Marine Pollution Bulletin 90 (2015) 115–120

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Responses of estuarine nematodes to an increase in nutrient supply: An in situ continuous addition experiment R.C. Ferreira a, A.B. Nascimento-Junior b, P.J.P. Santos b,c, M.L. Botter-Carvalho d, T.K. Pinto a,e,⇑ a

Programa de Pós-graduação em Diversidade Biológica e Conservação nos Trópicos, UFAL, Maceió, AL, Brazil Programa de Pós-Graduação em Biologia Animal, UFPE, Recife, PE, Brazil c Departamento de Zoologia, UFPE, Recife, PE, Brazil d Universidade Federal Rural de Pernambuco, Av. Dom Manoel de Medeiros s/n, Dois Irmãos, 52171-900 Recife, PE, Brazil e Unidade de Ensino Penedo/Campus Arapiraca, UFAL, Penedo, AL, Brazil b

a r t i c l e

i n f o

Article history: Available online 8 December 2014 Keywords: Meiofauna Mangrove Inorganic enrichment Fertilizers Sugar cane

a b s t r a c t An experiment was carried out on an estuarine mudflat to assess impacts of inorganic nutrients used to fertilize sugar-cane fields on the surrounding aquatic ecosystem, through changes in the nematode community structure. During 118 days, nine quadrats each 4 m2 were sampled six times after the beginning of fertilizer addition. The fertilizer was introduced weekly in six areas, at two different concentrations (low and high doses), and three areas were used as control. The introduction of nutrients modified key nematode community descriptors. In general, the nematodes were negatively affected over the study period. However, Comesa, Metachromadora, Metalinhomoeus, Spirinia and Terschellingia were considered tolerant, and other genera showed different degrees of sensitivity. Nutrient input also affect the availability and quality of food, changing the nematode trophic structure. The use of inorganic fertilizer should be evaluated with care because of the potential for damage to biological communities of coastal aquatic systems. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Eutrophication is one of the most severe and widespread forms of anthropogenic impacts on aquatic environments (Ristou et al., 2012). The occurrence of hypoxia/anoxia as a result of eutrophication in coastal and estuarine areas is increasing worldwide, mainly as a result of human activities, such as the drainage of agricultural products into waterways and the release of industrial wastewater (Wolanski, 2007; Wu, 2002). These activities are the main sources of input of both organic and inorganic nitrogen and phosphorus (Gaudes et al., 2013), the main compounds related to eutrophication processes, into marine environments (GESAMP, 1990). Inorganic fertilizers used on sugar-cane fields are composed primarily of these two elements. It is assumed that slightly over 50% of fertilizers used in agriculture is consumed by plant biomass, with the remainder carried into rivers and estuaries (McLusky and Elliott, 2004). The limited availability of oxygen (hypoxia/anoxia) arising from eutrophication has been associated, in estuarine and marine areas, with changes in these systems that can lead to the ⇑ Corresponding author at: Av. Beira Rio, s/n, Centro, Penedo, AL CEP 57200-000, Brazil. Tel.: +55 82 98284774. E-mail address: [email protected] (T.K. Pinto). http://dx.doi.org/10.1016/j.marpolbul.2014.11.012 0025-326X/Ó 2014 Elsevier Ltd. All rights reserved.

decline of different biotic compartments, such as benthic communities (Modig and Olafsson, 1998; Rosenberg et al., 2002). Among benthic organisms, the potential usefulness of nematodes for evaluating disturbances caused by humans is related to a number of characteristics of this taxon, such as close association with sediment, high abundances, low mobility, low dispersal ability and a short lifespan, resulting in a high capacity to reflect local events (Coull and Chandler, 1992; Souza et al., 2004). This indicator potential of nematodes is associated with the fact that sediments act as deposits for contaminants, and are strongly affected by the input of nutrients of allochthonous origin. Several studies have examined the response of nematode communities to conditions of hypoxia/anoxia induced in different ways, such as: controlling the oxygen level in the water (Steyaert et al., 2007; Modig and Olafsson, 1998); covering the sediment to exclude oxygen (Van Colen et al., 2009); adding contaminants such as heavy metals and organic carbon (Gyedu-Ababio and Baird, 2006); adding sucrose and organic matter (Gambi et al., 2009); and adding marine algae (Ascophyllum nodosum) (Schratzberger and Warwick, 1998). Most of these studies were conducted in temperate regions, with relatively few in tropical regions. In Cuba, Armenteros et al. (2010) induced a state of hypoxia/anoxia through the addition of Spirulina microalgae powder.

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Understanding the problems caused by human intervention in aquatic environments is essential in order to develop measures to mitigate these impacts. The present study evaluated, through a 118-day in situ experiment, the response of nematode genera to induced hypoxia/anoxia, through continuous exposure to different concentrations of inorganic fertilizer used on sugar-cane fields. 2. Materials and methods 2.1. Study area The study area is located in the coastal estuarine complex of the Ilha de Itamaracá region (7°46.184’S and 34°52.926’W), on the north coast of the state of Pernambuco, approximately 50 km from the city of Recife (Fig. 1). This part of the coast is distinguished by high primary productivity and faunal diversity (CPRH, 2001). The tidal flat area where the experiment was conducted is about 300 m from the waterline during average low water spring tides. 2.2. Experimental design and treatments The experiment was conducted in nine quadrats of 4 m2, 3 m apart. Six of these areas were enriched weekly with inorganic fertilizer (NPK TREVO: 20% nitrogen, as ammonium sulfate; 10% phosphorus, as water-soluble P2O5; and 20% potassium, as watersoluble K2O). Three of these areas received 750 g/4 m2 (low dose, LD) and three were treated with 1500 g/4 m2 (high dose, HD). The remaining three areas were not enriched and were considered as control (C) areas. Granulated fertilizer was applied directly on the sediment surface. The amounts of fertilizer were calculated to increase nutrient concentrations in the sediment, disregarding solubility in overlying water, by 12.5% and 25% weekly at the low dose and 25% and 50% weekly at the high dose for N and P respectively, compared to the concentrations analyzed before the experiment. The samples were collected at low tides on six occasions: day 0 (05/10/2005) when fertilizers were first added, day 26 (31/10/2005), day 43 (17/11/ 2005), day 71 (15/12/2005), day 91 (04/01/2006) and day 118 (31/01/2006).

A detailed description of the area and the environmental parameters measured during the experiment is available in the studies of Santos et al. (2009) and Botter-Carvalho et al. (2014). They reported that the sediment conditions before the experiment were similar in the three treatment areas, with fine-sediment percentage ranging from 16.9% to 31.3%, organic matter-content 2.04% to 6.65% and oxidation–reduction potentials below 84 mV. During the experiment, some differences were noted: the HD treatment showed the highest total-nitrogen concentration (1.8 mg/kg), organic-matter content (6.8%), and chlorophyll-a concentration (20 lg/cm2), and the lowest oxidation–reduction potential (359 mV). The total-phosphorus concentrations increased over time and between treatments, and Dunnett’s test revealed differences between the control and HD treatments on days 71 (p = 0.017), 91 (p = 0.010) and 118 (p = 0.010). 2.3. Nematofauna To assess the nematode fauna, a sample was taken in each treatment area (LD, HD and C) with the aid of a corer with inner area of 6.15 cm2. The samples were fixed in the field with 4% saline formalin. The samples were processed according to Elmgren (1976), involving wet washing in a geological sieve with apertures of 0.5 mm and 0.063 mm. The first 60 nematodes (from the sample) were removed for mounting on permanent slides and identification at the genus level (Warwick et al., 1998; Deprez, 2005). Nematodes were classified in trophic groups according to Wieser (1953). 2.4. Data analysis The STATISTICA v.10 statistical program was used to compare richness, diversity and number of genera between days and treatments, with analysis of variance (multifactorial ANOVA) and post hoc Tukey test for pairwise comparison. To assess the multivariate responses in the structure of the nematode community in relation to treatment and days, a multidimensional scaling (MDS) analysis was used to represent the similarity matrix, based on the Bray-Curtis index applied to genus densities. To test for significant

Fig. 1. Location of study area, Ilha de Itamaracá and Canal de Santa Cruz, Pernambuco, Brazil (adapted from Botter-Carvalho et al., 2014).

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differences between these factors, PERMANOVA (Permutational Multivariate Analysis of Variance) was applied to the same data. The Primer 6.1.15 Permanova + 1.0.5 statistical package was used.

which had the lowest densities (Fig. 3). In the control area, the densities remained high until the last day, although the dominant genera changed from the early days of the experiment, when Spirinia was the most numerous, to the end, when Terschellingia became dominant (Fig. 3(a)). In the LD treatment area, dominance also changed between these genera on the last two days of the experiment (Fig. 3(b)). In the HD treatment area, these two genera, which were co-dominant at the beginning of the experiment, virtually disappeared by the end (Fig. 3(c)). The multidimensional scaling (MDS) analysis of the data indicated a tendency to form groups of samples; on most days the samples from the HD treatment area were grouped together and separated from other treatments, with the exception of days 0 and 26 (Fig. 4). PERMANOVA analysis of the data for the density of nematode genera indicated that the variability was statistically significant, with differences for the separate factors and the interaction between them (F = 3.28; p < 0.001) (Table 1). On day 0 the treatments did not differ significantly, while significant differences were observed between control  HD and LD  HD areas from day 26 onward. From day 91, the LD and control areas also differed significantly. On the last day of the experiment, day 118, no significant differences were found between the HD and LD areas, although there was considerable dissimilarity between the samples from these treatments (>83%) (Table 2). The trophic structure of the nematode community was characterized by selective deposit feeders (1B), which were the most abundant trophic group, followed by non-selective deposit feeders (1A), epistrate feeders (2A) and predators and omnivores (2B), in that order. The trophic composition was similar in the LD and C areas during the experiment; however on days 91 and 118 the abundance of group 2B gradually increased (Fig. 5(a) and (b)). In the HD treatment, the abundance of group 1A decreased and the abundance of group 2B decreased on days 71 and 91 (Fig. 5(c)).

3. Results

20

Nutrient enrichment can change the dynamics of populations and consequently the structure of communities (Gaudes et al., 2013). The data obtained in this study indicated that the introduction of nutrients led to changes in key community descriptors, negatively affecting nematodes through decreases in density, diversity and genus richness over 118 days. The medium dose of 2.50

(a)

15 10 5 0

4. Discussion

Diversity (H')

Number of Genera

The nematode community was represented by 36 genera belonging to 19 families. Of these, Xyalidae was recorded only in the control area, and Anoplostomatidae, Microlaimidae and Neotonchidae were recorded only in the control and LD areas. Sphaerolaimidae was recorded only in the LD treatment area. No family was exclusive to the HD treatment area, which contained the fewest families (14). The lowest number of genera was recorded in the HD treatment area at the end of the experiment, with only six genera recorded on day 118. The number of genera did not change in the LD treatment area, with 18 genera recorded on each day. In the control area, this number ranged from 21 (day 0) to 17 (day 118) (Fig. 2(a)). Terschellingia, Spirinia, Pesudochromadora and Comesa were the most abundant genera in the experiment. Marylynnia, Daptonema and Polygastrophora were recorded only in the control area, while Sphaerolaimus, Prochromadorella and Eurystomina were found exclusively in the LD treatment area, and Rhips exclusively in the HD treatment area. Chromadorina and Endeolophos were found in both enriched-treatment areas. The highest values for diversity were found in the control area, followed by the LD and HD treatment areas in descending order. The highest and lowest equitability values were recorded in the HD treatment and the control area, respectively (Fig. 2(b) and (c)). Despite these trends, the variance analysis applied to the univariate index values revealed a significant difference only for the number of genera (F = 15.08, p = 0.004). The Tukey’s test for comparison of means showed that these differences were significant both for treatment areas at day 118, where the HD treatment differed from both the LD and C treatments (p = 0.04 and p = 0.007, respectively), and for days in the same treatment, where the number of genera of HD on day 26 differed significantly from the number of genera on day 118 (p = 0.007) (Fig. 3). The density values of nematode genera in the replicates ranged from 1.31 to 5516 ind.10 cm2, with the highest densities found in the control area, followed by the LD and HD treatment areas in descending order. The densities decreased over the duration of the experiment in the enriched-treatment areas, especially in HD,

0

26

43

71

91

2.00 1.50 1.00 0.50 0.00

118

(b)

0

26

Days

Eveness (J')

2.00

43

71

91

118

Days C

(c)

LD

HD

1.50 1.00 0.50 0.00

0

26

43

71

91

118

Days Fig. 2. Mean values of the univariate indices calculated: number of genera (a), Shannon’s diversity (loge) (b) and Pielou’s evenness (c), in control (C), low-dose (LD) and highdose (HD) treatment areas on all days of experiment.

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ind.10cm-2

5000

6000

(a)

5000

ind.10cm-2

6000 4000 3000 2000 1000

(b)

4000 3000 2000 1000

0

0

26

43

71

91

0

118

0

26

Terschellingia

ind.10cm-2

5000 4000

43

71

91

118

Days

Days Spirinia

Comesa

Metachromodora

Pseudochromadora

(c)

3000 2000 1000 0

0

26

43

71

91

118

Days Fig. 3. Mean densities (ind.10 cm2) of the most numerous nematode genera during the experimental sampling days (day 0, 26, 43, 71, 91 and 118). (a) Control (C); (b) Low dose (LD); (c) High dose (HD).

2D Stress: 0,05

0

118

118 0 0

43 118 26 26 43 71

43

91

71 91

26

71

91

Fig. 4. Multidimensional scaling (MDS) applied to untransformed density data for nematode genera, in the three treatments ( = C 4 = LD j = HD) over the experimental sampling days (day 0, 26, 43, 71, 91 and 118).

Table 2 PERMANOVA p-values for pairwise comparisons (C = control, LD = Low Dose, HD = High dose). Day

Dose

Mean dissimilarity

t

p

0

C-LD LD-HD C-HD

65.65 43.26 62.20

1.17 0.75 1.42

0.29 0.65 0.15

26

C-LD LD-HD C-HD

36.56 72.36 76.58

1.23 3.15 4.03

0.23 0.008 0.003

43

C-LD LD-HD C-HD

55.23 59.64 58.22

0.85 0.85 1.23

0.53 0.50 0.25

71

C-LD LD-HD C-HD

36.92 90.21 93.24

1.55 4.21 55.96

0.11 0.001 0.000

67.79 80.46 91

3.31 3.87 C-LD

0.008 0.004 0.003

98.40

LD-HD C-HD 2.03

0.04 0.26 0.03

91

118 Table 1 Results of Permutational Multivariate Analysis of Variance (PERMANOVA) applied to untransformed data for density of nematode genera.

Day Dose Day  Dose

F

p

5.36 10.15 3.28

0.0001 0.0001 0.0001

LD nutrients used in the treatment appears to have had a similar effect to the classical considerations of Pearson and Rosenberg (1978) in relation to the response of benthic organisms to input of organic material, where intermediate levels of disturbance at the beginning of the experiment seem to benefit the biota, leading to maximum density and diversity. The effects of the introduction of fertilizers were perceptible not only through univariate descriptors, but also through multivariate aspects of the community, as indicated by significant PERMANOVA results and the trends found by multidimensional scaling analysis, through which changes in the composition, density and genus richness in relation to the introduction of inorganic fertilizers were evident.

C-LD LD-HD C-HD

The degree of tolerance to disturbance varies among species (Warwick, 1988) and the responses of benthic organisms to impacts are very situation-specific, as they do not depend only on the type of impact and the characteristics of the taxa. Responses to disturbances are also associated with the characteristics of the environment affected and the ‘‘pool’’ of local species and their different ecological interactions. The nematode genera recorded in the present study responded differently to the introduction of fertilizer. Some genera, such as Comesa, Metachromadora, Metalinhomoeus, Spirinia and Terschellingia, were tolerant, occurring in all treatments and throughout the experiment. Others such as Halichoanolaimus, Ptycholaimellus, Sabatieria, Thalassomonhystera, Viscosia and Paracyatholaimus, which disappeared at some point in the experiment in one or in both of the enriched-treatment areas, were sensitive. Although higher densities of some genera, such as Spirinia and Comesa, were recorded at low treatment doses, none of the genera recorded in the experiment seems to have really benefited from the introduction of

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5000

5000

(a) ind.10cm-2

ind.10cm-2

(b)

4000

4000 3000 2000

3000 2000 1000

1000 0

0 0

26

43

71

91

0

118

26

Days 5000

43

71

91

118

Days

(c)

1A

1B

2A

2B

ind.10cm-2

4000 3000 2000 1000 0 0

26

43

71

91

118

Days Fig. 5. Mean density (ind.10 cm2) of trophic groups in treatment areas during the experimental sampling days (day 0, 26, 43, 71, 91 and 118): (a) control (C); (b) Low dose (LD); (c) High dose (HD). (1A = selective deposit feeder, 1B = non-selective deposit feeder, 2A = epistrate feeder, 2B = predator).

fertilizer, regardless of the dose used, as all decreased in density over time. Species of Spirinia are widely recognized as being resistant to environmental changes (Schratzberger et al., 2006; Steyaert et al., 2007), with some species (e.g., Spirinia parasitifera) capable of increasing their density in environments with intermediate concentrations of nutrients (Armenteros et al., 2010). Several authors have reported high densities of the genus Sabatieria in muddy sediments with low oxygen concentrations, and consider them to be tolerant to hypoxic conditions (Wetzel et al., 2002; Lampadariou et al., 1997), resistant to pollution (Bongers, 1990) and to various types of environmental changes (Schratzberger et al., 2006; Steyaert et al., 2007), as was the case with Comesa and Spirinia in the present study. However, in this experiment Sabatieria proved to be sensitive to the dosage used in the HD treatment area, disappearing from day 3 onward. While most studies consider members of Sabatieria to be opportunistic and tolerant to situations of hypoxia, some authors (Armenteros et al., 2010; Steyaert et al., 2007) found that the density of Sabatieria decreased with the reduction of oxygen, with losses of as much as around 40% of their total numbers (Van Colen et al., 2009). Several genera were considered sensitive, but to different degrees; some, such as Halichoanolaimus and Ptycholaimellus, disappeared at both low and high doses, indicating greater sensitivity; whereas others, such as Thalassomonhystera, Paracyatholaimus and Sabatieria, disappeared only at high doses and were tolerant to low doses. Ptycholaimellus and Halichoanolaimus disappeared in the early days of the experiment in the HD treatment area; however, in low nutrient concentrations, they persisted until days 71 and 91, respectively. Species of Halichoanolaimus have been recorded from estuaries ranging from unpolluted to heavily impacted (Essink and Keidel, 1998), where densities vary according to the type of environmental stress, decreasing in conditions of anoxia (Steyaert et al., 2007), and when subjected to different concentrations of nutrients (Schratzberger and Warwick, 1998). In some cases their densities were not altered in the presence of nutrients such as phosphorus and nitrogen originating from marine fish farms (Mirto et al., 2002).

Similar results were observed for Thalassomonhystera in microcosms enriched with different levels of organic matter (low, medium and high doses), where they thrived in medium dosages (Schratzberger and Warwick, 1998). In field studies comparing polluted and unpolluted estuaries, Paracyatholaimus was observed at all sampling stations, and is considered resistant in environments with high concentrations of nutrients (Essink and Keidel, 1998). Terschellingia, Pseudochromadora, Metachromadora and Viscosia were tolerant to low oxygen conditions, with density levels inversely proportional to the fertilizer dose. Similar behavior has been observed in the intertidal zone, where species of Terschellingia and Metachromadora were subjected to induced hypoxia/anoxia in microcosms (Steyaert et al., 2007). Several species of Terschellingia commonly occur in high abundance in estuaries and intertidal zones, and are most abundant in muddy sediments (Warwick and Gee, 1984; Steyaert et al., 2005). Terschellingia is successful in sediments with different oxygen concentrations (oxic/anoxic), showing little or no variation in density when subjected to hypoxia/ anoxia (Steyaert et al., 2007). The success of this genus in environments with low oxygen concentrations may be enabled by the species’ low respiration rates and slow mobility (Warwick and Price, 1979), which reduce oxygen demand. The results for Metachromadora were also expected, since this genus succeeds in environments with a limited oxygen supply (Armenteros et al., 2010). Despite the decrease in densities of these genera in the enriched sediments, the results of this study reinforce the premise that members of both Terschellingia and Metachromadora are highly resistant to hypoxia/anoxia. Species of Metachromadora may be benefited in anoxic environments by the capacity of adults to retain their offspring in their bodies during the juvenile stage, and once released these individuals are more likely to survive in disturbed environments (Steyaert et al., 2007); and also because the genus has, like Terschellingia, a low respiration rate (Warwick and Price, 1979). This study showed that an excess of nutrients in an estuarine environment may cause marked changes in the structure of nematode communities, in general damaging the biota, decreasing

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densities, and leading to the disappearance of various genera. These changes in the composition and density of genera are also associated with changes in the availability of the dominant food resources. The addition of fertilizer most probably altered the availability of food and the trophic structure of the nematode community in both treatment areas, with overgrowth of mainly diatoms and cyanobacteria observed in the LD and HD treatment areas, respectively (ML Botter-Carvalho and PVVC Carvalho, personal observations). Therefore, inducing a condition of hypoxia/anoxia through the input of nutrients affected not only the physiological characteristics and the ability of the animals to tolerate low oxygen availability, but also the availability and quality of food. Using the classification of Wieser (1953) for trophic groups, the genera considered tolerant were classified as epistrate feeders and nonselective deposit feeders. According to Moens and Vincx (1997), who defined most nematode trophic groups based on feeding observations, Terschellingia and Metalinhomoeus are classified as bacteria feeders. Their physiological characteristics, combined with an abundance of preferred food, are responsible for the ability of these genera to tolerate the excessive input of nutrients and the consequent changes in the conditions of an estuarine sedimentary environment. Deposit feeders generally are highly abundant in silt and clay sediments; this is related to the plasticity of their diet, which allows them to consume different food items of various sizes (Tietjen, 1969), a characteristic associated with opportunistic behavior. In the final days of the experiment, the density of predators increased in both enriched-treatment areas, and there was also a large increase in the density of Viscosia (2B), which was classified as a facultative predator by Moens and Vincx (1997), but is frequently considered to be opportunistic (Warwick and Gee, 1984). The results show the effectiveness of using the nematode community in an intertidal zone to detect short-term disturbances caused by inorganic-nutrient enrichment. The nematode community together with other estuarine benthic biota compartments, as found by Botter-Carvalho et al. (2014) for macrofauna using the same experimental design in the same area, was negatively affected by the continuous addition of fertilizers. The nematofauna responded to the different doses of inorganic nutrients in different ways, resulting in a decrease in the complexity of the community structure, due to a loss of diversity and abundance and changes in the proportion and dominance of trophic guilds. This suggests that the use of inorganic fertilizers to maximize crop production should be viewed with caution, and their use closely monitored because of the potential for damage to biological communities and coastal aquatic systems. Acknowledgements The authors wish to express their thanks to the CNPq for financial support (grant no. 477972/2004-7), for a research fellowship to P.J.P. Santos (No. 305609/2004-1). The authors are grateful to Janet W. Reid for English language revision. References Armenteros, M., Pérez-García, J.A., Ruiz-Abierno, A., Díaz-Asencio, L., Helguera, Y., Vincx, M., Decraemer, W., 2010. Effects of organic enrichment on nematode assemblages in a microcosm experiment. Mar. Environ. Res. 70, 374–382. Bongers, T., 1990. The maturity index: an ecological measure of an environmental disturbance based on nematode species composition. Oecologia 83, 14–19. Botter-Carvalho, M.L., Carvalho, P.V.V.C., Valença, A.P.M.C., Santos, P.J.P., 2014. Estuarine macrofauna responses to continuous in situ nutrient addition on a tropical mudflat. Mar. Pollut. Bull. 83 (1), 214–223. Coull, B.C., Chandler, T., 1992. Pollution and meiofauna: field, laboratory, and mesocosm studies. Oceanogr. Mar. Biol. Annu. Rev. 30, 191–271.

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