas coral reef and the crude oil loading terminal of the Mexican Oil Company (PEMEX).

For each of the 30 oil tankers, the port of origin and date of charge of the ballast water were recorded based on the ballast water notification form A20/RES.868 from the IMO (1997). Three tanks per ship were sampled, giving consideration to the origin and residence time of the water in individual tanks. When the water in all tanks had the same residence time, the tanks were sampled randomly. However, for the purposes of this paper, the values of all variables, except those for bacteria, were the mean value from three tanks per ship. Each sample was obtained directly from the manholes or hatches to each tank, using a 1.5 L Van Dorn bottle at a depth of 4 m. Temperature (°C) and oxygen concentration (mg/l) were obtained using a YSI-51-B (±0.1) meter. Each sample was deposited in a 500 ml sterile container, placed in a cooler at 4 °C, and transported to the laboratory at CINVESTAV Mérida the same day they were obtained from the tanker. 3. Microbiology To determine water quality in terms of the number of total and fecal coliform bacteria, we used the most probable number (MPN) technique, following the standard method suggested by Fujioka (2002). Additionally, Escherichia coli, Enterococcus and V. cholerae were isolated from those samples positive for fecal coliforms by following the protocols recommended by OPS/OMS (1988) and Fujioka (2002). The colonies of S. marcescens and Sphingomonas WP type II were isolated based on their morphological characteristics after culture in TSA + 2% NaCl and McConkey and Difco marine agar. Sphingomonas WP type II colonies were identified following the procedures described by Kilbane et al. (2002). Once isolated, the bacteria were re-inoculated in TSA, and presumptive tests (Gram, oxidase-citochrome, movility and catalase) were performed. Finally, for specific taxonomic identification each one of the isolated colonies was inoculated for 48 h in ID32 GN bands for the MiniApi system (Biomerioux) (Patterson et al., 2002). For the isolation of Beggiatoa spp., water samples were inoculated in marine agar, incubated at 37 °C for 24–48 h, and re-inoculated in agar enriched with vitamins, minerals, and sodium acid (0.5%) at 0.001 g/l, following the procedures of Strohl and Larkin (1978). For Desulfovibrio spp. isolation, tubes enriched with postgate B agar were used; anaerobic conditions were maintained by using mineral oil as a sealant. The presence of Desulfovibrio was confirmed by the black color and H2S smell, as well as the method

of Difrancesco and Mehrnoush (2004). To isolate Phormidium corallyticum, 200 ml samples obtained from each tanker were filtered using sterile cellulose acetate membranes (Millipore) with a 45 lm pore size. The membranes were placed in agar for cyanobacteria ASN-III (Rippka, 1988). Colonies were isolated and re-inoculated in TSA agar and processed as suggested by Holt et al. (1994) and Urmeneta et al. (2003). 4. Physicochemical parameters To quantify suspended solids, water was filtered using microfiber glass fiber filters. For each filtered sample, the following measurements were obtained based on the methods of Strickland and Parsons (1972): pH (NBS scale), turbidity (ICM model 11150 meter), salinity (Kahlsico RS-9 induction meter), conductivity (YSI33 meter), ammonium, nitrite, nitrate, phosphate, silicates, total alkalinity and total carbon dioxide. We also calculated the Water Quality Index (WQI) (Canadian Council of Ministers of the Environment, 2001). This index provides a convenient way of summarizing complex water quality data. The index has five categories: excellent: 95–100; good: 80–94; regular: 65–79; marginal: 45–64; and poor: 0–44. 5. Primary productivity parameters Immediately after collection, the water was filtered using an ester-cellulose Millipore filter (4.5 cm diameter, pore size of 0.45 lm). The filtrate volume was standardized to 1.5 L, deposited in 500 ml sterile containers and transported on ice to the laboratory. The concentrations of chlorophylls and carotenes were determined by spectrophotometric methods, using an extraction with 90% acetone (Jeffrey et al., 1999). To determine the eutrophic condition, the method of Vollenweider et al. (1998), which is based in the TRIX index, was used:

TRIX ¼ ½log10 ðChl-a  D%O  DIN  SRPÞ  a=b; where Chl-a = Chlorophyll-a (lg/l); D%O = absolute value of the saturated oxygen concentration with respect to 100%; DIN = dissolved inorganic nitrogen concentration (lg/l); SRP = soluble reactive phosphorous concentration (lg/l); a = –1.5; b = 1.2; scaling factor in order to have an index with values between 0 and 10. The index has the following categories: 0–4: oligotrophic; 4–6: mesotrophic; 6–8: eutrophic, >8: distrophic.

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6. Statistical analysis

8. Microbiology

Water productivity was analyzed using a non-parametric oneway analysis of variance to compare the four water sources. Significant differences at a 95% confidence level were established using the Kruskal-Wallis test (Zar, 1984). Due to the absence of Mexican standards for these variables, we followed the strategy of Warwick (1988, 1993) and Warwick and Clarke (1993), which suggests a comparison of the samples under study with reference samples from the surrounding environment. Multivariate statistical analyses were used to determine possible associations between physicochemical variables and the presence of bacteria and concentrations of total and fecal coliform. We used individual oil tankers as our unit of replication and Redundancy Analysis (RDA), the constrained form of PCA, using CANOCO software (ter-Braak, 1996). We used RDA based on the suggestions of ter-Braak and Smilauer (1998), since the lengths of the ordination axes were less than two standard deviations from the mean, as shown previously by Detrended Correspondence Analysis (DCA). Monte Carlo tests were used to determine the significance of the canonical axes for both bacteria and physicochemical variables. The normality of the transformed variables was determined using rankit plots (Sokal and Rohlf, 1995).

Twenty-six of the 30 vessels sampled (87%) contained total coliform bacteria (ranging from 4 to 240 MPN/100 ml), and 27% had fecal coliform bacteria. Both water (9 MPN/100 ml) and sediment (19 MPN/100 ml) samples from the reference sites tested positive for total coliforms but negative for fecal coliforms (Table 2). The water sample from vessel B8 was the only one that tested negative for both total and fecal coliforms. Seven samples (23% of the tankers) tested positive for E. coli, and one was positive for E. coli 0157 (B21). The water and sediment samples from the reference sites were negative for E. coli. Samples from 25 of the tankers tested positive for Enterococcus spp. (3– 163 cfu/ml). The values for water samples from the reference site were between 11 and 33 cfu/ml. The sediment samples from the B8 tanker and the reference sites had 19 cfu/ml and 67 cfu/ml, respectively. V. cholerae 01 was present in tanker B2, Sphingomonas spp. in tanker B8 and S. marcescens in tanker B14 (Table 2). All other water and sediment samples tested were negative for these or any other coral pathogenic bacteria under study.

7. Results Table 1 lists the 30 tankers sampled. With the exception of one vessel from Mexico (B16) and one from the Bahamas (28), all vessels came from ports on the US Gulf Coast. Only two vessels (from the USA and the Bahamas) reported exchange of coastal water for oceanic ballast water. The residence time of ballast water varied from 1 to 6 days. Table 1 Code and port origin of the 30 crude oil tankers sampled at the Cayo Arcas offshore crude oil loading terminal of the Mexican Oil Company (PEMEX) between 18th June and 6th July, 2005 Tanker code

Ballast water origin

# Tanks per ship

Water volume in sampled tanks (mean ± SD)

 Time of water residence (days)

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15 B16 B17 B18 B19 B20 B21 B22 B23 B24 B25 B26 B27 B28 B29 B30

Mississippi River, LA, USA Baytown, TX, USA Houston, TX, USA Sunoil Nederland, TX, USA Freeport, USA Loop, USA Lake St Charles, LA, USA Corpus Christi, TX, USA Mississippi River, LA, USA Corpus Christi, TX, USA Port Arthur, TX, USA Port Arthur, TX, USA Sunoil Nederland, TX, USA Port Arthur, TX, USA Pascagoula, LA, USA Tuxpan, Ver. Mex. Lake St Charles, LA, USA Houston, TX, USA Houston, TX, USA Pascagoula, LA, USA Chalmette, LA, USA Lake St Charles, LA, USA Pascagoula, LA, USA Houston, TX, USA Port Arthur, TX, USA Corpus Christi, TX, USA Freeport, Bahamas Pascagoula, LA, USA Mobile, AL, USA Galveston, TX, USA

12 12 10 12 13 12 10 16 10 10 12 10 11 14 12 16 12 8 10 13 8 12 13 11 14 16 13 12 10 10

2950 ± 443 3775 ± 0 6071 ± 453 3519 ± 0 6005 ± 465 3867 ± 317 6825 ± 2296 2293 ± 6 9336 ± 73 3375 ± 068 2583 ± 6 8721 ± 98 6204 ± 563 2741 ± 344 2749 ± 100 1311 ± 354 7537 ± 2160 3508 ± 1556 5510 ± 33 6005 ± 465 4011 ± 1533 2893 ± 251 3538 ± 854 2603 ± 3 2604 ± 6 2268 ± 111 7923 ± 657 2686 ± 28 4552 ± 1753 3789 ± 0

2 2 3 3 6 2 3 3 3 3 3 3 5 2 2 1 3 2 2 4 3 3 2 3 3 2 3 2 2 3

9. Physicochemical parameters The physicochemical parameters for ballast water and reference sites are shown in Table 3. There was a significant negative correlation between salinity and nitrate levels (r = –0.73; n = 32; p < 0.05). There were also significant correlations between dissolved inorganic nitrogen (DIN), salinity (r = –0.66; n = 32; p < 0.05), nitrites (r = 0.59; n = 32; p < 0.05) and nitrates (r = 0.96; n = 32; p < 0.05); phosphates and DIN (r = 0.82; n = 32; p < 0.05); and silicates and both salinity (r = –0.92; n = 32; p < 0.05) and nitrates (r = 0.63; n = 32; p < 0.05). There was also a significant correlation between pH and oxygen concentrations (r = 0.54; n = 32; p < 0.05). Water quality index (WQI) results ranged from 40.80 to 95.70; the highest value (95.2) was found in vessel B27. The samples from the reference sites had a mean of 88.0 (good condition); eight of the vessels had WQI values in this category (80–94, Table 3). There were also 12 vessels with WQI values in regular condition (65–79), five in marginal condition (45–64) and four in poor condition (0–44, vessels B3, B7, B9 and B21). Tankers 7, 9 and 21 were significantly different from all others (ANOVA one-way, F(1,90) = 69.92, p < 0.05). 10. Primary productivity parameters The chlorophyll composition of the ballast water and the reference sites is shown in Table 4. The concentration of pigments ranged between 0.69 and 2.61 mg/m3. The mean value of the phytoplanktonic biomass, expressed as Chl-a (mg/m3), was between 0.29 and 1.11 mg/m3. There were significant differences in the concentrations of Chl-a and pheopigments, depending on the origin of the ballast water. The largest Chl-a and pheopigment concentrations (>0.5 mg/m3) were found in ballast water from rivers and coastal zones, while lower concentrations of Chl-a (0.3 mg/ m3) were found in ballast water from oceanic waters and from the reference sites. The concentrations for pheopigments were significantly lower (1–2 mg/m3) for ballast water coming from oceanic waters and from the two reference sites than those from coastal or riverine waters (Table 4). The relative and absolute concentrations of pheopigments (2.21 mg/m3) were higher than those of the Chl-a (0.95 mg/m3). The TRIX indices of the ballast water and the two reference sites were between 0.32 and 0.84. The values showed that the ballast water had different origins: oligotrophic (80% of the ships), eutro-

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Table 2 Microbiology of ballast water in the 30 tankers sampled at the Cayo Arcas oil loading offshore terminal of the Mexican Oil Company (PEMEX) between 18th June and 6th July, 2005 Tanker code

Water Origin

Total Coliforms

Fecal Coliforms

E. coli 0157

Other E. coli

Enterococcus sp.

Vibrio cholerae

Serratia marcescens

Beggiatoa spp.

Desulfovibrio spp.

Phormidium corallyticum

Sphingomonas spp.

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15 B16 B17 B18 B19 B20 B21 B22 B23 B24 B25 B26 B27 B28 B29 B30

Coastal River River River Oceanic River River Coastal River Coastal River River River River River Coastal River River River Coastal River River Coastal River River Oceanic Oceanic Coastal Coastal Oceanic

0 9 4 9 93 240 93 0 23 23 4 9l 0 93 43 4 9 240 23 20 150 4 15 43 43 0 0 7 4 0

0 4 0 0 0 1 43 0 9 9 0 0 0 0 0 0 0 7 9 0 75 0 0 0 0 0 0 0 0 0

– – – – – – – – – – – – – – – – – – – – + – – – – – – – – –

– + – – – – + – + + – – – – – – – + + – – – – – – – – – – –

– 32 ufc/ml 16 ufc/ml – 8 ufc/ml 32 ufc/ml – – – 74 ufc/ml 33 ufc/ml 3 ufc/ml 66 ufc/ml 5 ufc/ml 3 ufc/ml 24 ufc/ml 9 ufc/ml 22 ufc/ml 35 ufc/ml 53 ufc/ml 163 ufc/ml 35 ufc/ml 154 ufc/ml 33 ufc/ml 57 ufc/ml 5 ufc/ml 28 ufc/ml 30 ufc/ml 21 ufc/ml 31 ufc/ml

– + – – – – – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – + – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – + – – – – – – – – – – – – – – – – – – – – – –

CA1 CA2

Reef Reef

9 9

0 0

– –

– –

33 ufc/ml 11 ufc/ml

– –

– –

– –

– –

– –

– –

0 19

0 0

– –

– –

19 ufc/g 67 ufc/g

– –

– –

– –

– –

– –

– –

Sediments B8 S Coastal CS1 Reef

A positive sign indicates the presence of a given species, while a negative sign indicates its absence. Codes for tankers are the same as in Table 1. ufc = colony producing units.

phic (16%) and mesotrophic waters (4%). The reference sites had values indicating oligotrophic characteristics (Table 4). 11. Redundancy analysis The RDA accounted for 44.3% of the total variance and was highly significant for the first and all four canonical axes (F = 4.19; P = 0.0024; 4999 permutations). The relevant environmental variables retained by the analysis were: ammonium, dissolved oxygen, phosphates, salinity, silicates, temperature, total CO2 and turbidity. The variance inflation factor for all variables ranged from 1.39 (for ammonium) to 10.90 (for salinity). Fig. 2 shows that ammonium, dissolved oxygen, salinity and temperature were negatively associated with total and fecal coliforms, E. coli 0157 and all other E. coli, while temperature and total CO2 were negatively associated with Chl-a, S. marcescens and Enterococcus spp. In contrast, Sphingomonas sp. was positively associated with each of these last two variables. 12. Discussion The results demonstrate that the ballast water discharged near the Cayo Arcas coral reef contained bacteria that have the potential to be pathogenic for humans and coral reefs. Also, regular to poor water quality was found in 70% of the tankers, most of them coming from coastal waters with low salinity, high nutrients and low oxygen concentrations. In contrast, samples obtained from both reference sites in the coral reef did not demonstrate the presence

of pathogenic bacteria beyond total coliforms, and the water quality index of such samples was relatively good (88) in both cases. Thus, the results do not conclusively demonstrate a significant deleterious effect of ballast water discharge upon the Cayo Arcas coral reef. Nevertheless, this reef is very close to the Campeche Canyon, which produces an important and persistent change in the direction of the oceanic currents (S-NW) from the normal Gulf of Mexico pattern (N-SW) (Monreal-Gómez et al., 2004). This increases the risk of contamination of the coral reef with ballast water, due to the reef’s proximity to the PEMEX mooring buoys. Consequently, there is a significant need for microbiological and sedimentological deposition studies of the Cayo Arcas coral reef. Furthermore, it is important to consider the origin of the water in assessing the potential impact of ballast discharges. This impact could be low if the origin of the ballast water is oceanic (depending on its biogeographical origin), low to medium if the origin of the water is coastal and medium to high if the origin of the water is riverine. The viability of the biological component in riverine ballast water is rather low, as demonstrated by the low number of species and individuals recovered (Olvera-Novoa, 2005). The low abundance of phytoplankton (<100 cells/ml) supports this assumption. Consequently, we would expect this viability to be seriously compromised in the ocean waters surrounding the Cayo Arcas coral reef. The total and fecal coliform levels were below both the maximum limit allowed in water for human use (200 MPN/100 ml), as established by Mexican regulations (NOM-127-SSA1, 1994), and the limit established for wastewater discharged in national

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Table 3 Physicochemical parameters of ballast water among the 30 tankers sampled Tanker code

T, °C

Sal, UPS

Cond, lS/cm

pH –

Alk, TCO2, meq/L mmol/L

Turbi, NTU

DO2, mg/L

S.O2, % Amon, lmol/l

Nitrite, lmol/l

Nitrate, lmol/l

lmol/l lmol/l

Silicate, lmol/l

TSS, mg/L

OSS, mg/L

ISS, mg/L

WQI –

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15 B16 B17 B18 B19 B20 B21 B22 B23 B24 B25 B26 B27 B28 B29 B30

30.93 31.20 31.60 32.32 30.33 28.12 29.75 30.47 29.45 31.20 30.57 32.67 31.80 31.05 30.67 30.00 30.80 30.00 31.27 29.87 30.30 30.88 30.18 31.87 31.83 31.52 30.95 31.38 30.37 32.68

28.77 10.40 13.80 8.78 30.37 29.27 0.17 29.48 0.23 32.90 20.13 14.43 9.80 20.33 28.77 35.73 22.20 15.37 14.93 28.27 0.45 23.45 29.08 16.67 20.10 34.25 33.68 29.38 25.30 33.25

32500 14000 16833 12000 34000 32333 667 32800 833 36733 24667 18800 13500 24900 30167 39400 24500 17667 16767 28433 1000 23567 28667 17500 21633 33967 33567 29750 26967 34233

7.31 7.49 7.19 7.69 7.38 7.76 8.20 7.67 8.34 7.84 7.85 7.67 7.57 7.48 7.32 7.91 7.87 7.70 7.60 7.67 8.55 7.48 7.63 7.77 7.61 7.93 8.06 7.99 7.77 7.94

2.45 1.86 2.45 2.03 2.55 2.41 2.92 2.80 2.96 2.47 1.63 1.28 0.86 1.54 1.71 2.23 2.23 2.55 2.52 2.34 2.63 2.16 2.26 2.41 1.97 2.63 2.55 2.29 2.16 2.46

2.68 1.97 2.77 2.11 2.78 2.49 2.94 2.91 2.96 2.53 1.68 1.34 0.90 1.64 1.87 2.27 2.28 2.65 2.64 2.43 2.60 2.30 2.36 2.49 2.07 2.68 2.58 2.34 2.23 2.50

8.47 8.60 14.77 11.73 4.93 2.97 7.87 3.73 6.80 3.40 6.47 5.20 4.50 6.90 6.20 2.33 4.27 9.10 10.77 4.67 16.45 5.47 5.20 4.67 3.43 3.20 2.17 6.75 5.10 4.47

2.18 0.89 1.24 2.25 2.46 2.81 4.22 3.05 3.37 2.91 3.17 2.57 2.23 2.97 2.89 2.15 2.93 3.20 3.40 3.37 4.28 2.82 3.85 3.50 4.94 6.74 6.25 4.65 4.74 5.73

34.29 12.78 18.14 32.58 38.61 42.23 55.79 47.84 44.33 47.01 47.33 38.47 32.11 44.77 45.26 34.67 44.38 46.12 50.03 52.07 57.15 43.18 59.97 52.44 75.36 110.46 101.17 73.97 72.59 95.11

5.21 0.62 0.63 0.68 2.64 0.38 0.49 7.25 0.31 2.84 0.42 2.41 0.42 0.49 13.02 0.21 0.87 0.38 0.52 6.21 0.51 0.30 7.92 0.35 0.59 1.62 0.65 39.55 11.27 10.63

0.74 0.02 25.64 0.23 2.21 0.00 0.05 1.24 0.09 0.05 7.88 3.60 0.04 8.93 1.02 0.04 13.39 30.49 16.13 1.13 0.14 10.60 1.22 9.23 4.56 0.61 0.11 0.84 8.00 0.19

2.99 24.62 93.04 59.71 19.72 9.91 97.39 23.22 98.02 27.25 27.10 19.10 26.06 17.60 8.36 4.21 21.04 91.66 94.24 46.12 98.80 29.26 18.03 88.34 23.66 19.41 8.37 4.53 13.57 3.87

8.94 1.74 25.26 0.92 119.31 12.58 60.63 1.68 24.58 1.13 10.28 0.60 97.93 4.51 31.72 2.05 98.42 3.28 30.15 0.91 35.40 1.36 25.12 1.12 26.53 1.02 27.01 1.14 22.40 2.56 4.47 0.26 35.30 1.94 122.54 10.68 110.89 14.39 53.46 1.80 99.45 4.17 40.16 2.01 27.16 1.56 97.92 13.05 28.81 1.20 21.64 1.54 9.13 0.34 44.93 1.34 32.84 0.57 14.69 0.00

28.28 124.60 97.34 124.90 30.49 37.94 123.05 45.19 119.45 30.85 64.18 91.42 133.52 55.03 24.09 8.13 65.62 70.39 86.08 28.19 132.02 83.11 24.50 86.52 77.91 56.16 18.07 28.92 49.27 26.18

8.87 4.67 7.40 5.87 5.20 4.53 3.47 3.93 3.33 6.67 8.40 10.00 6.20 5.47 5.40 4.13 5.47 6.73 3.67 1.60 6.50 1.80 2.07 3.20 4.27 1.13 1.53 2.10 4.87 1.13

2.73 2.27 3.07 2.40 2.80 2.73 1.60 2.53 1.87 2.67 2.07 2.07 3.67 1.27 2.53 1.80 1.87 2.07 1.13 0.67 1.10 0.33 0.27 0.67 1.27 0.73 0.93 0.80 2.80 0.93

6.13 2.40 4.33 3.47 2.40 1.80 1.87 1.40 1.47 4.00 6.33 7.93 2.53 4.20 2.87 2.33 3.60 4.67 2.53 0.93 5.40 1.47 1.80 2.53 3.00 0.40 0.60 1.30 2.07 0.20

83.4 68.1 46.5 61.6 73.5 75.6 37.3 77.7 42.0 81.0 78.4 74.5 67.8 76.2 82.9 87.6 71.9 47.8 58.3 82.5 40.8 71.8 85.0 64.3 82.8 89.5 95.7 79.4 79.2 90.6

R1 R2

29.05 36.20 40000 29.00 36.20 40000

8.02 2.57 8.10 2.59

2.60 2.61

2.00 1.80

3.72 3.85

59.16 8.96 61.26 11.53

0.00 0.00

3.32 2.17

26.73 34.77

47.20 2.20

10.60 1.00

DIN,

12.28 13.70

Phos,

0.00 0.00

36.60 88.0 1.20 88.0

T, temperature; Sal, salinity; Cond, conductivity; Alk, alkalinity; TCO2, total carbon dioxide; Turbi, turbidity; DO2, dissolved oxygen; S.O2, oxygen saturation; Amon, ammonium; DIN, dissolved inorganic nitrogen; Phos, phosphates; TSS, total suspended solids; ISS, inorganic suspended solids; OSS, organic suspended solids and WQI, water quality index. Codes for tankers are the same as in Table 1.

Mexican waters (1000–2000 MPN/100 ml on average, NOM-001ECOL, 1996). However, Enterococcus spp. was present in 25 of the 30 tankers sampled, and five tankers exceeded the limits established by the US EPA (>35 cfu/100 ml). Furthermore, the two reference sites also presented Enterococcus with values similar to those observed in ballast water. It was observed that ballast water of riverine origin had higher coliform values when compared with waters of coastal and oceanic origin, in addition to being the only source in which E. coli 0157 was detected. This could be mainly due to the sensitivity of coliform bacteria to salinity, depth, sunlight and other oceanic environmental factors. Alternatively, urban settlements in coastal and riverine zones may promote the presence of these bacteria, mainly due to inadequate sewage disposal (Griffin et al., 1999; Lipp et al., 2002). The high concentration of total coliforms (>200 MPN/100 ml in some tankers, Table 2) in the ballast water is not only the reflection of fecal material but also of a high quantity of particulated material derived from continental soil or plant decomposition. Thus, although the sampled tankers had flags from 13 countries, 93% of them reported the loading of ballast water from the coastal and riverine water of the United States. In the USA, there are clearly facilities present for sewage treatment. Still, the presence of both total and fecal coliforms suggests that these treatment systems are not sufficient to kill all possible pathogens. In the cases of E. coli 0157, V. cholerae 01 and Enterococcus spp., it was evident that the ballast water from which they were isolated had a high concentration of organic material coming from human settlements. The origin of the tanker containing V. cholerae was Baytown, TX. However, even with the information provided on the A20/RES.868 form, it can be difficult to track the geographical

origin of the ballast water as ballast water exchange can be made at different ports. Thus, we are faced with two important problems: the need for a rapid, reliable and cheap way to diagnose the presence of pathogenic organisms in ballast water, and the need for a more efficient way to track the origin of the water in each ballast tank. This is an extremely important public health issue, since the cholerae outbreak in Mexico from 1991 to 1992 started in ballast water (McCarthy and Khambaty, 1994; Whitby et al., 1999). Of the bacteria related to coral diseases, S. marcescens and Sphingomonas spp. are associated with white pox and the white plague type II, respectively (Bythell et al., 2004; Sutherland and Ritchie, 2004). Even though the prevalence of both bacteria was low (3% each), it is possible that their presence in ballast water poses a potential risk for the Cayo Arcas coral reef. This is especially important if we take into account the large number of tankers arriving at the oil loading facilities throughout the year. In the only published record about coral diseases in Cayo Arcas, JordánDahlgren and Rodríguez-Martínez (2004) found three diseases: black band, yellow band and thin dark line. Though they did not find white pox or white plague type II, the conditions for the arrival and establishment of these diseases are clearly present. The low prevalence of S. marcescens and Sphingomonas spp. could be due to the fact that it is far more difficult to detect these bacteria in water than in the mucus covering coral reefs. This phenomenon has been shown for Enteroccocus and fecal coliforms in Floridian coral reefs (Lipp et al., 2002; Patterson et al., 2002). Further support for our interpretation of the link between ballast water and coral reef diseases comes from other published records (Richardson, 1998; Rosenberg and Loya, 2004 and works therein). In addition,

M.L. Aguirre-Macedo et al. / Marine Pollution Bulletin 56 (2008) 1570–1577 Table 4 Trophic status and chlorophyll concentrations (mg/m3) of ballast water for the 30 tankers arriving at the PEMEX crude oil loading facilities between 18th June and 6th July, 2005, and two reference sites at Cayo Arcas coral reef Tanker code

Water origin

Chl-a

Chl-b

Chl-c

Car

Trix

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15 B16 B16 B18 B19 B20 B21 B22 B23 B24 B25 B26 B27 B28 B29 B30

Coastal River River River Oceanic River River Coastal River Coastal River River River River River Coastal River River River Coastal River River Coastal River River Oceanic Oceanic Coastal Coastal Oceanic

1.23 0.61 1.78 1.03 0.38 0.34 1.51 1.68 1.85 0.71 0.90 0.97 1.24 0.52 1.15 0.33 0.55 1.65 1.65 0.80 1.28 1.16 1.05 1.57 0.43 0.24 0.23 0.94 0.75 0.11

0.23 0.28 0.16 0.31 0.26 0.27 0.24 0.13 0.13 0.08 0.03 0.08 0.14 0.08 0.21 0.15 0.23 0.39 0.23 0.18 0.21 0.08 0.09 0.06 0.25 0.08 0.20 0.18 0.11 0.21

1.41 1.00 1.71 1.01 0.74 0.80 1.58 1.75 1.50 0.81 0.73 1.07 1.24 0.55 0.73 0.51 0.74 2.18 1.26 0.99 1.61 1.14 1.04 1.56 0.76 0.37 0.66 0.63 0.63 0.38

0.19 0.08 0.12 0.10 0.08 0.08 0.10 0.19 0.17 0.11 0.11 0.16 0.10 0.09 0.16 0.06 0.09 0.14 0.12 0.12 0.08 0.12 0.12 0.14 0.08 0.06 0.03 0.14 0.07 0.03

2.43 1.77 7.31 3.17 0.71 0.54 5.85 3.37 6.19 1.26 1.60 2.12 2.57 1.35 2.75 0.48 1.72 7.75 7.96 1.84 5.66 2.80 1.96 5.73 1.24 0.42 0.34 2.18 1.64 0.19

R1 R2

Reef Reef

0.34 0.24

0.24 0.08

0.46 0.37

0.07 0.10

0.84 0.32

Codes for tankers are the same as in Table 1.

Fig. 2. Redundancy analysis of the bacteria (presence/absence data) and total and fecal coliforms (MPN/100 ml) of ballast water of the 30 vessels sampled at the Mexican Oil Company’s (PEMEX) Cayo Arcas oil loading terminal between 18th June and 6th July, 2005. Abbreviations for physicochemical variables are as follows: AMON, ammonium; CO2TOT, total carbon dioxide; O2DIS, dissolved oxygen; PHOSPHA, phosphates; TEMP, temperature; TURBI, turbidity; SAL, salinity and SILI, silicates. Codes for biological variables (those in bold) are as follows: Chl-a, chlorophyll-a; Enteroco, Enterococcus spp.; EColi015, Escherichia coli 0157; FecColif, fecal coliforms; OtherEco, other E. coli species; Serratia, Serratia marcescens; Sphingom, Sphingomona spp.; TotColif, total coliforms and Vibrioch, Vibrio cholerae 01. The numbers associated with triangles are the tankers’ reference numbers in Table 1.

several bacterial species important to human public health are associated with coral diseases. This is the case with both black band disease and white plague type II (Kaczmarsky et al., 2005). The results show that most tankers filled their ballast tanks in rivers and coastal zones, as indicated by the low salinity and

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high turbidity values in Table 3. The nitrite concentrations in the ballast water were far higher than those at the reference sites, and the values found for B18 (30.49 lmol/l, Table 3) are considered toxic for aquatic organisms when compared to the normal range for that area (Boyd, 1990). The negative correlation between nitrate and silicates with respect to salinity indicates that this water had brackish and freshwater origins. Also, the positive correlations among turbidity, ammonium, nitrate and dissolved inorganic nitrogen (DIN) indicate that ballast water is enriching the waters surrounding the mooring buoys of PEMEX with these nutrients. The significant positive correlation between phosphates and DIN suggest that both these components had the same origin: coastal waters with high concentrations of nutrients. In the case of the total suspended solids (TSS) (organic and inorganic fractions) in ballast water, none of the tankers was above the limits considered normal for marine biota (25 mg/l, Boyd, 1990). A high rate of deposition of TSS is considered harmful for aquatic biota, since TSS can bury sedentary organisms such as corals and interfere with their feeding rates. In the case of turbidity, reference sites 1 and 2 and the ballast water had values of 1.04, 1.6 and 6.2 ± 3.7 NTU (for all 30 tankers), respectively. The range of values for turbidity in ballast water was 1.6–20.5 NTU for all 30 tankers (Table 3), a range that can be considered low, especially for the reference sites. The WQI values confirmed that water with low salinity showed the worst quality. The water from tankers B3, B7, B9 and B21 had the lowest WQI values, a product of low salinity, high turbidity and high nitrite concentrations. All the physicochemical parameters of the water samples from the reference sampling sites in the coral reef indicated normal oceanic water conditions, as did the 88% water quality index value (Table 3). It is possible to suggest that the ballast water discharged by the tankers is an important source of nutrients for the Cayo Arcas coral reef area. However, the most important question is whether or not the sediments can reach the reef. Because water currents were not measured during the sampling dates, such a question cannot be answered conclusively. Consequently, it will be necessary to undertake further oceanic current studies on a local scale (the mooring buoy and coral reef zone), as well as TSS deposition studies in the coral reef area. The phytoplankton biomass, expressed as Chl-a in ballast water, corresponded to an environment considered as oligotrophic (low productivity). There was also large heterogeneity among tankers (Table 4), which could be associated with factors such as the residence time of the ballast water in the tanks, light availability, nutrients and feeding rate. The relative and absolute concentration of pheopigments was larger than that of the chlorophylls, suggesting a high feeding rate in the tanks and low light availability. These conditions, in turn, would lead to a poor physiological status of phytoplankton cells. Similar conditions have been reported by Drake et al. (2002) with respect to phytoplankton in ballast water tanks. The eutrophic condition of the ballast waters and their Chl-a concentration were similar to that found at the reference sites. Because of this similarity, there is a fairly low risk that these water discharges would have a negative effect on the Cayo Arcas coral reef. However, this interpretation should be taken with caution, since the sampling design did not include temporal data or a large enough spatial coverage to be conclusive. Still, most of the tanks with riverine and coastal water were eutrophicated. This in turn, enhances the potential for a change of environmental conditions (oligotrophic to eutrophic) in the coral reef area. Consequently, the total potential impact of the discharge of ballast water from different origins would range from an increase in the Chl-a concentration to changes in the eutrophic conditions of the coral reef. The

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M.L. Aguirre-Macedo et al. / Marine Pollution Bulletin 56 (2008) 1570–1577

composition of phytoplankton species in ballast water was also rather different (Olvera-Novoa, 2005) from that of the reference sites in the reef. Thus, it is possible that some species would be able to invade this new habitat. The RDA suggested that the salinity at the discharge site could kill most of the E. coli present in the ballast water (Fig. 2) as demonstrated in experimental studies (Carlucci and Pramer, 1960; Anderson et al., 1979). Therefore, a short time after the discharge of ballast water, it would be expected that most total and fecal coliforms, as well as most E. coli, would die. These results suggest that, at least for these bacteria, it would not be necessary to apply expensive water treatments to ballast water before its release, as suggested elsewhere (Zhang and Dickman, 1999; Waite et al., 2003; Bai et al., 2005). In contrast, both S. marcescens and Sphingomona spp. had nearly no association with salinity, but were associated with temperature and total CO2. Thus, these bacteria would likely be able to survive in marine conditions, and the limiting factor in their survival would be related to the time they remain in the ballast water tanks. In this way, our findings agree with those of Drake et al. (2002), who found that bacteria are able to survive in ballast water tanks for up to five days. Clearly, this information provides additional evidence for the risk associated to the ballast water discharge near the reef. In light of these results, it is essential to establish a permanent monitoring program with respect to white pox and white plague and their possible association with ballast water discharges. However, it is also important to take into account that white pox is one of the most frequent and widely distributed coral reef diseases in the Caribbean (Weil, 2004). Consequently, any such program must determine the origin of the bacteria producing the disease, perhaps by utilizing radioactive isotopes for tracing. Furthermore, it would be desirable to establish a compulsory open-ocean exchange of ballast water and a preventative ballast discharge in oceanic water before arrival at the offshore crude oil loading terminal at Cayo Arcas. However, we should also consider that oceanic species represent a major risk of invasion due their affinity for the environmental conditions of the Cayo Arcas reef. In Mexico, there is no knowledge of the extent to which microorganisms arriving from oceanic (or coastal) water through ballast represent a risk of invasion (and impact) at the site of discharge. This is probably not because they are poor invaders but rather because detailed baseline studies are lacking. Therefore, it is urgent to establish baseline field studies to identify such a risk. This is important if we consider that, for the present paper, the capacity of each tanker was between 19,778 and 56,163 m3, for a total of 1,256,570.6 m3 of ballast water transported in the 30 sampled oil tankers during this 15-day study. Thus, even when microbial viability decreases through the tanker’s voyage, as observed for coastal microbes, the sheer volume of ballast exchanged may lead to the introduction of new species, as has been reported for oceanic species during the open-ocean exchange of ballast water (Ruiz et al., 2000a; Drake et al., 2002). To evaluate the risk associated with ballast water discharge, it is necessary to carry out studies on the potential invasion of bacteria as well as the zoo- and phyto- plankton communities. Also, it is necessary to consider the effect of distance from the ballast water discharge point and the dilution of that water as a potential source of coral reef disease, especially for the elkhorn coral, Acropora palmata, and the Montastrea species complex (see Rosenberg and Loya, 2004 and references therein). In conclusion, to care for the health of the Cayo Arcas reef, it would be advisable to establish a long-term follow-up program using bioindicators such as the phyto- and zoo- plankton community structure, Chl-a/pigments ratio, diatoms/dinoflagellates ratio, the presence of red tide species and the association between physicochemical parameters and phytoplankton.

Acknowledgments Special thanks to Dr. Uriel Ordoñez-López, Francisco Puc-Itza, Jorge A. Dominguez-Maldonado, Gregory Arjona Torres and José de la Cruz Cámara-Ramos for sampling and field work; Jorge Trejo, Ileana Osorio-Moreno, Elizabeth Real-De-León, Geraldine GarcíaUribe, Jorge Güemez-Ricalde, and Monica Leal-Corona for their help with laboratory analysis; and Dr. Mark E. Torchin from Smithsonian Tropical Research Institute, Panama, and Dr. Gregory M. Ruiz from the Smithsonian Institution, USA, for their comments on an early draft of the manuscript. This study was financed by PEMEX-GSIPAC-RMNE (Contract No. 412005814, ‘‘Caracterización de Agua de Lastre, Cayo Arcas, 2005). References Anderson, I.C., Rhodes, M.W., Kator, H.I., 1979. Sublethal stress in Escherichia coli: a function of salinity. Applied and Environmental Microbiology 38, 1147–1152. Bai, X.Y., Zhang, Z.T., Bai, M.D., Yang, B., Bai, M.B., 2005. 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Ballast water as a vector of coral pathogens in the Gulf ...

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Common dolphin Photo-ID Volunteer Position in the Hauraki Gulf ...
The volunteer position entails assisting during field surveys and analysis of common ... Subsequent analysis of photo-identification data in the lab, including ...