M Schratzberger
email: .m.schratzberger@cefas.co.uk

K Warr

S I Rogers

Functional diversity of nematode communities in the southwestern North Sea

Keywords: Meiofauna, Free-living nematodes, Community function, Biological traits analysis, North Sea

A range of biological traits of nematode species were combined to identify patterns in the functional composition of their assemblages collected at 19 soft-bottom stations in the southwestern North Sea with the primary aim to determine which environmental variables control communities. We used 19 categories of five biological traits thought or known to represent an important ecological function. These were related to buccal morphology, tail shape, body size, body shape and life history strategy. Data on trait membership was provided by biological information on species and genera. A total of 79 different trait combinations were recorded.

Results from correlation analyses revealed several significant relationships between traits. Some trait combinations were shared by different species and genera, and the ratio of realised versus total number of possible trait combinations of < 1 suggested that some trait combinations were not represented by the nematode fauna from this region. The functional composition of nematodes was strongly linked to median particle diameter and silt content of the sediment and water depth. The approach adopted and our attempts at defining and analysing functional attributes of nematode communities raised a number of conceptual and methodological issues which are discussed.

Introduction

Sedimentary habitats cover most of the ocean bottom and therefore constitute the largest single ecosystem on earth in spatial coverage. Benthic organisms which occupy these habitats make a significant contribution to the regulation of carbon, nitrogen and sulphur cycling, water column processes, pollutant distribution and fate, secondary production and transport and stability of sediments [START_REF] Snelgrove | The importance of marine sediment biodiversity in ecosystem processes[END_REF]. Four of every five bottom-living multicellular animals on earth are nematodes [START_REF] Bongers | Nematode community structure as a bioindicator in environmental monitoring[END_REF]. Whereas the importance of parasitic nematodes has been recognised for many decades, this is not the case for the free-living species, especially those of aquatic environments. They remain poorly understood, despite the fact that they are extremely abundant and diverse, often numbering millions per square metre in sediments, and occur in more habitats than any other metazoan group [START_REF] Heip | The ecology of marine nematodes[END_REF].

The traditional methods of nematode community analyses, deriving diversity and community structure from species abundance data, do not take account of the diverse biology and autecological requirements of the taxa. Natural-history information on nematode species is scant, so studies which require information on nematode ecology have used a functional group approach [START_REF] Thistle | The nematode fauna of a deep-sea site exposed to strong near-bottom currents[END_REF]. Species in functional groups share morphological traits that are thought or known to represent an important ecological function [START_REF] Chalcraft | Mapping functional similarity of predators on the basis of trait similarities[END_REF].

Studies on macrobenthic invertebrates have shown that linking taxonomic and functional diversity, i.e. pooling species from different taxonomic entities into functionally similar groups, can reveal different relationships between assemblages (e.g. [START_REF] Bremner | Assessing functional diversity in marine benthic ecosystems: a comparison of approaches[END_REF][START_REF] Boström | Environmental variables explaining structural and functional diversity of seagrass macrofauna in an archipelago landscape[END_REF]. This suggests that taxonomic and functional analyses should complement each other when deriving general descriptions of benthic diversity, and that using only taxonomic analyses to infer the effects of environmental variables and human activities on biota may omit key functional attributes [START_REF] Frid | Using biological characteristics to develop new indices of ecosystem health[END_REF][START_REF] Bremner | Assessing functional diversity in marine benthic ecosystems: a comparison of approaches[END_REF].

There are several morphological features of free-living nematodes thought to be related to important ecological functions. In his articles on the ecology of nematodes from European and American coasts, [START_REF] Wieser | Die Beziehung zwischen Mundhöhlengestalt, Ernährungsweise und Vorkommen bei freilebenden marinen Nematoden[END_REF][START_REF] Wieser | The effect of grain size on the distribution of small invertebrates inhabiting the beaches of Puget Sound[END_REF] showed that the buccal structures of marine nematode genera allow a relatively easy classification into feeding guilds which has since found application in a variety of marine habitats (Giere, 1993 and references therein). [START_REF] Thistle | The nematode fauna of a deep-sea site exposed to strong near-bottom currents[END_REF] noted that nematode tails could be important in locomotion and reproduction and proposed a functional grouping of species based on tail shapes. There is inevitably some bias involved in deriving functional characteristics from assumed links to morphology, however, evidence from previous workers suggests that the functional group approach can be valid (e.g. [START_REF] Thistle | Nematode tail-shape groups respond to environmental differences in the deep sea[END_REF].

The fitness of a population is a function of physiological as well as ecological features, some of which clearly depend on organism size, such as metabolic rates, the tolerance to chemical stress, the ability to move or migrate and vulnerability to predation [START_REF] Peters | The ecological implications of body size[END_REF][START_REF] Soetaert | Size and shape of ocean margin nematodes: morphological diversity and depth-related patterns[END_REF].

Nematode length and width are robust parameters that are easily determined using nondestructive methods and describe important functional attributes of nematode species and genera [START_REF] Vanhove | Deep-sea meiofauna communities in Antarctica: structural analysis in relation with the environment[END_REF][START_REF] Soltwedel | The size structure of deep-sea meiobenthos in the North-Eastern Atlantic: nematode size spectra in relation to environmental variables[END_REF][START_REF] Tita | Size spectra, body width and morphotypes of intertidal nematodes: an ecological interpretation[END_REF][START_REF] Soetaert | Size and shape of ocean margin nematodes: morphological diversity and depth-related patterns[END_REF][START_REF] Vanaverbeke | Nematode biomass spectra as descriptors of functional changes to human and natural impact[END_REF][START_REF] Vanaverbeke | Changes in morphometric characteristics of nematode communities during a spring phytoplankton bloom deposition[END_REF]. Analysing the nematode fauna from a 15 m deep sandy bottom in the northern part of Öresund, Jensen (1987a) found that species, living in deeper sediment layers were significantly more slender than their oxybiotic, surface-dwelling congeners. He suggested that this morphotype represents adaptations to life in oxygen poor and sulphide rich sediments which is in contrast to previous assumptions that the body length and shape reflect adaptations exclusively to the physical constraints of interstitial space. [START_REF] Bongers | The maturity index: an ecological measure of environmental disturbance based on nematode species composition[END_REF] developed a grouping of nematode genera according to their life history strategies to assess the condition of terrestrial and freshwater habitats. This classification, relating the more r-selected 'colonisers' to the more K-selected 'persisters' has been applied less frequently to marine nematodes [START_REF] Bongers | Interpretation of disturbance-induced maturity decrease in marine nematode assemblages by means of the Maturity Index[END_REF][START_REF] Fraschetti | Structural and functional response of meiofauna rocky assemblages to sewage pollution[END_REF], partly due to a lack of empirical support for the classification of some marine genera and the absence or rarity of extreme colonisers and persisters in most marine habitats.

The reduction of a generally high species diversity into a smaller number of single functional groups, suggesting a very limited functional diversity, risks underestimating the true functional complexity of nematode communities [START_REF] Thistle | Nematode tail-shape groups respond to environmental differences in the deep sea[END_REF]. In this paper we address this by combining a range of biological traits of nematode species and genera describing buccal morphology, tail shape, body size, body shape and life history strategy to identify patterns in the functional diversity of nematodes from the southwestern North Sea. Taxonomic and functional aspects of nematode communities at 19 stations were investigated. The following questions were posed:

(1) Which environmental factors explain the number and composition of nematode functional groups?

(2) What is the degree and importance of redundancy, i.e. multiple species/genera per biological trait?

(3) How useful is the functional group concept and the biological traits approach in the study of nematodes in benthic soft-bottom ecosystems compared to their taxonomic properties?

Materials and Methods

Study site and sample collection

Sediment samples were collected at 19 stations in the southwestern North Sea (Fig. 1). All locations were sampled in May 2000, except of station 5, 6 and 9 where samples were collected in May 2001. All stations studied were located away from anthropogenic point-source impacts.

The closest dredgings disposal site, for example, was located approximately 14 km west off station 1 while the nearest oil and gas well was found about 750 m off station 3. range ring (using SEXTANT TM software and DGPS position fixing). From each deployment, two sub-samples, one for particle size and organic carbon content analysis and one for the study of meiofauna, were collected with a perspex corer (3 cm diameter) to a depth of 5 cm. Only two valid replicates could be retrieved from station 4, 14 and 15. All meiofauna samples were fixed in 5 % formaldehyde in 63 µm filtered seawater and samples for particle size and organic carbon content analysis were frozen to a temperature of -20 °C pending analysis.

Sample processing

One replicate sample collected for the analysis of environmental variables was processed per station. Sediment granulometry was determined by a combination of dry sieving and laser sizing to give the full particle size distribution [START_REF] Dyer | Coastal and estuarine sediment dynamics[END_REF]. Organic carbon content of the sediment fraction < 63 µm was determined with a Leeman CE 440 elemental analyser.

Meiofauna samples were initially washed onto a 63 µm sieve to remove the fine silt fraction and the formalin. After decanting the samples five times onto a 63 µm sieve, meiofauna was extracted with Ludox TM 40 with a specific gravity of 1.15 [START_REF] Somerfield | Meiofauna in marine pollution monitoring programmes. A laboratory manual[END_REF]. The extraction process was repeated three times. The extracts were evaporated slowly in anhydrous glycerol and evenly spread on microscope slides for identification and counting of nematodes under a highpower microscope (Olympus BX 51) with Nomarski interference contrast illumination. All nematodes in each sample were analysed to species or genus level.

Data processing

Treatment of raw data

A total of 169 nematode species were recorded in the southwestern North Sea [START_REF] Schratzberger | Patterns of nematode populations in the southwestern North Sea and their link to other components of the benthic fauna[END_REF]. Nematode species and genera were classified according to their buccal morphology, tail shape, adult length, adult shape and life history strategy.

Buccal morphology:

Free-living marine nematodes display a high diversity in the structure of their mouth cavity.

Based on these morphological characteristics, [START_REF] Wieser | Die Beziehung zwischen Mundhöhlengestalt, Ernährungsweise und Vorkommen bei freilebenden marinen Nematoden[END_REF] devised a classification of feeding types including selective deposit feeders consuming bacteria and small-sized organic particles (1A), non-selective deposit feeders also feeding on organic deposit but targeting larger-sized particles (1B), epigrowth feeders scraping food off surfaces similar to macrobenthic grazers (2A), and predators feeding on nematodes and other small invertebrates (2B). Although various authors have later revised and modified this classification [START_REF] Romeyn | Food selection and consumption by estuarine nematodes[END_REF]Jensen, 1987b;[START_REF] Moens | Observations on the feeding ecology of estuarine nematodes[END_REF][START_REF] Moens | Use of carbon and energy sources by nematodes[END_REF], nematode genera were assigned to [START_REF] Wieser | Die Beziehung zwischen Mundhöhlengestalt, Ernährungsweise und Vorkommen bei freilebenden marinen Nematoden[END_REF] feeding guilds since this remains the only scheme available that considers free-living marine nematodes from a variety of habitats.

Tail shape:

In addition to the functional grouping of feeding types, other aspects of nematode morphology can provide alternative functional group classifications. In their study of deep sea nematodes from the North Atlantic, [START_REF] Thistle | The nematode fauna of a deep-sea site exposed to strong near-bottom currents[END_REF] pioneered a scheme based on tail shape. Tail types are diverse and variable and tail and buccal morphology together have proven to be an effective method to discriminate nematode communities [START_REF] Thistle | Nematode tail-shape groups respond to environmental differences in the deep sea[END_REF]. Tail shapes for nematode species recorded in the southwestern North Sea were taken from the taxonomic description of species and genera. Nematodes were assigned to four tail shape groups, common in free-living marine nematodes from coastal environments (short/round, elongated/filiform, conical, clavate).

Adult length and adult shape: Similar to tail shape, the total length and the length-width ratios for adult nematode species were deduced from the taxonomic literature. Although simple measures of length and width cannot do justice to the wide morphological variation amongst free-living nematodes, they can offer a unique opportunity for evaluating the impact of environmental variables on their populations and assemblages [START_REF] Soetaert | Size and shape of ocean margin nematodes: morphological diversity and depth-related patterns[END_REF]. The length-width ratio is a measure of a nematode's body shape with long/thin animals having high ratios, stout animals low ratios. [START_REF] Soetaert | Size and shape of ocean margin nematodes: morphological diversity and depth-related patterns[END_REF] suggested that three morphologies have developed over evolutionary time-scales: Stout, slender and long/thin. Slender and long/thin nematodes are able to move swiftly through the sediment, but small, juvenile stages are vulnerable to predation. The stout morphotype, in contrast, may have evolved towards reducing this predation pressure on the small individuals but these adaptations undoubtedly bring about reduced mobility. Nematode species were assigned to four length groups (< 1 mm, 1-2 mm, 2-4 mm, > 4 mm) and three shape categories (stout with a length-width ratio < 18, slender with a length-width ratio of 18-72 and long/thin with a lengthwidth ratio > 72) which are thought to conform as closely as possible to likely functions.

Life history strategy:

A number of attempts to investigate the life cycles of marine nematodes by examining the age structure or size frequency throughout the year are reported in the literature [START_REF] Warwick | Ecological and metabolic studies on free-living nematodes from an estuarine mud-flat[END_REF]. Analyses of this kind are successful only if the reproductive cycles of individuals in the population are synchronised and the number of annual generations is relatively small to avoid confusion through excessive overlap in generations [START_REF] Wieser | Growth and metabolism in a marine nematode Enoplus communis Bastian[END_REF]. However, culture experiments have shown that many small species have short generation times of usually about one month or less [START_REF] Gerlach | On the importance of marine meiofauna for benthos communities[END_REF][START_REF] Heip | The ecology of marine nematodes[END_REF]. Nematodes were allocated to life history groups according to [START_REF] Bongers | The maturity index: an ecological measure of environmental disturbance based on nematode species composition[END_REF] and [START_REF] Bongers | Interpretation of disturbance-induced maturity decrease in marine nematode assemblages by means of the Maturity Index[END_REF][START_REF] Bongers | Proposed changes of c-p classification for nematodes[END_REF] where genera are classified on a five-point scale from colonisers (inter alia short life-cycle, high reproduction rates, high colonisation ability, tolerant to various types of disturbance) to persisters (inter alia long life-cycles, low colonisation ability, few offspring, sensitive to disturbance). In contrast to terrestrial environments, extreme colonisers (coloniser-persister score of 1) and persisters (coloniser-persister score of 5) are virtually absent in marine systems.

Biological traits analysis

A biological traits analysis, following the approach described for macrobenthic communities [START_REF] Frid | Using biological characteristics to develop new indices of ecosystem health[END_REF][START_REF] Bremner | Assessing functional diversity in marine benthic ecosystems: a comparison of approaches[END_REF] was carried out to assess the functional structure of nematode communities in the southwestern North Sea. Five traits were used, buccal morphology, tail shape, adult length, adult shape and life history strategy. There were a total of 19 categories of these traits, as described above. A biological traits matrix was constructed by assigning to each nematode species/genus its affinity to each trait category (Appendix). The biological traits matrix was then raised by the relative species abundance to give abundance-weighted traits matrices for each station.

One of the problems associated with assigning species/genera to biological trait categories is that all species allocated to a particular biological trait may not be equivalent in the degree to which they express this trait [START_REF] Petchey | Functional diversity (FD), species richness and community composition[END_REF]. Species within a particular trait group, however, are more similar in the extent to which they display a particular trait [START_REF] Chalcraft | Mapping functional similarity of predators on the basis of trait similarities[END_REF]. Biological traits analysis relies on the ecological importance and accuracy of measurement of all the different functionality measures that are selected for use. In this analysis, we apply equal weighting to all traits due to the lack of information on their relative importance, and only use traits that were considered potentially important by the authors of primary literature.

Analysis of nematode distribution patterns

Non-metric multi-dimensional scaling (MDS) ordination using the Bray-Curtis similarity measure was applied to relative abundance data to compare spatial patterns in the taxonomic and functional composition of nematode communities at the 19 sampling locations. Separate resemblance matrices were created based on the composition of the nematode communities with respect to species and genus identity, buccal morphology, tail shape, adult length, adult shape and coloniser-persister score. A further resemblance matrix was created based on the abundanceweighted biological traits matrix to summarise patterns in the functional structure of nematode assemblages of all species. Analysis of similarities (ANOSIM) was used to test for significant taxonomic and functional differences between stations and the Similarity Percentages (SIMPER) procedure was applied to identify the trait groups that were primarily responsible for the functional differences observed between locations.

Resemblance matrices derived from the species and genus abundance matrix, the single functional group matrices and the biological traits matrix were used in a second-stage MDS analysis to examine similarities between each of the first-stage MDS matrices [START_REF] Somerfield | Taxonomic levels, in marine community studies, revisited[END_REF]. By means of this technique, Spearman rank correlations (r s ) calculated between pairs of similarity matrices are used as a basis for a second-stage MDS ordination. The proximity of the points in the resultant MDS ordination indicates the extent to which different analysis options capture the same information.

Five environmental parameters were included in the data analyses (median particle diameter, total organic carbon and silt content, sorting coefficient and water depth) together with a derived measure of sediment heterogeneity following [START_REF] Ward | Studies on the free-living marine nematodes of Liverpool Bay. II. Influence of sediment composition on the distribution of marine nematodes[END_REF]:

h = QD∅ QD∅ = Sorting coefficient Md∅ x % silt Md∅ = Median particle diameter [phi]
The concentrations of trace metals in the sediment were low at all stations and an unlikely cause of biological effects (see [START_REF] Schratzberger | Patterns of nematode populations in the southwestern North Sea and their link to other components of the benthic fauna[END_REF]. Trace metal concentrations were therefore omitted from the correlation analyses.

The relationship between the structure of nematode assemblages and environmental variables was explored by calculating Spearman rank correlations (r s ) between similarity matrices derived from the faunal data (based on Bray-Curtis similarity) and matrices derived from various subsets of environmental data (based on normalised Euclidean Distance), thereby defining suites of environmental variables which best explained the biotic structure (BIOENV procedure). A permutation test was applied to assess the significance of these relationships.

All statistical analyses were performed using the software packages Statgraphics Plus version 3.3

and Primer version 6.1.5 [START_REF] Clarke | Change in marine communities: an approach to statistical analysis and interpretation[END_REF][START_REF] Clarke | PRIMER version 6. User Manual[END_REF].

Results

The study area comprised a stable, sedimentary environment increasing significantly in depth to the north and east. Most of the sediments collected consisted of moderately to poorly sorted muddy sands. The finest and most uniform substrates with the highest silt content (station 1 and 13) were found in the northern part of the study area while the coarsest, most heterogeneous sediments with lowest silt content (station 14 to 19) were located in the south (Table 1).

Functional attributes of nematode communities in the southwestern North Sea are listed in Table 2 and3 A total of 79 trait combinations were represented amongst the nematode fauna of the southwestern North Sea (Appendix). The biological traits matrix revealed several notable relationships between traits. For example, large-bodied nematodes generally had a high coloniserpersister score while that for smaller species was low. Equally, in contrast to the generally smallsized selective deposit feeders, predators were usually large. While ecologically implausible trait combinations such as, for example, small body size combined with a K-selected life history strategy (i.e. high coloniser-persister score) were absent from the nematode communities of the southwestern North Sea, the traits matrix revealed some combinations of functional groups to which no species belonged (e.g. no epigrowth feeders with rounded tails), although these combinations are known from other environments. The spatial pattern of mean number of biological trait groups mirrored the pattern of mean species and mean genus number (Fig. 2).

Assemblages collected at the 19 stations differed both taxonomically and functionally (Table 4) but the ordinations of taxonomic groups did not match the ordinations based on functional groups and traits (Fig. 3). In the ordinations based on the relative abundance of nematode species and genera, stations in the northern part of the study site tended to cluster to the left while the southern stations were grouped to the right. This geographic separation was less pronounced in the ordinations derived from functional characteristics of nematode communities. We observed a high dominance of non-selective deposit feeders (1B) and epigrowth feeders (2A) at station 13 and 17 respectively and nematode communities collected at station 1 and 13 were highly dominated by individuals with an elongated/filiform tail. In contrast to other functional groups, the separation of nematode assemblages based on their adult length and shape was comparatively low with Bray-Curtis similarities between stations exceeding 80 %. High proportions of colonisers (coloniser-persister score of 2) prevailed at station 13, whereas more K-selected species (coloniser-persister score of 3 and 4) were abundant at other stations. Station 13 diverged notably from other locations when a wider range of biological traits expressed by species and genera was considered (Fig. 3). Two trait combinations were primarily responsible for the difference between this comparatively deep, silty site and other sampling stations. The trait combination selective deposit feeder/clavate tail/1-2 mm/slender/coloniser-persister score 2 dominated station 13, whereas species combining a similar length, shape and colonisation potential with an epigrowth feeding habit and a conical tail occurred in high proportions at other locations. These two discriminating trait combinations were the most species-rich. Eighteen species belonging to the comesomatids, xyalids, linhomoeids and axonolaimids shared the same trait combinations in the first group while a total of 15 desmodorid, microlaimid, chromadorid and cyatholaimid species were abundant in the latter. Forty eight species each had a combination of trait categories that was unique only to them (Appendix). A high proportion of one of these trait groups (represented by Leptonemella aphanothecae), combining a selective deposit feeding habit, conical tail, relatively long and thin body with a high coloniser-persister score of 4, distinguished nematode communities at station 12 from those at other locations.

The similarity of the spatial groupings of the 19 sampling stations is illustrated by a second-stage MDS based on Spearman rank correlations (r s ) of the eight underlying similarity matrices (Fig. 4). The ordination revealed that -The information in a particular functional group was not a simple reflection of the information in another group (i.e. matrices based on single functional groups such as buccal morphology, tail shape etc. clustered separately from each other).

-Clumping of species and genera into combined biological traits increased the relative importance of the taxonomic community composition (i.e. the biological traits matrix clustered closest to matrices derived from the species and genus composition).

-Ecological information captured by adult length, adult shape and life history strategy was less site-specific and thus differed notably from information contained in other functional groups.

With the exception of tail shape, which was significantly related to the silt content of the sediment, none of the relationships between functional groups and measured environmental factors was significant at p < 0.05 (Table 5). Nematode distribution patterns based on proportions of species and genera were strongly linked to sediment heterogeneity, a derived measure combining sorting coefficient, median particle diameter and silt content. The composition of assemblages in terms of biological traits was best explained by a combination of factors related to sediment granulometry (median particle diameter and silt content) and water depth (Table 5). The number of species, genera and trait groups generally increased with increasing water depth and decreased with increasing mean particle diameter and sediment heterogeneity.

Discussion

Functional diversity, the extent of functional differences among the species in a community, is an important determinant of ecosystem processes. A common measure of functional diversity is the number of functional groups represented by the species in a community [START_REF] Petchey | Functional diversity (FD), species richness and community composition[END_REF]. In this article we described nematode assemblages on the basis of the functional attributes of species and genera, and showed how the functionally-defined assemblages varied spatially in response to environmental variables. To our knowledge, very few attempts have been made to understand the environmental factors which determine functional diversity of meiobenthic nematodes at regional scales. We posed three questions in the introduction related to practical and philosophical issues surrounding the assessment of functional diversity in nematode communities.

These questions are addressed in the light of the outcome from the analysis of faunal and environmental data collected at 19 stations in the southwestern North Sea.

Which environmental factors explain the number and composition of nematode functional groups?

In a previous article [START_REF] Schratzberger | Patterns of nematode populations in the southwestern North Sea and their link to other components of the benthic fauna[END_REF] gave a detailed account of the species distribution patterns, diversity and community structure of nematode assemblages collected at the 19 sampling stations. Assemblages showed a characteristic pattern in the multivariate ordination, with stations grouped in two big clusters, one consisting of the deeper, fine sediment, northern stations (station 1 to 13), the other including the shallower, coarser sediment southern stations (station 14 to 19). These groups exhibited clear differences in species diversity, and stations with similar sediment type and water depth were also most similar taxonomically. Sediment-related factors were also important for morphological characteristics of species and genera that have been shown to be of functional importance.

Of all single functional groups, spatial dissimilarities in tail shape and buccal morphology were the most pronounced. Tail morphology showed a clear link to the silt content of the sediment.

Species with elongated/filiform tails were disproportionally abundant at station 1 and 13 where the siltiest sediments occurred. Rieman (1974) reported that some individuals with a long, filiform tail have a partly sessile existence in which tail morphology plays a crucial role. He suggested that this is a special adaptation to fine sand and muddy sediments where only an incomplete interstitial system exists. In these sediments, the tail would enable animals to retract from dead-end interstitial passageways that are too narrow to allow the worm to turn around and escape.

In contrast to tail morphology, there was no evidence of association between the relative abundance of buccal morphology groups and variation in measured environmental variables. We therefore hypothesise that other, unmeasured sediment-related environmental variables might be important for the functional structure of assemblages (e.g. input of organic material). The association between trophic structure of nematode communities and the carbon content in the sediment was weak, and chlorophyll a content in the sediment was not measured in our study.

However, it has been shown that during the deposition of a phytoplankton bloom in the southern North Sea between April and June, the chlorophyll a content in the water column closely follows pigment concentrations in the bottom water [START_REF] Vanaverbeke | Changes in morphometric characteristics of nematode communities during a spring phytoplankton bloom deposition[END_REF]. In 2000 and 2001, the chlorophyll a content in the vicinity of some of our sampling stations was determined at a depth of 4 m between May and August (N. Greenwood unpubl. data). Lowest chlorophyll a concentrations were recorded at station 13, highest at station 17 and these were clearly linked to a low and high prevalence of epigrowth feeding nematodes respectively.

At station 13, a low median particle diameter, high silt content and water depth were indicative of a relatively high consistency of physico-chemical factors. These environmental conditions were coupled with a dominance of species combining a selective deposit feeding habit, clavate tail, intermediate-sized, slender body with a high colonisation potential. Sediments with high silt content generally show a high portion of deposit-feeders [START_REF] Heip | The ecology of marine nematodes[END_REF], and these often score 2 or 3 on the coloniser-persister axis [START_REF] Bongers | Interpretation of disturbance-induced maturity decrease in marine nematode assemblages by means of the Maturity Index[END_REF][START_REF] Bongers | Proposed changes of c-p classification for nematodes[END_REF]. It is therefore possible that a relatively un-specialised feeding group of nematodes, characterised by short life cycles and a high colonisation ability was able to exploit prevailing environmental conditions at this station.

What is the degree and importance of redundancy, i.e. multiple species/genera per functional group?

The 169 nematode species identified in the southwestern North Sea had 79 different combinations of biological traits, and 33 species shared the two most common combinations of traits. To some extent, this pattern has been caused by the allocation of nematodes to only 19, partly interrelated, functional groups. The functional groups used represent the major trophic and morphological categories available to the phylum, and each could be sub-divided further to provide more species with unique combinations of traits. Although it is unlikely that further sub-division of existing traits would be meaningful, it is unclear whether the inclusion of additional traits, for example related to the physiology (e.g. respiration rates, [START_REF] Warwick | Ecological and metabolic studies on free-living nematodes from an estuarine mud-flat[END_REF] of species, would provide greater differentiation in the functional roles of nematodes.

We observed a strong link between species and genus counts and the number of functional traits supporting the contention that, under a constant set of environmental conditions, an increase in species richness (i.e. number of species) should lead to increased functional diversity.

Spatial patterns in the functional structure of assemblages were primarily related to differences in the two most species-rich trait combinations, shared by 18 and 15 species respectively. These trait combinations included both dominant and low-abundance species with different taxonomic affiliation. [START_REF] Walker | Plant attribute diversity, resilience, and ecosystem function: the nature and significance of dominant and minor species[END_REF] hypothesised that many of the low-abundance species are analogues of the dominants with respect to their ecological functions. They differ in terms of their capabilities to respond to environmental stresses and disturbance, thereby conferring resilience on the community. Environmental conditions thus influence the importance of functional complementarity in structuring communities [START_REF] Hooper | Effects of biodiversity on ecosystem functioning: a consensus of current knowledge[END_REF].

Concepts of niche differentiation imply that functional characteristics of coexisting organisms must differ at some level, but the identification of which and how many species act in a complementary way in complex communities is just beginning [START_REF] Hooper | Effects of biodiversity on ecosystem functioning: a consensus of current knowledge[END_REF]. There may be species that can be lost from some ecosystems without substantial alteration of ecosystem function, as two species (for example Nannolaimoides effilatus and Terschellingia gourbaultae, see Appendix) can show similarities in the way they feed, reproduce and move within the habitat.

However, they probably do not carry out these activities in exactly the same way or at the same time, and the functional significance of these differences, not recorded by this analysis, is likely to depend on the species and ecosystem in question [START_REF] Snelgrove | Getting to the bottom of marine biodiversity: sedimentary habitats[END_REF].

To date, few studies have tested the concept of functional redundancy in free-living nematode communities [START_REF] Mikola | Relating species diversity to ecosystem functioning: mechanistic backgrounds and experimental approach with a decomposer food web[END_REF][START_REF] De Mesel | Influence of bacterivorous nematodes on the decomposition of cordgrass[END_REF]. These investigations into the contribution of species to ecosystem processes revealed effects of individual species rather than functional groups (e.g. feeding types). A more reliable approach to assessing the functional structure of nematode communities might therefore be to consider combinations of biological traits expressed by organisms (i.e. biological traits analysis) rather than relying on single functional groups. [START_REF] Postma-Blaauw | Within-trophic group interactions of bacterivorous nematode species and their effects on the bacterial community and nitrogen mineralization[END_REF], for example, showed that differences in life history strategies between nematode species of the same trophic group is of importance for their communal effect on soil ecosystem processes.

How useful is the functional group concept and the biological traits approach in the study of nematodes in benthic soft-bottom ecosystems compared to their taxonomic properties?

Results from the ANOSIM analyses indicated that spatial differences of combined biological traits were more pronounced than dissimilarities based on the proportions of individuals from single functional groups. Although the biological traits approach is no more powerful than traditional taxonomic methods in detecting spatial differences between communities, it is of additional ecological importance. The outcome from multivariate analyses confirmed that the ecological information captured by the biological traits matrix was not a simple reflection of the information contained in single functional groups. Consequently, combining a number of functionality measures in one biological traits analysis provided ecological information that could not have been discerned from the separate analysis of functionally important morphologies. For example, although station 13 showed a characteristic spatial pattern with respect to some functional groups, the functional differences between this and other locations could be most reliably linked to measured environmental variables only when the biological traits approach was applied.

Functional diversity is an important component of biodiversity, yet in comparison to taxonomic diversity, methods of quantifying functional diversity are less well developed [START_REF] Petchey | Functional diversity (FD), species richness and community composition[END_REF]. Our results from the second-stage MDS showed that assigning species and genera to biological traits provided additional insights to those from traditional taxonomic analyses. This suggests that measures based on a phylogenetic classification do not alone capture all of the important, heritable differences in nematode attributes [START_REF] Walker | Plant attribute diversity, resilience, and ecosystem function: the nature and significance of dominant and minor species[END_REF]. Improving our understanding of diversity-function relationships across ecosystems will require a categorisation of species attributes that can be related to function. Consequently, obtaining a greater knowledge of the functional roles of nematode species will be the key to improve the sensitivity and interpretation of biological traits analyses of marine benthic communities. Relative abundance (mean ± SE) of nematodes in adult length, adult shape and life history categories at 19 stations in the southwestern North Sea.

C-p = coloniser-persister score [START_REF] Bongers | Interpretation of disturbance-induced maturity decrease in marine nematode assemblages by means of the Maturity Index[END_REF][START_REF] Bongers | Proposed changes of c-p classification for nematodes[END_REF]

Figure captions Fig. 1 .

 captions1 Figure captions

Fig. 2 .

 2 Fig. 2. Mean (± SE) number of species, genera and biological trait groups of nematode

Fig. 3 .

 3 Fig. 3. Non-parametric multi-dimensional scaling (MDS) ordination based on the relative

Fig. 4 .

 4 Fig. 4. Non-parametric multi-dimensional scaling (MDS) second-stage ordination of inter-matrix

  Fig.1

  2A) whereas proportions of selective deposit feeders (1A) and predators (2B) were comparatively low at most stations. Conical and clavate were the most prevalent tail shapes at the majority of stations (Table2). Average adult lengths of nematodes ranged from 0.35 mm to 12

	mm with the majority of all recorded individuals (76 %) occurring in the two intermediate length
	classes (1-2 mm and 2-4 mm). Three nematode morphotypes were present in the sediments. The
	majority of nematodes were slender (82 %) while stout animals accounted for 6 % and long/thin
	animals for 12 % of all nematodes identified. Over 81 % of all identified individuals attained a
	coloniser-persister score of 2 or 3. Extreme persisters (coloniser-persister score of 5), in contrast
	were either absent or rare (Table 3).

. Assemblages were dominated by non-selective deposit feeders (1B) and epigrowth feeders (

Table 1

 1 Location of and environmental parameters at 19 stations in the southwestern North Sea

	Station	Latitude	Longitude	Depth	Median	Silt	Sorting	Sediment	C
				[m]	[µm]	[%]	coefficient	heterogeneity h	[%]
	1	55.00 N	1.00 W	78	110	15.6	2.3	0.05	no data
	2	55.00 N	0.00	76	184	7.3	1.8	0.10	3.0
	3	54.75 N	0.50 W	64	143	3.1	1.0	0.11	4.8
	4	54.75 N	0.50 E	78	122	2.8	1.1	0.12	3.5
	5	54.75 N	1.50 E	26	167	1.1	1.4	0.50	3.7
	6	54.75 N	2.50 E	40	127	3.5	1.2	0.12	2.8
	7	54.50 N	0.00	67	160	2.1	1.1	0.19	5.2
	8	54.50 N	1.00 E	61	212	1.3	1.0	0.35	4.9
	9	54.50 N	2.00 E	20	173	1.0	1.4	0.56	3.8
	10	54.25 N	0.50 E	66	210	1.3	1.0	0.34	4.6
	11	54.25 N	1.50 E	54	157	3.3	1.2	0.13	2.9
	12	54.00 N	1.00 E	44	145	2.5	1.0	0.14	3.5
	13	54.00 N	2.00 E	76	117	14.5	1.7	0.04	1.9
	14	53.75 N	1.50 E	60	374	0.8	1.4	1.18	2.3
	15	53.50 N	2.00 E	23	316	0.7	0.7	0.62	3.4
	16	53.25 N	2.50 E	32	202	1.4	0.8	0.25	2.2
	17	53.00 N	1.00 E	16	262	1.5	1.0	0.33	no data
	18	52.75 N	2.50 E	42	339	0.9	1.4	1.02	1.6
	19	52.25 N	2.50 E	45	337	0.3	1.4	3.20	2.5

Table 3

 3

Table 4

 4 Results from ANOSIM analyses: Global R of differences between nematode assemblages collected at 19 stations in the southwestern North Sea. All R-values were significant at p < 0.01.

	Station		Adult length			Adult shape			Life history strategy
		> 1 mm	1-2 mm	2-4 mm	> 4 mm	stout	slender	long/thin	c-p 2	c-p 3	c-p 4	c-p 5
	1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19	29 ± 5 18 ± 3 34 ± 6 19 ± 6 13 ± 1 24 ± 3 26 ± 2 24 ± 2 9 ± 2 21 ± 8 13 ± 2 13 ± 10 17 ± 7 35 ± 9 25 ± 3 11 ± 2 31 ± 2 39 ± 13 24 ± 5	55 ± 4 57 ± 7 57 ± 5 54 ± 13 65 ± 2 52 ± 3 53 ± 4 48 ± 6 74 ± 7 47 ± 10 62 ± 5 45 ± 17 66 ± 10 58 ± 9 68 ± 2 67 ± 10 55 ± 7 50 ± 10 65 ± 3	12 ± 1 21 ± 5 8 ± 2 22 ± 11 20 ± 2 22 ± 2 18 ± 5 26 ± 8 17 ± 5 29 ± 14 22 ± 5 40 ± 23 15 ± 3 7 ± 0 6 ± 1 21 ± 12 11 ± 3 10 ± 3 10 ± 6	5 ± 3 5 ± 1 1 ± 1 5 ± 3 1 ± 0 3 ± 1 3 ± 1 2 ± 1 0 ± 0 3 ± 3 3 ± 1 2 ± 1 2 ± 0 0 ± 0 2 ± 0 1 ± 1 3 ± 2 0 ± 0 1 ± 0	4 ± 1 2 ± 1 8 ± 2 4 ± 2 1 ± 1 12 ± 4 11 ± 6 4 ± 2 0 ± 0 11 ± 11 6 ± 2 8 ± 8 2 ± 0 6 ± 5 5 ± 0 5 ± 2 8 ± 3 13 ± 3 7 ± 7	86 ± 3 90 ± 2 89 ± 2 80 ± 8 85 ± 4 69 ± 2 77 ± 5 74 ± 4 91 ± 3 69 ± 4 85 ± 3 58 ± 15 93 ± 1 91 ± 6 90 ± 1 86 ± 5 83 ± 3 81 ± 3 86 ± 9	10 ± 3 8 ± 2 4 ± 16 ± 9 14 ± 3 19 ± 2 13 ± 3 22 ± 5 9 ± 3 19 ± 7 9 ± 3 34 ± 20 5 ± 0 3 ± 0 5 ± 2 9 ± 4 9 ± 0 6 ± 1 6 ± 3	47 ± 7 56 ± 5 56 ± 13 48 ± 9 62 ± 5 44 ± 4 47 ± 7 51 ± 6 57 ± 7 43 ± 9 56 ± 5 48 ± 16 80 ± 4 57 ± 0 56 ± 1 49 ± 17 52 ± 7 48 ± 8 53 ± 15	38 ± 2 33 ± 6 33 ± 10 26 ± 1 15 ± 6 28 ± 2 37 ± 7 24 ± 5 21 ± 4 38 ± 11 30 ± 5 17 ± 6 13 ± 3 35 ± 4 32 ± 5 25 ± 10 32 ± 5 33 ± 8 20 ± 3	14 ± 8 11 ± 3 10 ± 4 25 ± 10 22 ± 2 26 ± 2 15 ± 6 25 ± 6 18 ± 2 17 ± 6 14 ± 1 35 ± 19 5 ± 1 8 ± 4 12 ± 4 24 ± 9 15 ± 3 18 ± 8 27 ± 13	1 ± 1 1 ± 1 1 ± 1 1 ± 1 2 ± 1 2 ± 1 1 ± 1 0 ± 0 3 ± 2 1 ± 1 1 ± 0 0 ± 0 2 ± 1 1 ± 0 1 ± 0 2 ± 2 1 ± 1 2 ± 2 0 ± 0

Table 5

 5 Results from BIOENV analyses: Spearman rank correlation (r s ) and significance level (p) between averaged faunal data and mean environmental variables.

		r s	p	Environmental variable
	Species	0.652	0.01	Sediment heterogeneity
	Genus	0.689	0.01	Sediment heterogeneity
	Buccal morphology	0.254	0.31	---
	Tail shape	0.543	0.01	Silt content
	Adult length	0.359	0.06	---
	Adult shape	0.076	0.84	---
	Life history strategy	0.318	0.15	---
	Biological traits matrix	0.431	0.03	Median, silt content, depth

Acknowledgements

We thank members of the Centre for Environment, Fisheries and Aquaculture Science and the officers and crew of RV ' Cirolana' for practical assistance in the collection of samples. Claire Mason conducted the particle size analyses. Comments of two anonymous referees significantly improved an earlier version of the manuscript. The work was supported by the Department for Environment, Food and Rural Affairs (research project AE002 and AE1148).

Appendix

Biological traits matrix. 1A = selective deposit feeders, 1B = non-selective deposit feeders, 2A = epigrowth feeders, 2B = predators [START_REF] Wieser | Die Beziehung zwischen Mundhöhlengestalt, Ernährungsweise und Vorkommen bei freilebenden marinen Nematoden[END_REF]. S/r = short/round, e/f = elongated/filiform, co = conical, cla = clavate. Adult length is shown in mm. St = stout, sl = slender, l/t = long/thin. C-p = coloniser-persister score [START_REF] Bongers | Interpretation of disturbance-induced maturity decrease in marine nematode assemblages by means of the Maturity Index[END_REF][START_REF] Bongers | Proposed changes of c-p classification for nematodes[END_REF]

Appendix continued Biological traits matrix. 1A = selective deposit feeders, 1B = non-selective deposit feeders, 2A = epigrowth feeders, 2B = predators [START_REF] Wieser | Die Beziehung zwischen Mundhöhlengestalt, Ernährungsweise und Vorkommen bei freilebenden marinen Nematoden[END_REF]. S/r = short/round, e/f = elongated/filiform, co = conical, cla = clavate. Adult length is shown in mm. St = stout, sl = slender, l/t = long/thin. C-p = coloniser-persister score [START_REF] Bongers | Interpretation of disturbance-induced maturity decrease in marine nematode assemblages by means of the Maturity Index[END_REF][START_REF] Bongers | Proposed changes of c-p classification for nematodes[END_REF]


Keywords: meta-analysis, aquaculture impact, water column, dissolved nutrients, fish, shrimp, bivalve, polyculture

Environmental effects of aquaculture loadings have often been reviewed descriptively, and thus have not provided quantitative estimates of the overall response in the water column.

Meta-analytical reviewing techniques allow the contextualisation of quantitative effects in the domain of current literature. In the present paper, more than 50 peer-reviewed articles were analysed and about 425 study cases used to test whether worldwide cultivations have a differential effect on dissolved nutrient levels. Meta-analysis feasibility depends on obtaining an estimate of the effect size from every study and the most common measure of effect size (Hedges' d) is the difference between means of controls and impacts standardised by dividing by the pooled standard deviation. Across all study cases, irrespective of cultivation and organism type, the cumulative effect size was large and significant (d > 0.8) for ammonium, nitrite and nitrate, medium (0.8 > d > 0.5) for dissolved phosphorus, and not significant (d < 0.2) for silicates. Effects were mainly correlated with the degree of openness in water bodies, and ammonium and the other nitrogen forms were the most highly informative descriptors of effects in the area surrounding farms, even though weakness in statistical approach was highlighted. The results partially contradict the common view that effects of aquaculture and associated environmental patterns are well defined throughout the current literature.

INTRODUCTION

Since aquaculture was first considered an important anthropic activity with potential detrimental effects on the environment [START_REF] Naylor | Nature's subsidies shrimp and salmon farming[END_REF]), a number of descriptive reviews on this issue have been published. Aquaculture facilities have effects on both the water column [START_REF] Modica | The effect of mariculture facilities on biochemical features of suspended organic matter (southern Tyrrhenian, Mediterranean)[END_REF]) and on sediments (Kalantzi & Karakassis 2006) along many different axes of variability. Numerous questions have therefore arisen as to how, where and when aquaculture has had measurable effects on the environment. It has often been noted [START_REF] Gowen | The ecological impact of salmonids farming in coastal waters: a review[END_REF][START_REF] Iwama | Interactions between aquaculture and the environment[END_REF][START_REF] Enell | Environmental impact of nutrients form Nordic fish farming[END_REF][START_REF] Iversen | Fish farming in Denmark: environmental impact of regulative legislation[END_REF][START_REF] Wu | The environmental impact of marine fish culture: towards a sustainable future[END_REF][START_REF] Beveridge | Cage aquaculture, 2th edn[END_REF][START_REF] Bardach | Aquaculture, pollution and biodiversity[END_REF][START_REF] Carpenter | Non-point pollution of surface waters with phosphorus and nitrogen[END_REF][START_REF] Naylor | Effect of aquaculture on world fish supplies[END_REF][START_REF] Black | Environmental impacts of aquaculture[END_REF][START_REF] Fernandes | The scientific principles underlying the monitoring of the environmental impacts of aquaculture[END_REF], Islam et al. 2004[START_REF] Pillay | Aquaculture and the environment, 2th edn[END_REF][START_REF] Islam | Nitrogen and phosphorus budget in coastal and marine cage aquaculture and impacts of effluent loading on ecosystem: review and analysis towards model development[END_REF]) that the type of cultivated organisms (e.g. fish, shrimps, bivalves), the locations of cultivation (i.e. ecosystems and water types such as fresh, mixed [brackish] and marine waters), the cultivated biomass, the number of species, the quality and quantity of supplied food or management practices (i.e. intensive, semi-intensive, extensive) are the prime factors in determining the extent of effects on the environment, in that they represent the direct causal factors of eutrophication risk. Many authors have pointed out that aquaculture is an anthropogenic activity, which has only limited effects on the environment compared to other forms of pollution (e.g. Islam & Tanaka 2004). Others have proposed conceptual nutrient mass balance models to extrapolate the level of nitrogen and phosphorus discharged from a hypothetical culture system (e.g. [START_REF] Islam | Nitrogen and phosphorus budget in coastal and marine cage aquaculture and impacts of effluent loading on ecosystem: review and analysis towards model development[END_REF]). Nevertheless, only rarely has there been an attempt in the primary literature to estimate the how, where and when of environmental effects of aquaculture. The present review, using meta-analytic techniques, aims to provide a quantitative estimate of aquaculture effects using data amassed from current peer-reviewed literature, and is directed at understanding whether dissolved nutrients in the water column are generally affected by aquaculture facilities. [START_REF] Gurevitch | A meta-analysis of competition in filed experiment[END_REF] pointed out that meta-analysis, as opposed to other review techniques, offers major advantages for research synthesis in ecology. Indeed, meta-analysis is a quantitative tool available to ecologists who wish to obtain general knowledge about the magnitude of a certain effect, whether that effect is different among contrasting categories of studies and how much variation is explained both within and among categories. The specific aims of the present meta-analysis are to estimate: (1) the degree of heterogeneity of results reported from aquaculture studies investigating the effects on dissolved nutrient levels; (2) the cumulative effect of aquaculture loadings on nitrogen, phosphorus and silica across the peer-reviewed literature; (3) the differential effects of aquaculture loadings on each form of the dissolved nutrients; (4) which factors mainly affect the variability of cumulative dissolved nutrient levels and lastly (5) whether possible deviation from common natural patterns induced by aquaculture loadings is generally similar as a function of the ecosystem and cultivated organism types.

MATERIALS AND METHODS

Literature search, meta-analysis criteria and data eligibility. Data on the effects of aquaculture loadings on the water column through dissolved nutrients as descriptors were obtained from a literature search using mainly the Aquatic Science and Fisheries Abstracts (ASFA) and some other databases, such as Bio-One or Zoological Records available on-line on the internet. The scope of this search ranged between 1980 and the present, and when grey literature, internal reports or unpublished data were not readily available on-line, I personally contacted a number of authors to obtain their publications, though to no great degree (22%) of success. The search was initially carried out using prominent or substantial key-words such as "aquaculture and impact". This method revealed a very large number of publications (11,656 published by January 2006) stemming from all possible sources, most of which were immediately discarded, either because of their low accessibility to a large audience (internal institutional reports or symposium proceedings and/or language barriers; some of these reports were written only in French, Italian, Chinese, Japanese, Russian etc). Many of the documents published prior to 1985 were ruled out due to the physical difficulty of obtaining these papers from authors or libraries, despite the fact that 450 studies among those published in peer-reviewed journals had been initially retained. Due to these hindrances, therefore, I focused only on widespread and easily accessible sources, such as those published in peerreviewed journals after 1985. Whilst the potential loss of useful data found in grey literature and internal sources is an important meta-analytic concern, it is hoped that considering only peer-reviewed articles ensured consistently high quality data, such that are often not found in other (perhaps less established) sources. Although major journals tend to publish only significant results, thereby generating a potential publication bias and distorting the direction of true effect [START_REF] Hedges | Statistical methods for meta-analysis[END_REF], the peer-review process is the best method to reduce the likelihood of potential quality biases in reviewing.

Most of the contemporary peer-reviewed articles, however, reported the words "aquaculture" and "impact" in contexts not relevant to the present meta-analysis. I thus reduced the scope of the search, using keywords such as "aquaculture and impact and nutrient", "aquaculture and impact and ammonium", "…and nitrite" etc., which resulted in a database of about 100-120 peer-reviewed articles, which were then checked for against the required criteria for metaanalysis. Unlike descriptive reviews, meta-analysis requires the quantitative measure of variance to be stated by each study [START_REF] Hedges | Statistical methods for meta-analysis[END_REF]. I therefore obtained the means for the control and treatment groups, their standard deviations and their sample sizes [START_REF] Hedges | Statistical methods for meta-analysis[END_REF] in order to calculate meta-analytic statistics. In the present meta-analysis, control groups are represented by data collected from distinct areas identified by each author, where effects of aquaculture facilities were not present. The treatment group is represented by data collected from areas directly affected by farm loadings. Thus, the first screening of these studies allowed me to include all papers reporting at least means, deviations and sample sizes for both controls and farmed areas. In 30-40% of the studies it was not possible to extrapolate deviations or sample sizes, and they were therefore excluded from this meta-analysis.

Meta-analysis methodology. Meta-analysis feasibility [START_REF] Hedges | Statistical methods for meta-analysis[END_REF][START_REF] Cooper | The handbook of research synthesis[END_REF][START_REF] Rosemberg | MetaWin: Statistical software for Metaanalysis[END_REF][START_REF] Scheiner | Design and analysis of ecological experiments, 2th edn[END_REF]) depends on obtaining an estimate of the effect size (i.e. the magnitude of the effect of interest) from every study. The effect of interest in the present paper was the differential effect exerted by each organism (shrimps [SHR], fish [FISH], bivalves [BIV] and polyculture [POLY]) cultivated in different water bodies (i.e. ecosystem; freshwater, mixed or marine conditions) on dissolved nutrients (NH 4 , NO 2 , NO 3 , PO 4 , SiO 2 ). The most common measure of effect size is the difference between means of the controls and farmed areas, standardised by dividing by the pooled standard deviation [START_REF] Cohen | Statistical power analysis for the behavioural sciences[END_REF]. This standardised mean difference, Hedges' d (hereafter called simply d), is conventionally considered to be 'large' for values of 0.8 or higher (i.e. the farmed area group mean is eight tenths of a standard deviation greater than that of the control group), 'medium' for values of 0.5 and 'small' when d equals 0.2 [START_REF] Cohen | Statistical power analysis for the behavioural sciences[END_REF]. The usual method is to provide the 95% confidence intervals (CI) for d as well. When CI overlaps zero, there is no significant difference between controls and farmed areas.

Another fundamental part of meta-analysis is to calculate the cumulative effect size representing the overall magnitude of the effect present in all studies. When the calculated CI of the cumulative effect size does not bracket zero, it is considered to be significantly different from zero (in the case of the present topic, for example, a significant CI would indicate significant evidence that farm loadings would have a certain effect on dissolved nutrients). The magnitude scale for the cumulative effect size is that proposed by [START_REF] Cohen | Statistical power analysis for the behavioural sciences[END_REF]. In addition, to calculate the degree of heterogeneity among case studies and to estimate whether effect size d was homogenous among studies, I used the Q statistics (Q tot ; [START_REF] Hedges | Statistical methods for meta-analysis[END_REF].

The meta-analysis approach used here was similar to that reported in [START_REF] Gurevitch | A meta-analysis of competition in filed experiment[END_REF] and [START_REF] Hedges | Statistical methods for meta-analysis[END_REF]. I first tested whether all studies shared a common effect size, but having established that the hypothesis of equality among effect sizes was rejected (i.e. studies were highly heterogeneous not differing only for the sampling errors), the data was analysed in groups. In so doing, I estimated the singular overall effect of aquaculture on each nutrient, the effect on each cultivated organism and, where the deviation from a common natural pattern was different, the ecosystem factor was also tested.

Means and sample size data were taken from publication tables and figures. Data from figure format were captured from plots using TechDig (rel. 2.0d; R. B. Jones 1998) of which the error margin was estimated at around 0.2-0.5%. Once all the data had been obtained and entered into a MS Excel spreadsheet, the entire dataset was standardised for the type of deviation (standard deviation or standard error) and the units of measure (µM or mg l -1 ). Most of the papers reported means and standard deviations, while others reported means and standard errors (as a function of their experimental design). Some papers expressed the concentration in water of each nutrient in µM and others in mg l -1 . In the present meta-analysis, I transformed all deviations to standard deviations using the calculator included in the MetaWin 2.0 software [START_REF] Rosemberg | MetaWin: Statistical software for Metaanalysis[END_REF], while all concentrations were transformed into mg l -1 . Since one of the major concerns of a meta-analyst is the publication bias (i.e. the selective publication of articles showing certain types of results in preference to those showing other types of results, substantially increasing the risk of distortion of the true direction of the effect) the d normal quantiles were plotted versus the standardised mean effect [START_REF] Rosemberg | MetaWin: Statistical software for Metaanalysis[END_REF]. The normal quantile plot also allowed possible deviations of the studied cases to be examined [START_REF] Wang | Using the normal quantile plot to explore meta-analytic data sets[END_REF].

Furthermore, the Rosenthal index enabled the fail-safe number to be estimated, i.e. the number of non-significant, unpublished, low accessible or missing studies that would need to be added to a meta-analytic dataset in order to change the results of the meta-analysis from significant to non-significant [START_REF] Rosenthal | The file drawer problem and tolerance for null hypothesis[END_REF][START_REF] Rosemberg | MetaWin: Statistical software for Metaanalysis[END_REF]. All calculations were carried out using MS Excel and MetaWin 2.0 [START_REF] Rosemberg | MetaWin: Statistical software for Metaanalysis[END_REF].

RESULTS

Overview of literature. A total of 52 articles published between 1982 and 2005 in peerreviewed journals (Table 1) were included in the present meta-analysis. This resulted in 427 independent cases wherein all variables were considered together (NH 4 , NO 2 , NO 3 , PO 4 , SiO 2 ). Although the total number of studies represented substantially less than 10% of all accessible and useful information published on this topic in the last two or three decades, it provided sufficient information and gave a reliable estimate of the true effect of aquaculture loadings on the water column. The Rosenthal Index was calculated to be in the order of 7,053. This high Rosenthal value confirmed that the observed results, even with some publication bias, could be treated as a reliable estimate of the true effect. In addition, the possible risk of publication bias or possible deviation of meta-analysis structure were investigated by means of the normal quantile plot (Fig. 1). The plot indicated no deviations from meta-analysis assumptions showing that meta-analytic results were not invalidated by issues in the publication query or by general bias.

The overall effect size of aquaculture loadings on the nutrient pool. When all case studies were analysed together, aquaculture facilities appeared to have an overall effect on dissolved nutrients. This was evident from the cumulative mean effect size (d = 0.45) which, according to [START_REF] Cohen | Statistical power analysis for the behavioural sciences[END_REF], indicated a medium effect of aquaculture loading on the dissolved nutrients in the water column. It was significantly different from a zero effect because its confidence limits did not bracket zero (95% CI = 0.22-0.57). The total heterogeneity (Q tot ) among all studied cases was very high, in the order of 3,563.0 (df = 426; p<0.05) suggesting that variance among effect sizes was greater than expected by sampling error. Thus, assuming this meta-analytic outcome, the studies were broken into groups to test possible influences on the direction of the effect size raised by other factors such as the type of cultivated organisms or ecosystems.

General factors affecting the variability of the dissolved nutrients. Nutrient species were differentially influenced by aquaculture facilities across all studies (Table 2). Ammonium appeared to be the nutrient most affected by loadings from aquaculture facilities irrespective of organisms and ecosystem; nitrites and nitrates and then phosphorus were other compounds to be significantly influenced, while silicates did not show any significant effect (Fig. 2a) as the d value turned out negative and confidence limits bracketed zero.

The grand mean effect size was calculated as a function of each organism type irrespective of the nutrient type and ecosystem. The mean size effect was large, in the order of 0.94 (95% CI = 0.80-1.1; p<0.05), while the total heterogeneity was 816.59 (p<0.05). Almost all organisms had an effect on the water column, and polycultures in particular appeared to have a major effect on the dissolved nutrients, followed by fish and shrimps. Bivalves did not show any significant influence on the dissolved nutrient levels as the d value turned out negative and confidence limits bracketed zero (Table 3; Fig. 2b).

The overall effect of organism type on nutrients appeared to be significant in each ecosystem analysed (Table 4; Fig. 2c). Indeed, the meta-analysis suggested that the effect was higher in freshwater environments than in mixed waters and lowest in marine waters. All these effects were significantly higher than zero.

From a differential point of view, the valid shrimp cases regarding only mixed waters showed that they affected mainly ammonium and nitrite, while other nutrients were not influenced (Fig. 2d). It was not possible to make a comparison between shrimp farming in mixed waters and marine or freshwater conditions. Fish appeared to have a major effect in fresh waters, where all nutrients significantly responded to farm loadings. In marine waters only ammonium and nitrites appeared to be significantly affected, while in mixed waters, few cases were available and these showed no significant effect elicited by fish on the sum of the dissolved nutrients (Fig. 3). Valid cases of bivalve cultivations could only be studied in marine waters regarding ammonium, nitrates and phosphorus (Fig. 4). Bivalves appeared to have no effect among nutrient levels. Polyculture appeared to have the biggest effect size among all cultivation types in marine waters and, although only a few cases were available, it was possible to estimate the effect on ammonium, which was very high (d = 6.84; 95% CI = 4.06-9.61; p<0.05). In contrast, in fresh waters, polyculture had an apparent effect on ammonium, nitrites and phosphorus, but these effects were not significant (Fig. 5). Finally, a correlation between the size effect on biomass expressed as total biomass (tonnes) and the number of species cultivated was attempted. In both cases, there was no relationship (p>0.05) with biomass or the number of species.

DISCUSSION

The ecological effects of aquaculture on water column quality have still not received sufficient critical examination (sensu [START_REF] Islam | Nitrogen and phosphorus budget in coastal and marine cage aquaculture and impacts of effluent loading on ecosystem: review and analysis towards model development[END_REF]. This view is supported by the high heterogeneity of studies available in the current literature. High values of heterogeneity are not common throughout the ecological literature [START_REF] Cote | Effects of marine reserve characteristics on the protection of fish populations: a meta-analysis[END_REF]) except for some rare cases [START_REF] Gurevitch | A meta-analysis of competition in filed experiment[END_REF]. This seems to demonstrate, in contrast with other ecological topics, that the present topic may be affected strongly by several sources of variability producing high levels of heterogeneity in studying the response of dissolved nutrients in the water column to aquaculture loadings.

Features of cultivated organisms (fish, shrimps and molluscs) and their metabolic processes, ecosystem type (mixed, marine and fresh water), typology of cultivation (ponds, cages in open waters, land-based etc.), influence from receiving aquatic ecosystems (e.g. hydrodynamics of water bodies and water residence time), and management practices (e.g. annual biomass productions, feed supply etc.) have been invoked as major factors affecting the area surrounding farms [START_REF] Islam | Nitrogen and phosphorus budget in coastal and marine cage aquaculture and impacts of effluent loading on ecosystem: review and analysis towards model development[END_REF]. These high and complex sources of variability lead only to a very fragmented panorama, from which one can generalise only with great difficulty about the phenomenon of environmental effects of aquaculture. Nevertheless, the results from the present meta-analysis quantitatively contextualise, in the domain of the current literature, the effects of aquaculture loadings on the surrounding water column. The overall direction of the size effect is indubitably corroborated; i.e. farm loadings stemming from different types of cultivated organisms and in different locations appear to induce a significant deviation from common natural patterns (as measured by each authors in their respective chosen controls) of almost all dissolved descriptors in a water column. Although this result may no longer be an assumption in the current literature, it could provide a synthesis of the direction of aquaculture effects, not least because some authors do not use a common scientific methodology or direction in their research effort, or in the way they report their data and results (sensu [START_REF] Riley | Statistical aspects of aquaculture research: pond variability and pseudoreplication[END_REF][START_REF] Karakassis | Ecological effects of fish farming in the Mediterranean[END_REF][START_REF] Ling | Statistical power in comparative aquaculture studies[END_REF]. To some extent, the present meta-analysis provides confirmation of this perspective, because not all factors usually cited among the main causal factors appeared to have a true effect across the analysed literature.

The cumulative effect. When examining the cumulative results, all nutrients commonly used to describe possible eutrophication risks induced by aquaculture appeared to be affected by loadings, except for silicates. Ammonium was the nitrogen form that more often deviated under cultivation conditions, followed by nitrates and nitrites and lastly by phosphorus. The differing chemical behaviour and the origin of each nutrient could explain these results [START_REF] Valiela | Marine Ecological Processes[END_REF][START_REF] Hargreaves | Nitrogen biogeochemistry of aquaculture ponds[END_REF][START_REF] Quian | Comparison of nutrients release among some maricultured animals[END_REF]. For example, as ammonium is mainly produced by excretion of organisms (both fish and shrimps) and in shallow waters (shallow basins, ponds, estuaries) from sediment-flux derived from the mineralisation of organic matter (e.g. food), it could be expected that it would be the first nutrient greatly affected by aquaculture loadings. More than 80% of nitrogen excretion by shrimps [START_REF] Paez-Osuna | Fluxes and mass balances of nutrients in a semi-intensive shrimp farm in North-Western Mexico[END_REF]) and fish, however, is represented by ammonium [START_REF] Wood | Nitrogen excretion in some marine teleosts[END_REF][START_REF] Tanaka | Kinetics of nitrogen excretion by cultured flounder Paralichthys olivaceus[END_REF]. Phosphorus originating from decomposition of organic matter and metabolic activities has usually been invoked as the nutrient mainly affected after ammonium [START_REF] Beveridge | Cage aquaculture, 2th edn[END_REF] by aquaculture loading, which is an important limiting factor to primary productivity in most aquatic environments [START_REF] Valiela | Marine Ecological Processes[END_REF]. Nevertheless, across the current literature the phosphorus pool appeared less affected with respect to nitrogen forms. Silicates mostly of terrigenous-continental origin [START_REF] Valiela | Marine Ecological Processes[END_REF] did not seem to be affected by aquaculture loadings.

The effect of type of organism and ecosystem. The major factors generating differences among studies in determining differences in dissolved pool appeared to be (1) the type of cultivated organism and (2) the ecosystem where they are cultivated, irrespective of management practices and site-specific features.

Accordingly, in most of the studied cases, types of organism depicted the same direction of the effect on dissolved nutrients, although each organism affected specific nutrients differently, depending on the ecosystem where they are cultivated and the intrinsic biology of each organism (metabolic processes, excreta quantity etc).

The size effect of fish and shrimps appeared to be significantly higher, comparable among each other and effective in determining a nutrient-rich environment in farmed areas regarding the common natural patterns as measured in controls. By contrast, bivalve case studies, although reduced in number (n = 22), did not produce a significant effect. Differences in metabolic processes and biological features among these cultivated organisms combined with the type of management practices could explain these differences. Furthermore, dissolved waste release, for example, showed higher results in these experiments in fish than in bivalves, while shrimps were in the middle [START_REF] Quian | Comparison of nutrients release among some maricultured animals[END_REF]. This observation supports the direction of size effect (see Fig. 2) because the pattern of size effect could appear to correlate with the quantity of excreta release, which is a group-specific factor. However, this pattern is also linked to the type of management practice. Fish are mostly cultivated under intensive conditions and feed is provided by an external source; cultivation of shrimps is often carried out under semi-intensive conditions [START_REF] Paez-Osuna | The Environmental impact of shrimp aquaculture and the coastal pollution in Mexico[END_REF]) and the quantum of feed provided is smaller than for fish. In contrast, bivalves, such as suspensivores, collect their food directly from the environment without any other external input [START_REF] Hickman | The mussel Mytilus: ecology, physiology, genetics and culture[END_REF]). Thus, the direction of the size effect can be further affected by the quality or the quantity of feed supplied to each type of organism [START_REF] Enell | Environmental impact of nutrients form Nordic fish farming[END_REF][START_REF] Beveridge | Cage aquaculture, 2th edn[END_REF][START_REF] Islam | Nitrogen and phosphorus budget in coastal and marine cage aquaculture and impacts of effluent loading on ecosystem: review and analysis towards model development[END_REF]) and moreover can be linked indirectly to the cultivated biomass. However, the dependence between the total cultivated biomass and the number of species was tested and there was no significant relationship, which is surprising, because in many reviews these two factors are those most often invoked to explain differences in dissolved nutrient levels among farmed areas and controls.

Polyculture seems to be a separate case, having been cited on many occasions as a practice able to increase the environmental sustainability of aquaculture and economic incoming [START_REF] Troell | Integrated mariculture: asking the right questions[END_REF][START_REF] Neori | Integrated aquaculture: rationale, evolution and state of the art emphasizing seaweed biofiltration in modern mariculture[END_REF]). The high magnitude of the measured size effect shows that the combination of different organisms coming from different trophic levels cultivated together did not minimise differences between controls and farmed areas with respect to monoculture. Such a result is quite puzzling but is very likely strongly divergent due to the low number of studies analysed from very different types of papers (Costa-Pierce 1998, [START_REF] Neori | Sustainable integrated system for culture of fish, seaweed and abalone[END_REF][START_REF] Wahab | Effects of adding the small fish punti (Puntius sophore Hamilton) and/or mola (Amblypharyngodon mola Hamilton) to a polyculture of large carp[END_REF] which resulted from the low accessibility of other data meeting all the requirements of reliability to be analysed with meta-analytic procedures. Thus, although reliable papers used in this meta-analysis were very few and highly heterogeneous, I wanted to analyse them to emphasise the scarcity of reliable information existing on this topic across the peer-reviewed literature.

Ecosystem type seems to be the other main causal factor determining differences among studies. The effect of aquaculture appeared highly location-specific, linked to width of the water body and consequently to the hydrodynamic regime, rather than to the cultivated biomass or number of cultivated species or type of species. In contrast, the direction effect seems, in general, inversely correlated to the size of the water body (sensu [START_REF] Fernandes | The scientific principles underlying the monitoring of the environmental impacts of aquaculture[END_REF]) and indirectly to hydrographic conditions [START_REF] Sarà | Use of stable isotopes to investigate dispersal of waste from fish farms as a function of hydrodynamics[END_REF]): the higher the degree of enclosure of water bodies, the bigger the effect size (i.e. effects of cultivation in fresh waters were greater than effects in mixed waters, and these in turn were greater than the effects in marine waters). Consequently, it was possible to extrapolate that nutrient loadings could be a function of degree of enclosure of water bodies, that indirectly determines both the flushing rate levels and the intrinsic chemical features of waters [START_REF] Pillay | Aquaculture and the environment, 2th edn[END_REF]). In closed lakes, allochthonous nutrient loadings coming from farms amass to the autochthonous dissolved pool that in limited hydrodynamics (sensu Sarà in press, [START_REF] Sarà | Use of stable isotopes to investigate dispersal of waste from fish farms as a function of hydrodynamics[END_REF]) can more easily induce a deviation of farmed areas from controls, thus altering the chemical equilibrium of the water column. In closed fresh waters, throughout the literature studied, the difference between controls and farmed areas was very high, and the highest for the resulting effect size. In mixed ponds or estuaries, where there is a concomitance of fresh-and marine waters, and where the circulation within ponds is often forced by gravity or pumps, allochthonous nitrogen and phosphorus loadings are often forced out of basins, which partially limits their accumulation.

Therefore, the direction of the effect size suggests a general significant difference between controls and farmed areas, although it was lower than in fresh waters. In marine open waters, the intrinsic feature of openness would appear to limit the accumulation of allochthonous nutrients, reducing differences between controls and farmed areas and the overall size effect throughout the literature. To better investigate the link between the degree of openness of water bodies in the sea (an important concern highlighted in many reviews; Enell 1995) and the extent of effects, I attempted to classify the structure of aquaculture areas in semi-closed, sheltered, open etc. for each study. The results obtained were highly subjective due to limited knowledge of each site, conditions of which could not be ascertained and, because of this, the attempt was abandoned.

Statistical issues. The present meta-analysis based on the quantitative estimates of variance

among all studies led me to exclude a high percentage of peer-reviewed papers not responding to meta-analytic criteria (~40%). In addition, most of the papers included in this meta-analysis reported the deviation tendency as standard deviations and only rarely as standard errors for means. This suggests that the accuracy of measures is quite low, and that, due to the limited reporting of standard errors, there is an overall weakness in experimental design across the literature [START_REF] Fernandes | The scientific principles underlying the monitoring of the environmental impacts of aquaculture[END_REF][START_REF] Ling | Statistical power in comparative aquaculture studies[END_REF]. The study of effects of aquaculture on the surrounding environment seems to suffer from this deficiency, i.e. authors appeared rather lax in designing their experiments. This is quite typical of ecological studies carried out in large experimental fields (sensu Oksasen 2001) and is a genuine cause for concern. In most of these cases, however, it is simply not possible to plan experimental designs with the same rigour as with those in small enclosures, aquaria, a laboratory or similar controlled conditions (sensu Oksasen 2001). The degree of replication in large field conditions (sensu Hulbert 1984) is doubtlessly affected by several variables and factors that very often can be controlled only with difficulty (sensu Oksasen 2001). In my view, this is the cause of the high variance between studies that is greater than the intrinsic sampling errors from each study. Throughout the scientific literature on this subject, it is clear that the effort involved in sampling has been huge; more often than not, however, it is not channelled into building a good experimental design [START_REF] Schmitt | Detecting ecological impacts. Concepts and applications in coastal habitats[END_REF], sensu Underwood 1997). The resulting situation is that in most of the studies, it is hard to detect the true effect of aquaculture facilities on the surroundings [START_REF] Schmitt | Detecting ecological impacts. Concepts and applications in coastal habitats[END_REF], which enhances the risk of statistical errors (sensu Hulbert, 1984;[START_REF] Underwood | Experiments in ecology. Their logical and interpretation using analysis of variance[END_REF].

Research priorities, new perspectives and trends. The results of the present meta-analysis, even when cautiously interpreted, raise many more questions than they have answered. At the start of my data collection, it seemed that the huge quantity of publications would lead to a clear view of aquaculture effects on the environment. However, although a large amount of data had been published about this topic, the difficulty in finding them, the overall lack of useful information and the weakness in experimental designs all limit the conveying of such knowledge to the audience. Consequently, although we can find several thousands of citations through the WWW, the number of papers offering useful, accessible and substantially prominent information is only small. This has serious implications for potential environmental repercussions. More effort should be directed towards understanding the extent to which, and in what way, this anthropogenic disturbance can affect water column quality. Some of the present findings, in particular, should be made priority issues in any future research. Some authors have suggested polyculture as a potential solution to some aspects of eutrophication, because the contextual cultivation of species from different trophic levels (e.g. algae and bivalves together with carnivorous fish) would reduce the impact that would emerge from the cultivation of only one carnivorous species [START_REF] Troell | Integrated mariculture: asking the right questions[END_REF]. Although theoretically this may be a satisfactory assumption, there is no evidence in the current literature, apart from a few (influential) in-yard and open field experiments, to support the notion that polyculture reduces environmental impacts. Thus, due to the scant information on this topic, greater effort should be directed towards examining this assumption that as yet is only theory.

Although bivalves are among the primary cultivated species in the world, involving large coastal populations and often political concerns [START_REF] Hickman | The mussel Mytilus: ecology, physiology, genetics and culture[END_REF], there is very limited information in the current literature about their potential impact on dissolved nutrient levels. This is very unusual, as they have received much attention from researchers over the last three or four decades, of which physiological aspects and, in particular, excretion rates have been the most investigated features (see for example [START_REF] Dame | Ecology of marine bivalves: an ecosystem approach[END_REF], or Wildish & Khristmanson 1997 as reviews).

Although the choice of descriptors of dissolved nutrients is widely consolidated, some studies still pay attention to silicates. However, they appear to be poor descriptors of behaviour of heterotrophic loadings in a water column. In contrast, nitrogen forms appeared to be the more highly informative descriptors of eutrophication risks in a water column, while in sensu stricto, the role of dissolved phosphorus in describing the risk of eutrophication results was limited only to small water bodies, contradicting the commonly held view that phosphorus is, in most cases, the best descriptor.

Lastly, the present meta-analysis was not aimed at investigating the consequential effects of dissolved pool changes on suspended biological communities (phytoplankton, free-living bacteria etc; Sarà in preparation). Thus, since a deviation from a common natural pattern of dissolved nutrients does not necessarily imply a direct response of the suspended biota (sensu [START_REF] Pitta | Natural versus mariculture induced variability in nutrients and plankton in the Eastern Mediterranean[END_REF], Sarà et al. 2006), the discovery of contextual reporting, within the same article, of both chemical and biological data, would possibly reveal the true detrimental effects of aquaculture loadings on the environment. Tables Table 1. The list of peer-reviewed papers published from 1982 to 2005 included in the present meta-analysis, reporting also countries (n = 30) where experiments were carried out, areas, type of ecosystem (mixed, freshwater and marine) and cultivated organism (SHR = shrimps; FISH = fish; BIV = bivalves; POLY = polyculture). The total cultivated species were 45 among fish (n = 24), shrimps (n = 11), bivalves (n = 7) and algae or plants (n = 3). The topological features of each study have been also reported (controlled = environment controlled-by-researchers with variable volumes from a few litres [mesocosm] Figure 3 The mean effect size of fish aquaculture in all ecosystems on each nutrient (NH 4 = ammonium; NO 2 = nitrite; NO 3 = nitrate; PO 4 = phosphorus; SiO 2 = silicates) (95% confidence limits were reported).

Figure 4 The mean effect size of bivalve aquaculture in marine waters on each nutrient (NH 4 = ammonium; NO 2 = nitrite; NO 3 = nitrate; PO 4 = phosphorus; SiO 2 = silicates) (95% confidence limits were reported).

Figure 5 The mean effect size of polyculture in fresh waters on each nutrient (NH 4 = ammonium; NO 2 = nitrite; NO 3 = nitrate; PO 4 = phosphorus; SiO 2 = silicates) (95% confidence limits were reported).

Figures

Figure 1

The

Figure captions Figure 1 Figure 2

 captions12 Figure captionsFigure1The normal quantile plot plotted using all studies (n = 427) Figure2General meta-analytical results with a) the mean effect size of all cumulated aquaculture facilities across all studies on each nutrient (NH 4 = ammonium; NO 2 = nitrite; NO 3 = nitrate; PO 4 = phosphorus; SiO 2 = silicates ALL = all nutrient cumulated together); b) the mean effect size of each organism type on dissolved nutrient levels across all studies and in all ecosystems cumulated together (SHR = shrimps; FISH = fish; BIV = bivalves; POLY = polyculture; ALL = all organisms cumulated together); c) the mean effect size of aquaculture facilities in each ecosystem analysed irrespective of dissolved nutrient type and organism across all studies and d) the mean effect size of shrimp aquaculture only in mixed water on each nutrient (NH 4 = ammonium; NO 2 = nitrite; NO 3 = nitrate; PO 4 = phosphorus; SiO 2 = silicates).

  impact of aquaculture on dissolved matter: a meta-

  Figure 3

Table 2 .

 2 to some hundred or thousand of m 3[enclosure]; open field = open environment mostly in the sea or in estuaries never controlled by researchers; pond = earthen basins usually used for shrimp cultivation) Effect of aquaculture facilities on each dissolved nutrient across all studies, in all ecosystems and for each type of organism (NH 4 = ammonium; NO 2 = nitrite; NO 3 = nitrate; PO 4 = phosphorus; SiO 2 = silicates ALL = all nutrient cumulated together; df = degree of freedom; d + = mean size effect; 95% CI = 95% confidence interval; P = probability level)

	#	STUDY	COUNTRY	AREA	ECOSYSTEM TYPE TOPOGRAPHY
	1 Alongi et al 1999	Vietnam	Pacific	Mixed	SHR Pond
	2 Alongi et al 2003	Malaysia	Pacific	Mixed	FISH Open field
	3 Azim et al 2003	Netherland	Inland	Freshwater	FISH Controlled
	4 Ball et al 1997	Ireland	Atlantic	Marine	BIV	Open field
	5 Bechara et al 2005	Argentina	Atlantic	Freshwater	FISH Controlled
	6 Biao et al 2004	China	Pacific	Marine	BIV	Pond
	7 Boaventura et al 1997	Portugal	Atlantic	Freshwater	FISH Open field
	8 Burford 1997	Australia	Pacific	Mixed	SHR Pond
	9 Burford et al 2003a	Australia	Pacific	Mixed	SHR Pond
	10 Burford et al 2003b	Belize	Atlantic	Marine	FISH Pond
	11 Chandra Das et al 2005	India	Indian	Freshwater	FISH Controlled
	12 Chin and Ong 1997	Singapore	Pacific	Freshwater	SHR Pond
	13 Christensen et al 2003	New Zealand	Pacific	Marine	BIV	Open field
	14 Costanzo et al 2004	Australia	Pacific	Mixed	SHR Pond
	15 Costa-Pierce 1998	California, USA	Pacific	Freshwater	POLY Controlled
	16 Cowan et al 1999	Thailand	Pacific	Mixed	SHR Pond
	17 De Casabianca et al 1997	France	Mediterranean Marine	BIV	Open field
	18 Figueredo and Giani 2005	Brasil	Inland	Freshwater	FISH Controlled
	19 Galope-Bacaltos et al 1999	Phillippines	Pacific	Freshwater	FISH Open field
	20 Green et al 2002	Egypt	Mediterranean Freshwater	FISH Controlled
	21 Guerrero-Galvan et al 1999	Mexico	Pacific	Mixed	SHR Pond
	22 Guo and Li 2003	China	Inland	Freshwater	FISH Open field
	23 Hopkins et al 1995	South Carolina, USA Atlantic	Mixed	SHR Open field
	24 Hussenot 2003	France	Atlantic	Marine	FISH Open field
	25 Islam et al 2004	Bangladesh	Indian	Mixed	SHR Pond
	26 Jackson et al 2004	Australia	Pacific	Mixed	SHR Pond
	27 Jones et al 2001b	Australia	Pacific	Mixed	SHR Pond
	28 La Rosa et al 2002	Italy	Mediterranean Marine	BIV	Open field
	29 Martin et al 1998	New Caledonia	Pacific	Marine	SHR Controlled
	30 McKinnon et al 2002	Australia	Pacific	Mixed	FISH Open field
	31 Merceron et al 2002	France	Atlantic	Marine	FISH Open field
	32 Motzkin et al 1982	Israel	Mediterranean Marine	FISH Pond
	33 Neori et al 2000	Israel	Mediterranean Marine	POLY Controlled
	34 Nordvarg and Johansson 2002	Finland	Atlantic	Marine	FISH Open field
	35 Ogilvie et al 2000	New Zealand	Pacific	Marine	BIV	Open field
	36 Paez-Osuna et al 1997	Mexico	Pacific	Mixed	SHR Pond
	37 Pietros and Rice 2003	Rhode Island, USA	Atlantic	Marine	BIV	Controlled
	38 Pitta et al 1999	Greece	Mediterranean Marine	FISH Open field
	39 Pitta et al 2005	Greece	Mediterranean Marine	FISH Open field

Table 3 .

 3 Effect of organism type on the sum of dissolved nutrients through all studies and in all ecosystems (SHR = shrimps; FISH = fish; BIV = bivalves; POLY = polyculture; ALL = all organisms cumulated together; df = degree of freedom; d + = mean size effect; 95% CI = 95% confidence interval; P = probability level)

	Organism df	d +	95% CI	P
	SHR	132 0.71 0.46-0.96 <0.05
	FISH	257 1.10 0.92-1.29 <0.05
	BIV	22 -0.03 -0.69-0.62 >0.05
	POLY	12	1.80 0.91-2.65 <0.05
	ALL	426 0.94 0.80-1.10 <0.05

Table 4 .

 4 Effect of aquaculture facilities in each ecosystem analysed irrespective of dissolved nutrient type and organism across all studies (ALL= all nutrient cumulated together; df = degree of freedom; d + = mean size effect; 95% CI = 95% confidence interval; P = probability level)

	Ecosystem	df	d +	95% CI	P
	Mixed waters 153 0.61 0.39-0.84 <0.05
	Freshwater	116 2.04 1.78-2.30 <0.05
	Marine waters 125 0.37 0.14-0.61 <0.05
	ALL	426 0.93 0.76-1.11 <0.05

Acknowledgements:

I thank all scientists who responded to this literature search, Dr. J. Gurevitch (State University of New York at Stony Brook, USA) for the primary input into this meta-analysis, Dr. D. Campobello (University of Manitoba, Canada) and Prof. John Dean (University of South Carolina, USA) for their continued reviewing of earlier versions of this manuscript and to Prof. A. Mazzola (University of Palermo, Italy), my mentor, who was a major impetus in the starting of my scientific career. Thanks are also due to the anonymous referees who amended an