Iranian Journal of Fisheries Sciences 17(4) 641-656 2018 DOI: 10.22092/ijfs.2018.116991

Assessing benthic health of hard substratum macrobenthic community using soft bottom indicators and their relationship with environmental condition

Mehdipour N.1*; Gerami M.H.2; Nemati H.3

Received: April 2016 Accepted: June 2016

Abstract This study aimed to assess ecological quality status of hard substratum macroinvertebrates communities of the with three ecological indices and their relationship with environmental factors. For this purpose, benthic communities of the Caspian Sea basin were studied seasonally during 2014 in 8 sampling sites. Temperature, salinity, dissolved oxygen, pH, nitrate, nitrite, silicate and phosphate were measured as environmental factors. The benthic classification indices AMBI (AZTI Marine Biotic Index), M-AMBI (Multivariate AMBI) and BENTIX (BENthic IndeX) were applied to assess the ecological status of the studied area. Results showed low dissimilarity based on species composition and abundance among seasons, while all seasons discriminated clearly based on environmental factors. In addition, AMBI index was more successful to assess ecological health of hard substratum in the Caspian Sea basin than M-AMBI and BENTIX. Furthermore, AMBI showed high sensitivity to environmental variation. Results indicated that temperature, nitrate, silicate, phosphate and nitrite were the most important factors in the composition and abundance fluctuation of hard substratum macroinvertebrates communities, respectively.

Keywords: Benthic health, AMBI, M-AMBI, BENTIX, Environmental factors, Caspian Sea

1-Iranian National Institute for Oceanography and Atmospheric Sciences, Iran. 2-Young Researchers and Elite Club, Shiraz Branch, Islamic Azad University, Shiraz, Iran. 3-Faculty of Biological Sciences, Shahid Beheshti University. *Corresponding author's Email: [email protected] 642 Mehdipour et al., Assessing benthic health of hard substratum macrobenthic community using…

Introduction stresses (Zubikarai et al., 2014; Hard substratum benthic communities Abbaspour et al., 2017; Ghorbanzadeh in coastal areas, especially those areas Zaferani et al., 2017). Therefore, these that located near or at urban and organisms are considered powerful industrial centers, are highly affected by indicators of environmental pollution. anthropogenic activates. (Fraschetti et The AZTI Marine Biotic Index al., 2001; Zalmon et al., 2011; Spaccesi (AMBI, also referred to as BC) and Rodrigues Capitulo, 2012). developed by Borja et al. (2000) Although these shores support a rich evaluates ecological health condition in biodiversity, but contaminations such as five categories based on the distribution heavy metals and/or bacteria, nutrients of individual abundances in benthic from organic or industrial pollution communities. The species were from diverse sources and from distributed in those groups according to sedimentation seriously affected the their sensitivity to an increasing stress diversity and functioning of these gradient (enrichment of organic matter) ecosystems (Terlizzi et al., 2002; De (Glémarec and Hily, 1981; Hily, 1984). Wolf et al., 2004; Piola and Johnston, M-AMBI (‘Multivariate AMBI’, Bald 2008). et al., 2005; Muxika et al., 2007) is a Hard communities are stressed multivariate index for assessing the environments; due to waste water ecological health and quality status of discharges that is close to the coastline. marine and transitional waters. It is These waters cause deterioration in based on benthic macroinvertebrates water quality and discourage the communities and integrates AMBI, a settlement of several organisms; that biotic index based on species affect these communities thriving in sensitivity/tolerance, with diversity and rocky-shores (Arévalo et al., 2007). richness (Sigovini et al., 2013). It aims Organic and nutrient enrichment due to to integrate the response of species domestic wastes is today one of the richness, the Shannon diversity index main reasons explaining the (Shannon and Weaver, 1949) and the deterioration of marine nearshore biotic index AMBI (Borja et al., 2000). ecosystems (Fletcher, 1996). In Simboura and Zenetos (2002) designed addition, seasonal oscillations in new index based on the AMBI Index environmental conditions dramatically (Borja et al., 2000) with a re- influence the macrobenthic combination of ecological groups that communities in coastal waters assigns different weighting coefficients (Reizopoulou et al., 2014). Benthic and results to the reduction of invertebrates and macroalgae are macrozoobenthic data in three wider sedentary long lives, easy sampling ecological groups (Simboura and organisms that many literatures Argyrou, 2010). published on their distribution in Anthropogenic disturbances and specific environments and on their habitat natural changes are the most response to different environmental important factors in reaction of aquatic Iranian Journal of Fisheries Sciences 17(4) 2018 643 organisms (Nouri et al., 2008; Saghali bottom benthic indices in assessing the et al., 2013). Benthic fauna's structure ecological health in these coastlines. is affected by environmental factors such as temperature, pH, dissolved Materials and methods oxygen and pollution (Sharma and Sampling was performed in 8 sites form Rawat, 2009; Saghali et al., 2013). southwest to southeast shores of the Variation in these factors cause changes Caspian Sea in Iranian waters basin. in rate of supply of organic matter and Sites were located to Astara (S1), consequently affect the composition Anzali (S2), Chamkhaleh (S3), Ramsar and abundance of marine organisms, (S4), Sisangan (S5), Babolsar (S6), such as macrobenthic communities Amirabad (67) and Khajeh Nafas (S8) (Erftemeijer and Herman, 1994; (Fig. 1; Table 1). Sampling was done at Bachelet et al., 2000). the midpoint of each season from spring Assessment of the to winter 2014. Temperature, salinity, macroinvertebrates communities’ health dissolved oxygen and pH were in soft bottom substratum has measured using the portable multi- progressed in recent years (Borja, 2005; meters (HACH 51154, USA) with three Borja, 2006; Kutser et al., 2006; Pinedo replicates in each site. Species area et al., 2007; Borja et al., 2008), but data curve method applied for sampling on hard substratum is limited. The aim from (Browne, 1996). 20×20 cm of this study was to examine the hard- quadrat (0.04 m2) was performed on substratum benthic community rocky substratum from supralittoral organism in 8 sites of the Caspian Sea zone to sample from macrobenthic rocky seawalls under different communities with three replicate and environmental conditions and relate it samples were preserved in 4% formalin. to ecological status indices. Moreover, In the laboratory, the macrofauna were the study explores the applicability of sorted, identified up to species level and three of the most commonly used soft counted (Freeman and Bracegirdle, 1971).

Figure 1: Map showing the sampling locations in the southern Caspian Sea (2014). 644 Mehdipour et al., Assessing benthic health of hard substratum macrobenthic community using…

Table 1: Geographical coordinates of the stations. Station N E

Astara 38°25´59.33” 48°52´52.77” Anzali 37°28´53.00” 49°27´20.55” Chamkhaleh 37°12´57.68” 50°16´33.22” Ramsar 36°55´45.75” 50°40´05.19”

Sisangan 36°35´02.49” 51°48´43.41” Babolsar 36°42´52.43” 52°39´35.40” Amirabad 36°51´30.55” 53°23´22.17” Khajeh nafas 36°57´49.43” 54°00´52.51”

Surface water samples were collected study; salinity from 0 to 36 ppm for simultaneously from rock pools in all Mediterranean lagoons and 0 to 16 ppm the selected sampling sites for analysis for Caspian Sea in this study; Oxygen the nutrient contents. Water nutrient from 0 to 14 mL L-1 for Mediterranean concentration was measured according lagoons and 0 to 11 mL L-1 for Caspian to photometric methods (Wood et al., Sea in this study; Nitrate from 0.1 to 1967; Strickland and Parsons, 1972). 300 µg L-1 for Mediterranean lagoons Phosphate analyzed by a modified and 0 to 600 µg L-1 for Caspian Sea in ascorbic acid reduction method and this study; pH from 7.5 to 9.8 µg L-1 for silicate assessed based on calorimeter Mediterranean lagoons and 8.3 to 8.9 with formation of molybdic acid µg L-1 for Caspian Sea in this study (Strickland and Parsons, 1972). Nitrite (López and Tomàs, 1989). The determined by colorimetric and ion reference values for M-AMBI were set chromatographic methods and nitrate as: Diversity=0 to 1.62, S=0 to 7 and was measured based on cadmium- AMBI=0.09 to 4, based on AMBI copper reduction to nitrite (Wood et al., calculation. To calculate the BENTIX 1967). (BENthic IndeX) (Add-in v.1.0 version) AMBI, M-AMBI and BENTHIX the software for MS Excel 2007 has biotic indices were calculated by been used downloaded free from: following methods: To calculate the http://www.hcmr.gr/en/articlepage.php? AMBI and M-AMBI, the free software id=141. Diversity indices (Margalef, (http://www.azti.es v.4) along with the Pielou, Shannon and Simpson) and guidelines from the authors (Borja and MDS analyses were carried out using Muxika, 2005) was used in this study. the PRIMER v5 software package, There are no proposed reference values developed in the Plymouth Marine for M-AMBI in the Caspian Sea; Laboratory. Canonical Correlation therefore, due to similarity of the Analysis (CCA) and Canonical Caspian Sea with Mediterranean Discriminant Analysis (CDA) analysis lagoons, (mean average of temperature: was assessed by R statistical packages 16 to 23 °C for Mediterranean lagoons (Version 3.13, CCA package). SIMPER and 13 to 31 °C for Caspian Sea in this analysis preformed to assess Iranian Journal of Fisheries Sciences 17(4) 2018 645 dissimilarities between seasons and parameters are illustrated in the CDA sites. In addition, Tow-Way- analysis (Fig. 2). It shows that all PERMANOVA with 9999 permutations seasons discriminated clearly. carried out to determine significant However, this separation was higher difference between environmental between summer with other seasons. In factors in different seasons and sites by addition, temperature and pH was the PERMANOVA 1.6-Anderson. most important factors in discriminant Results analysis. The differences among the eight sites regarding to the environmental

Figure 1: CDA analysis for environmental parameters in all sites (Sal= Salinity, Temp= Temperature, Phos= Phosphate, Sil= Silicate, Nitra= Nitrate, Nitri= Nitrite and O2= Oxygen).

Tests of dimensionality and 2 and Fig. 2, indicate that one of the standardized for the canonical four canonical dimensions are correlation analysis, as shown in Table statistically significant at the .05 level.

Table 2: CCA analysis between species and nutrients in all sites Dimension Correlation p value 1 0.5199 0.01230497 2 0.4258 0.25825982 3 0.2539 0.85435332 4 0.1136 0.94958338 Factors (Species and Nutrient) Dimension 1 Benthic Macrofauna Pontogammarus maeuticus -0.84062775 Balanus improvisus 0.07808775 Rhithropanopeus harrisii tridentatus 0.06355215 Simulium sp. -0.70477671 Chironomus albidus 0.79912391 lineatus 0.08378822 Nereis diversicolor -0.27048504 Tubificoides fraseri 0.26574585 Nutrient Nitrate -1.1022654 Nitrite 0.5875904 Phosphate -0.6865075 Silicate 0.9421993 646 Mehdipour et al., Assessing benthic health of hard substratum macrobenthic community using…

Fig. 3 shows CCA plot between with nitrate. In addition, feebly positive environmental factors and relationship was observed between macroinvertebrates community. Tubificoides fraseri and nitrate and According to the plot, Chironomus phosphate. albidus had strong positive relationship

Figure 2: Two dimensional CCA plot for benthic macrofaunal and nutrients variables (Phos=Phosphate, Nitrit=Nitrite, Nitra=Nitrate and Silic=Silicate).

For the Benthic macrofauna variables phosphates, nitrates etc.). Results dimension 1 was most strongly indicate that site 8 and 6 was grouped influenced by Pontogammarus clearly according to environment, while maeuticus. For the nutrient variables, site 1, 7 and 8 was clearly separated by nitrate was the strongest variable than benthic condition. Furthermore, other parameters. analyzing based on nutrient condition in Fig. 4 shows MDS ordination plot sites revealed that site 7, 2, 8, 3 and 6 based on environmental (e.g. salinity, was clearly grouped and discriminated temperature etc.), benthic communities from other sites. and nutrition conditions (e.g. Iranian Journal of Fisheries Sciences 17(4) 2018 647

Figure 4: Multidimensional scaling based on: A) environmental condition, B) benthic composition, and C) nutrients conditions in the southern Caspian Sea (2014).

Table 2 shows SIMPER analysis among responsible for differences between, as sites and time series. Data analysis well as dissimilarity within benthic indicated there were different guilds of assemblages. Mytilaster lineatus, P. benthic species at each of these eight maeuticus and Balanus improvises were communities, which are principally contributed to dissimilarity within all 648 Mehdipour et al., Assessing benthic health of hard substratum macrobenthic community using… compared sites and determined the eight sites. Based on the average values, community structure within benthic results showed that all indices were in a communities. uniform range. In fact, undistributed Table 3 lists the average values of the status in AMBI indices coincided with biotic classification indices (AMBI, M- good status in M-AMBI and high status AMBI and BENTIX) and the resulting in BENTIX indices, relatively. ecological quality status (EQS) for the

Table 3: Values of the classification metrics and respective EQS assessment in the all sites.

SD= Slightly disturbed, Un= Undisturbed, MD= Moderately disturbed, ED= Extremely disturbed, P= Poor, B=Bad, G= Good, H= High and EQS= Ecological quality status.

PERMANOVA test performed on varied between seasons in S1, S3 and nutrient data and results showed S7 (p<0.05). Phosphates and silicates seasonal changes in nutrient content showed significant seasonal oscillation with significant differences between only in site S8 (p<0.05). Silicate had each season (p<0.05). According to significant different amounts in summer PERMANOVA test results nitrate in comparison with autumn and winter significantly varied between seasons in in all sites (p<0.05). S1, S4 and S8 (p<0.05) and Nitrite

Table 4: Abundance of species during the study in the southern Caspian Sea (2014) Abundance (number) Ecological Scores Species S1 S2 S3 S4 S5 S6 S7 S8 AMBI BENTIX 15 38 13 44 24 45 42 49 Pontogammarus maeuticus IV 2 62 7 25 00 75 0 86 8 12 64 20 18 49 86 Balanus improvisus II 1 25 66 75 29 33 01 25 50 2 Rhithropanopeus harrisii tridentatus II 1 0 0 75 0 0 25 0 0 Iranian Journal of Fisheries Sciences 17(4) 2018 649

Table 4 continued: Simulium sp. IV 2 0 37 0 0 0 50 0 0 10 26 92 17 40 Chironomus albidus III 2 0 75 0 62 2 5 5 00 14 13 37 11 15 57 24 27 Mytilaster lineatus I 1 31 22 05 45 73 5 75 5 37 50 0 87 6 22 11 22 17 40 20 Nereis diversicolor III 2 0 50 5 50 5 5 0 0 Tubificoides fraseri V 2 50 0 0 0 0 0 0 0 15 14 43 11 25 32 69 96 Total -- -- 04 83 88 90 58 74 61 23 77 87 3 38 6

Discussion nutrient factor in freshwater ecosystems Trophic interactions are the most and it was significantly different among important factor in macrobenthic spatial seasons. However, Howarth et al. and temporal variation in coastal (2000) reported that in many coastal environment (Boaventura et al., 1999). marine systems the limiting nutrient is Koutsoubas et al. (2000) declared that usually nitrogen. In addition, Gao et al. frequent fluctuations in environmental (2011) declared that total nitrogen was parameters (daily, monthly or seasonal among the main environmental factors basis) would cause changes in the affecting the distribution of structure and distribution pattern of macrobenthos, and ammonium organisms. Furthermore, natural nitrogen, nitrate nitrogen, and disturbance often results in the instant chlorophyll a also had definite effects. destruction of great numbers of In addition, Lamptey and Armah (2008) individuals (Guelorget and Perthuisot, stated that spatial and seasonal 1992). These faunal communities’ variability in silicate resulted in habitat structural changes are principal factors heterogeneity among the sampling for assessing ecological health by stations and this heterogeneity among ecological indicators. the stations because of the Nutrient content is the main factor environmental variables possibly responsible for fluctuation in benthic created conditions that influenced the macrofaunal assemblages (Aller et al., abundance patterns of the macrobenthic 2001; Kuffner and Paul, 2001; Stief et fauna. Overall results showed that al., 2002; Sivadas et al., 2012; Amiri et seasonal changes of macroinvertebrates al., 2014). In this study CCA analysis communities were highly associated revealed that nutrient group most with environmental factors. strongly influenced by nitrate and Table 3 showed that all sites had silicate (Table 2) and Fig. 3 showed that acceptable ecological conditions based distribution of species was less on AMBI index. Overall sites, 3 associated with phosphate than other slightly disturbed and 5 undistributed nutrients. There is plenty of evidence status occasions were observed. that phosphate is the main limiting According to PERMANOVA results temperature, oxygen, phosphate, and 650 Mehdipour et al., Assessing benthic health of hard substratum macrobenthic community using… nitrite were the main factors that varied 4C), while S1 overlapped. These significantly between seasons in whole changes in ecological health assessment study area. In addition, all nutrient based on macroinvertebrates were also factors varied significantly between observed in Mediterranean basin. sites. MDS plot based on the benthic Reizopoulou et al. (2014) reported that communities, demonstrated BENTIX and M-AMBI underestimated discrimination between S1, S7 and S8 and AMBI overestimated the ecological with other sites (Fig. 4B). AMBI clearly status of Mediterranean coastal lagoons. separated these sites from the rest in the Borja et al. (2008) stated that the analysis, M-AMBI could not greatest number of disagreements when distinguish these sites and BENTIX comparing AMBI or M-AMBI with was successful in separating S1 and S7 other indices is found in low salinity from other sites (Table 3). However, M- locations. The Caspian Sea is an AMBI was successful to distinguish S1, enclosed inland body of water with S7 and S8 in seasonal analysis from average salinity of 13 ppm (Karbassi et other sites similarly to other indices. al., 2008) and is very low in contrast of Table 4 shows that S1, S7 and S8 had estuarine and marine salinity. The fewer total species than other sites. P. problems in assessing the benthic maeuticus and B. improvisus were ecological status in low salinity or dominant in S1 and S7, and S8, highly changing salinity habitats have respectively. High dominance of P. been discussed under the ‘Estuarine maeuticus with IV score in AMBI Quality Paradox’ (Dauvin et al., 2007; index, resulted changes in ecological Elliott and Quintino, 2007). In addition, status for these sites. The resulted Fig. 2 showed that all seasons were ecological status by M-AMBI diverge discriminated based on environmental from the results of AMBI due to the condition and the Caspian Sea basins diversity components included in the have highly variable ecosystems. method. Species scores are equal in Zubikarai et al. (2014) described two AMBI and M-AMBI formula; and reasons for variation in species richness changes in reference value for AMBI, between rocky substratum s. They Diversity and Richness; caused changes declared that reasons for these in calculation of M-AMBI in differences can be (i) lower discharge comparison of AMBI. In addition, B. or (ii) much higher wave energy improvises with score 1 in BENTIX between sites. However, equitable index, was dominant in S8 and resulted distribution of trophic groups in to high status classification for this site, environment and sampling area, while P. maeuticus with score 2 was indicate a healthier ecosystem dominant in S1 and S7 (Table 4). functioning and, as such, an Furthermore, analysis of nutrient improvement in the quality of the content and environmental condition environment (Bremner et al., 2006). showed that S7 and S8 were clearly Although many benthic indices were separated from other sites (Figs. 4 A, successfully validated during the last Iranian Journal of Fisheries Sciences 17(4) 2018 651 decade, most indices and assessment addition, AMBI showed high sensitivity scales were developed for local to environmental variation. Indeed, geographic regions, and often only for successful classification of these indices specific habitats within the region is highly relevant to regional ecological (Borja and Tunberg, 2011). In fact, conditions. For instance, Simboura and species composition and reference Reizopoulou (2008) studied the rocky conditions change naturally with deep and sedimentary shallow water ecoregion and habitat (Borja et al., body type three lagoonal sites located in 2009). Therefore, many studies have (Eastern Mediterranean) and been conducted to establish different stated that In the studied the rocky deep reference conditions for different areas the BENTIX index seems to give estuarine habitats before benthic a more biologically relevant and condition assessment (Weisberg et al., consistent with the environmental 1997; Borja et al., 2008; Teixeira et al., conditions classification, compared to 2008). However, authors set reference the AMBI assessment, while BENTIX value for M-AMBI based on AMBI was more successful in this study. calculation (by default. Furthermore, Furthermore, results indicated that these three indices were established to temperature, nitrate, silicate, phosphate apply for soft bottom communities and and nitrite were the most important employed for hard substratum factors in spatial and temporal communities in this study. variations of hard substratum Nevertheless, some of them have been macroinvertebrates communities, also successfully applied also for hard respectively. bottom communities as BENTIX index Further studies should be conducted in Bhosphorus Strait communities to determine reference value and (Kalkan et al., 2007) and was boundary limits for hard substratum successful to separate control and region especially in the southern discharge communities. Caspian Sea. It should be noted that Results of this study revealed the dominant species, food webs, habitat weakness of the biotic indices to reflect structure, life span and cycle, and discriminate among the reproductive rate and dispersal potential anthropogenic and natural stress in the are important factors that affect hard substratum ecosystems as well. In ecological quality status of ecosystems addition, species sensitivity, richness and should be considered in analysis. and diversity as benthic community traits do not seem to function well in Acknowledgments assessing the ecological quality status The financial support received from the in these ecosystems. However, AMBI Iranian National Institute for index was more successful to assess Oceanography and Atmospheric ecological health of hard substratum in Sciences for this research work is the southern Caspian Sea basin gratefully acknowledged. compared to M-AMBI and BENTIX. In 652 Mehdipour et al., Assessing benthic health of hard substratum macrobenthic community using…

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