8    Benthic Recolonisation


8.1    Background


The EIA conducted for this Project has indicated that benthic fauna are expected to recolonise the pits following capping with uncontaminated mud.  It is expected that recolonisation of the natural benthic assemblage will occur and eventually the benthic assemblage will resemble that of the surrounding areas.  Recolonisation may be achieved by larval recruitment, influx of juveniles or adults carried in water currents, or through the active swimming or crawling of individuals.  However, other natural (eg storm events, hypoxia, salinity fluctuations) or anthropogenic (eg pollution, dredging activities and fisheries operations) activities may hinder recolonisation of capped pits.  As a result, the factors contributing to the composition of the benthic assemblage may be difficult to determine.  It is also important for any recolonisation studies to be aware of any cap maintenance (or "topping up") activities which may also impact the resident benthic assemblages. 


In order to verify the recolonisation of marine biota on the capped pits, a benthic recolonisation programme is recommended.  The full details of the EM&A programme for benthic recolonisation are presented in the following sections.


8.2    Objective


The objective for this component of the Study is to monitor and report on the benthic recolonisation of the capped pits including the previous ones and specifically to determine the difference in infauna between the capped pits and adjacent sites.


8.3    Hypothesis


The impact hypothesis for this task is as follows:

Recolonisation is occurring at the capped pits such that assemblages at the capped pits become more similar to reference assemblages as time since capping increases.

The null hypothesis to be tested for this work component is as follows:

H0      There is no difference in the structure of benthic infaunal assemblages found at the capped pits at the active facility and adjacent reference areas.

8.4    Sampling Design


The sampling design of this task involves two treatments: capped pits and reference areas (Figures 8.4a and 8.4b).  The capped pit treatment shall involve collection of samples from the capped mud pits at the active facility.  The pits are anticipated to be capped at different times.  The number of samples to be collected at each station, the number of impact stations and their locations should be confirmed prior to the commencement of capping activities and agreed with EPD based on the detailed design of the disposal facility.


The second treatment shall involve sampling at different reference sites where the number of sample stations and the exact position of the sampling sites should be determined prior to the commencement of the capping activities and agreed with EPD based on the detailed design of the disposal facility.  The reference sites should be chosen to improve the balanced nature of the design (ie the number of reference sites should be the same as the impact sites).  Using multiple controls is an effective way of ensuring that the extremely variable nature of Hong Kong's marine benthos from one site to another does not overly influence or alter the results.  Current ecological theory suggests that the use of multiple control sites in sampling designs are statistically more robust and hence the conclusions more reliable ([1]) (2).

One grab sample shall be taken at each of the different stations clustered within each site.  The analysis (Cumulative Running Mean Test to determine optimum sample size for stabilising mean and standard error values) revealed that 12 samples would be the optimum number.  The technique of clustering stations within one site has been proven to be an effective way of testing hypotheses and removing the confounding effects of spatial variation from the interpretation.  The number of samples to be collected at each site and the exact locations of the sampling stations should be determined prior to the commencement of capping activities based on the detailed design of the disposal facility.


8.5    Statistical Treatment of Data


The data collected during the monitoring programme shall be analysed using two different but complementary approaches as detailed below. 


8.5.1    Univariate Analyses


ANOVA & MANOVA:  Simple, univariate measures shall be tested using an Analysis of Variance (ANOVA), and multivariate measures of community structure shall be tested using the Multiple Analysis of Variance (MANOVA).  Both ANOVA and MANOVA test the same null hypothesis using similar methods.  The method is essentially a comparison of the variability within a site to the variability between sites.  If the ratio of these two variances (that is, the between‑group‑variance over the within‑group‑variance) is large enough, then any differences observed are due to true differences that exist between the groups and not just to random variation.  ANOVA and MANOVA tests are based on several assumptions related to the underlying distribution of the data being analysed (ie normality, homogeneity of variances).  If the data deviate significantly from these assumptions, then these tests are considered to be inappropriate.  If this situation arises, alternative procedures (ie parametric tests with rank transformed data or nonparametric analogues such as Kruskal Wallis) which address similar hypotheses but do not require such stringent assumptions shall be adopted.  Observed differences between the sites and/or areas shall be tested using multiple comparison procedures such as the Student Newman Keuls (SNK) or Tukey test.


8.5.2    Multivariate Analyses


Multi Dimensional Scaling (MDS):  Multi dimensional scaling (MDS) shall also be used to depict the similarities between stations based on their benthic assemblages.  MDS is a method for creating a low dimensional picture of the relationships between stations in a complex, multi dimensional problem.  The Bray-Curtis distance metric shall be used for both the clustering techniques and the MDS.  The dendrogram from the cluster analysis, and the MDS ordination plot will provide complementary views of the same similarity information.  The data for MDS and cluster analyses should be standardised prior to analysis, to ensure that bias resulting from including data in different forms (eg percent data for silt clay composition, numerical data for abundances and biomass data in mg) does not occur.


8.6    Use of Data


The detailed statistical analyses described above shall be used to comprehensively explore the benthic assemblage patterns in the area of the active pits.  This exploration should lead to conclusions regarding the effectiveness of the cap material in promoting post-dredging benthic assemblages.  This information can be used to reassess the choice of capping materials or revise the procedures for placing the caps at the active facility. 


8.7    Data Collection Parameters


The benthic sediment samples collected during this task will be analysed for the following parameters: 


·                Percentage of silt/clay in the sediments;

·                Faunal Abundance;

·                Faunal Biomass;

·                Species Composition; and,

·                Trophic Structure.

8.8    Sampling Procedure and Equipment


The sampling team and vessel will be deployed and accurate positioning attained as described in Section 4.  The vessel will be equipped with adequate fixed sieve stations to facilitate rapid processing of samples and ensure the maximum number of samples are collected in each survey.  At each of the designated benthic sampling stations, seafloor sampling will be carried out with a modified Van Veen grab sampler (dimensions 30 cm H 30 cm H 30 cm) or similar instrument approved by EPD/AFCD.  One subsample of approximately 1 kg sediment shall be collected from each sample for analysis of particle size.  The remaining sediment from each sample shall be used for sorting.  Samples will be labelled and sieved through a 1 mm and 0.5 mm sieve and all residues and organisms retained, double-bagged and preserved in 4% buffered formalin in seawater.  A vital stain (eg Rose bengal) will be added to distinguish organic materials and organisms from other non-living residues.  The grab and utensils will be washed thoroughly with seawater after each deployment to avoid cross-contamination between samples.  On completion of the survey all samples will be transferred to the laboratory for sorting and identification.  All sediment sieving will be conducted by qualified marine scientists who will oversee and coordinate all field operations.


8.9    Laboratory Procedures


Upon arrival at the laboratory, all benthic samples should be re-inventoried and checked against chain-of-custody forms.  Sample rescreening should be performed after the samples have been held in formalin for a minimum of 24 hours to ensure adequate fixation of the organisms.  Individual samples from the 500 mm and 1 mm2 mesh sieves will be gently rinsed with fresh water into a 250 mm sieve to remove the formalin from the sediments.  Sieves will be partially filled while rinsing a specific sample to maximize washing efficiency and prevent loss of material.  All material retained on the 250mm sieve is placed in small fractions into a labelled petri dish and preserved with 70% ethanol.  The material is lightly agitated to ensure complete mixing of the alcohol with the sediments.  The sediment is then sorted to remove all animals and fragments.  Original labels will remain with the rescreened sample material.


Standard and accepted techniques shall be used for sorting organisms from the sediments ([1]).  Small fractions of a sample will be placed in a petri dish under a 10-power magnification dissecting microscope.  The petri dish will be scanned systematically and all animals and fragments removed using forceps.  Each petri dish will be sorted at least twice to ensure removal of all animals.  Organisms representing major taxonomic groups including Polychaeta, Arthropoda, Mollusca, and miscellaneous taxa will be sorted into separate, labelled vials containing 70 percent ethanol.  All sorted samples will be systematically checked to ensure compliance with QA/QC program requirements before proceeding to the taxonomic identification, enumeration, and biomass determination phases of the analysis.


Taxonomic identifications will be performed by regional taxonomic experts using stereo dissecting and high-power compound microscopes, to the family level except for dominants, which will be identified, where possible, to species.  The careful sampling procedure employed in the Study will minimise fragmentation of organisms, however should breakage of soft-bodied organisms occur, only anterior portions of organism fragments will be counted.  All fragments will be retained and weighed during biomass determinations, described below.  Rare or questionable taxa will be compared against reference collection specimens for confirmation and consistency of identification.  The nomenclature used in all reference collections referred to in this study should be cross checked and differences or discrepancies should be noted.  Biomass determinations will be made by taking the blotted wet mass of each taxonomic fraction.


8.10    Benthic Macro-Infauna and Taxonomic Identification


Sorting QA/QC will be performed using 25-power magnification by someone other than the original sorter.  Twenty percent of each sorted sample should be resorted to ensure 95 percent sorting efficiency.  A sample passes QA/QC if the number of organisms found during the QA/QC check does not represent more than 5 percent of the total number of organisms found in the entire sample.  If the number of organisms found is greater than 5 percent of the total number, the entire sample will be resorted.  Any samples where the identification of taxa is questionable will be sent out for independent reidentification by a qualified regional expert.  Reference collections developed during previous seabed and benthic studies in Hong Kong should be consulted as necessary. 

([1]) RJ Schmitt & CW Osenberg(1996) Detecting Ecological Impacts: concepts and applications in coastal habitats. Academic Press.

([3])     Holme, N. A. and A. D. McIntyre (eds) (1984) Methods for the study of marine benthos.  Blackwell Scientific Publications, Oxford (UK).