Marine Infauna Study. Burwood Beach WWTW HUNTER WATER August 2013

Transcription

1 Marine Infauna tudy Burwood Beach WWTW August 20 Infrastructure & Environment 3 Warabrook Boulevard ewcastle, W 2304 Australia PO Box 814 EWCATLE W 2300 Telephone: Facsimile: AB Copyright 20 WorleyParsons

2 YOPI The Burwood Beach Marine Infauna tudy was undertaken to assess the distribution of marine infauna along the effluent dispersion pathway, as a function of distance from the outfall. The study aimed to characterise changes in the infaunal communities that may be related to the discharge of treated wastewater effluent and biosolids from the Burwood Beach WWTW. The key objective of the Burwood Beach Marine Infauna tudy was to monitor changes in the distribution of marine infauna along the effluent dispersion pathway, as a function of distance from the outfall. Changes in the abundance, richness and diversity of infauna and in the dominance of opportunistic species were monitored. Infauna sampling was undertaken using a gradient sampling design with sites positioned at increasing distances from the outfall (10 m, 20 m, 50 m, 100 m, 200 m and 2,000 m) along two radial axis (approximately north-east and south-west). urveys were undertaken during December 2011, April 2012, October 2012 and April 20. Mixed model nested analyses of variance (AOVAs) were used to assess for differences between time, distance and sites in the abundance, richness and diversity of infauna, as well as the ratio of polychaetes to other taxa. Polygordid, dorvilleid and nereid polychaetes, as well as gammarid amphipods and nematodes were analysed separately, as these were the dominant taxa found across all surveys. For the majority of analyses there were inconsistent trends over the sampling events. For the ratio of polychaetes to other taxa, while there was a significant interaction found between time and site, there was an elevated ratio at sites close to the outfall (< 20 m) during most sampling events. The ratio of polychaetes to other taxa was significantly higher at sites close to the outfall (10 m or 20 m) in comparison to all other sites during December 2011, October 2012 and April 20. For each sampling event, multivariate analyses were also undertaken on infauna assemblages. During all sampling events, the MD plots showed that there was a slight gradient with distance from the outfall. There was also a directional influence within most distances, with the northern and southern sites clustered separately. Overall analyses were undertaken on the full dataset, to determine if there were differences over time, distance, direction or season. Time was found to be the most important factor influencing infaunal assemblages, with the December 2011 sampling event clustered separately to all other sampling events. Marine sediment sampling was also undertaken during December 2011 and October The particle size distribution of marine sediments was analysed using principle component analysis (PCA) and it was found that with minor exception, most sites were very similar and had a high proportion of sand ranging from 97-99%. All sites were found to have similar levels of total organic carbon (TOC), apart from elevated TOC in some samples taken within 10 m of the outfall, including two during December 2011 and one during October Document o : 104 Page ii

3 The findings of the Burwood Beach Infauna tudy suggest that for measures of abundance, richness and diversity there are no apparent trends with distance from the outfall that are consistent over the four sampling surveys or two seasons. In addition, there are no consistent trends seen for the dominant taxa groups. The ratio of polychaetes to other taxa was elevated at sites close to the outfall during three of the four sampling events and there was also sediment sampled within 10 m of the outfall (during the Burwood Beach ediment tudy) that was found to have elevated levels of TOC. These findings may indicate an impact of higher organic loading very close to the outfall (in comparison to all other sites) with a zone of impact < 20 m. imilar to the findings of others, there was significant temporal and spatial variability in the abundance and composition of infauna communities in the receiving environment surrounding the Burwood Beach WWTW outfall. As there were no consistent trends with distance from the outfall this high level of variability makes it difficult to determine the potential effects of increased flows on marine infauna communities in the receiving environment with any certainty. Document o : 104 Page iii

4 Disclaimer This report has been prepared on behalf of and for the exclusive use of Hunter Water, and is subject to and issued in accordance with the agreement between Hunter Water and WorleyParsons. WorleyParsons accepts no liability or responsibility whatsoever for it in respect of any use of or reliance upon this report by any third party. Copying this report without the permission of Hunter Water or WorleyParsons is not permitted. Document o : 104 Page iv

5 Internal and Client Review Record PROJECT BURWOOD BEACH REV DECRIPTIO ORIG REVIEW WORLEY- PARO APPROVAL DATE CLIET APPROVAL DATE A Draft issued for internal review Dr K ewton / Dr M Priestley H Houridis 5 March 2012 /A B Draft issued for client review Dr M Priestley Hunter Water / CEE 8 March 2012 C Draft issued for internal review Dr M Priestley Dr K ewton H Houridis 31 May 2012 D Draft issued for client review Dr K ewton Hunter Water / CEE 22 October 2012 E Draft issued for internal review Dr M Priestley Dr K ewton H Houridis 7 January 20 F Draft issued for client review Dr M Priestley Dr K ewton Hunter Water / CEE 7 January 20 G H Draft issued for internal review Dr M Priestley H Houridis / Dr K ewton Draft issued for client review Dr K ewton Hunter Water / CEE 17 June June 20 I FIAL DRAFT Dr K ewton / Dr M Priestley EPA August 20 Document o : 104 Page v

6 COTET 1 ITRODUCTIO Burwood Beach WWTW Treatment Process Environmental Protection Licence Conditions Characteristics of Current Effluent and Biosolids Discharges Effluent and Biosolids Flow Data Dilution Modelling / Dispersion Characteristics Biosolids Deposition Burwood Beach Marine Environmental Assessment Program Initial Consultation tudy Area cope of Works / tudy Objectives ull Hypothesis Review of Previous tudies Impacts of ewage Discharges on Infauna Assemblages Infauna Assessments at Burwood Beach METHOD Infauna ampling ites Temporal Assessment Field ampling Methods Laboratory and Data Analysis Laboratory Analysis Taxa Abundance, Richness and Diversity Polychaete Ratio ediment Characteristics tatistical Analysis Page vi : 104 FIAL DRAFT: August 20

7 3 REULT Univariate Analyses of Marine Infauna Abundance Richness Diversity Polychaete Ratio Polychaete Families Other Infauna Taxa ummary of AOVAs Power Analysis Multivariate Analyses of Infauna December April October April ummary of MD Marine ediments December October Multivariate Analyses of ediments DICUIO COCLUIO ACKOWLEDGEMET REFERECE Page vii : 104 FIAL DRAFT: August 20

8 Figures Figure 1.1 Location of Burwood Beach WWTW. Figure 1.2 Burwood Beach WWTW and outfall alignment. Figure 1.3 Effluent and biosolids flow data for the study period (July May 20). Figure 2.1 Location of all infauna sampling sites. Figure 2.2 ampling sites near to the outfall. Figure 2.3 Infauna sampling equipment. Figure 3.1 Abundance of all infauna taxa surveyed. Figure 3.2 Infauna taxa in high abundance. Figure 3.3 pecies richness (number of taxa) of all infauna taxa surveyed. Figure 3.4 pecies diversity (hannon wiener index) of all infauna taxa surveyed. Figure 3.5 Ratio of polychaete abundance to all other taxa abundance. Figure 3.6 Mean abundance of polychaete families surveyed. Figure 3.7 Mean abundance of dominant infauna (other than polychaetes) surveyed. Figure 3.8 MD analysis (square root transformation with Bray Curtis measure of similarity) of infauna assemblages for December Figure 3.9 MD analysis (square root transformation with Bray Curtis measure of similarity) of infauna assemblages for April Figure 3.10 MD analysis (square root transformation with Bray Curtis measure of similarity) of infauna assemblages for October Figure 3.11 MD analysis (square root transformation with Bray Curtis measure of similarity) of infauna assemblages for April 20. Figure 3.12 Overall MD analysis of infauna assemblages by distance. Figure 3. Overall MD analysis of infauna assemblages by sampling event. Figure 3.14 Overall MD analysis of infauna assemblages by direction. Figure 3.15 Overall MD analysis of infauna assemblages by season. Figure 3.16 Principal component analysis of particle size distribution in sediments sampled during December Figure 3.17 Principal component analysis of particle size distribution in sediments sampled during October Page viii : 104 FIAL DRAFT: August 20

9 Figure 3.18 MD analysis of particle size distribution in sediments during December 2011 and October 2012 represented by zone. Tables Table 1.1 Load limits for effluent and biosolids discharges. Table 1.2 ummary of physicochemical, metal/metalloid and organics data in effluent during Table 1.3 ummary of physicochemical, metal/metalloid and organics data in biosolids during Table 1.4 Effluent and biosolids flow data for the study period (July May 20). Table 1.5 Classification of zones based on prior effluent dilution modelling. Table 1.6 Examples of infauna monitoring programs undertaken in Australia and ew Zealand. Table 2.1 GP co-ordinates and depths of infauna sampling sites. Table 3.1 ummary of mixed model nested AOVAs for selected dependent variables of infauna taxa. Table 3.2 IMPER analysis results for December Table 3.3 IMPER analysis results for April Table 3.4 IMPER analysis results for October Table 3.5 IMPER analysis results for April 20. Table 3.7 ediment characteristics at each sampling site for December Table 3.8 ediment characteristics at each sampling site for October Appendices APPEDIX 1 - IFAUA ABUDACE (ITE AVERAGE) APPEDIX 2 - TATITICAL OUTPUT APPEDIX 3 - POWER AALYI Abbreviations AOVA CEE EPA Analysis of Variance Consulting Environmental Engineers Environment Protection Authority Page ix : 104 FIAL DRAFT: August 20

10 EPL MD MEAP OEH PCA PERMAOVA PD IMPER TOC WWTW Environmental Protection License Multi-Dimensional caling Marine Environmental Assessment Program Office of Environment and Heritage Principle Component Analysis Permutational Multivariate Analysis of Variance Particle ize Distribution Percentage imilarity Analysis Total Organic Carbon Wastewater Treatment Works Page x : 104 FIAL DRAFT: August 20

11 1 ITRODUCTIO 1.1 Burwood Beach WWTW The Burwood Beach Wastewater Treatment Works (WWTW) is located on the Hunter Central Coast of ew outh Wales (W) approximately 2.5 km south of the city of ewcastle (Figure 1.1). The plant treats wastewater from ewcastle and the surrounding suburbs, servicing approximately 185,000 people and local industry and has an average daily dry weather flow of 44 million litres of wastewater (44 ML/d). Over the next 30 years these flows are expected to increase to ML/d, even with water conservation measures in place Treatment Process The secondary treatment process at Burwood Beach consists of physical screening to remove large and fine particulates, biological filtration and waste activated sludge (biosolids) processing including aeration and settling stages. econdary treated effluent from Burwood Beach WWTW is discharged to the ocean through a multi-port diffuser which extends 1,500 m offshore, with diffusers at a depth of approximately 22 m (Figure 1.2). Approximately 2 ML/d of biosolids, which is surplus to treatment requirements, is also discharged to the ocean via a separate multi-port diffuser that extends slightly further offshore than the effluent outfall. Both outfalls have been operating in their current configuration since January Environmental Protection Licence Conditions The Environment Protection Licence (EPL) for Burwood Beach WWTW specifies limit conditions for the operation of the plant. These conditions provide an indication of the characteristics of the effluent and biosolids discharged into the ocean. Condition L1 specifies that the operation of the outfall must not cause or permit waters to be polluted (i.e. the licensee must comply with section 120 of the Protection of the Environment Operations Act 1997). Condition L2 specifies limits relating to total loads discharged to the ocean (including the effluent and biosolids). These limits are provided in Table 1.1. Condition 3 specifies limits to concentrations of suspended solids and oil / grease in the effluent discharged to the outfall. The three day geometric mean concentration limit for suspended solids is 60 mg/l and for oil / grease is 15 mg/l. Condition 4 sets volume and mass limits of effluent and biosolids discharged via the outfalls. The limit for effluent flow rate is 510 ML/d (to allow for higher flows in wet weather) and for biosolids the flow limit is 5 ML/d. Daily monitoring of flow is required. Page : 104 FIAL DRAFT: August 20

12 Figure 1.1 Location of Burwood Beach WWTW. Page : 104 FIAL DRAFT: August 20

13 Figure 1.2 Burwood Beach WWTW and outfall alignment. Page : 104 FIAL DRAFT: August 20

14 Table 1.1 Load limits for effluent and biosolids discharges. Parameter Load Limits kg/year kg/day Total suspended solids 4,717,189 12,924 Biochemical oxygen demand - - Total nitrogen 778,257 2,2 Oil and grease 341, Total phosphorous - - Zinc 3, Copper 2, Lead 1, Chromium Cadmium elenium Mercury Pesticides and PCBs Characteristics of Current Effluent and Biosolids Discharges The final treated effluent and biosolids from Burwood Beach WWTW has been monitored by Hunter Water for microbiological indicators of faecal contaminations and for a suite of metals/metalloids and organic chemicals. A summary of this data during the period is provided in Tables 1.2 (effluent) and 1.3 (biosolids) (data provided by Hunter Water 20) Page : 104 FIAL DRAFT: August 20

15 Table 1.2 ummary of physicochemical, metal/metalloid and organics data in effluent during Group Parameter (units) Period Median Mean Min Max td Error 75%ile 90%ile Physicochemical uspended solids (mg/l) UV254nm Transmittance (%T) ph Total dissolved solids (mg/l) Biological Oxygen Demand - total (mg/l) Chemical Oxygen Demand - Flocculated (mg/l) Grease - total high range (mg/l) Grease - total low range (mg/l) Ammonium nitrogen (mg/l ) itrate + nitrate oxygen (mg/l ) Total Kjeldahl itrogen (mg/l ) Total nitrogen (mg/l ) Total phosphorus (mg/l P) Metals / Metalloids ilver-ag-aa furnace (µg/l) ilver Ag-ICP (µg/l) Arsenic As-vga (µg/l) Cadmium Cd-furnace (µg/l) < < <5 4.7 < <2 2.7 < < < < <1 <1 <1 <1 - <1 <1 Page : 104 FIAL DRAFT: August 20

16 Group Parameter (units) Period Median Mean Min Max td Error 75%ile 90%ile Cadmium Cd-ICP (µg/l) Chromium Cr-furnace (µg/l) Chromium Cr- ICP (µg/l) Chromium Cr VI-furnace (µg/l) Copper Cu-furnace (µg/l) Copper Cu-ICP (µg/l) Mercury Hg-VGA ug/l) Manganese Mn-furnace (µg/l) Manganese-ICP (µg/l) ickel i-furnace (µg/l) ickel i-icp (µg/l) Lead Pb-furnace (µg/l) elenium e-vga (µg/l) Zinc Zn (µg/l) Zinc Zn-ICP (µg/l) Organics Aldrin (µg/l) α-bhc Bhc-a (µg/l) β-bhc-b (µg/l) α Chlordane (ug/l) 59 <1 0.5 <1 1 <1 <1 < < <1 0.7 < <1 0.7 < < < < <1 <1 <1 <1 - <1 < < < < <0.01 <0.01 <0.01 < <0.01 < <0.01 <0.01 <0.01 < <0.01 < <0.01 <0.01 <0.01 < <0.01 < < < <0.01 <0.01 Page : 104 FIAL DRAFT: August 20

17 Group Parameter (units) Period Median Mean Min Max td Error 75%ile 90%ile Chlordane (ug/l) λ Chlordane (µg/l) Chlorpyrifos Lindane (µg/l) DDT (ug/l) DDD (µg/l) DDE (µg/l) Diazinon (ug/l) Dieldrin (µg/l) Endosulfan (µg/l) Endosulfan-s (µg/l) Endosulfan-1 (µg/l) Endosulfan-2 (µg/l) Endrin (µg/l) Heptachlor (µg/l) HCB (µg/l) Heptachlor-epoxide (µg/l) Methoxychlor (µg/l) Parathion (ug/l) 90 < < <0.01 < < < <0.01 < < < <0.01 < < < <0.01 < <0.01 <0.01 <0.01 < <0.01 < <0.01 <0.01 <0.01 < <0.01 < <0.01 <0.01 <0.01 < <0.01 < < < <0.01 < < < <0.01 < < <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 < < < <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 < <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 < <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 < <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 < <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 < < < <0.1 <0.1 Page : 104 FIAL DRAFT: August 20

18 Group Parameter (units) Period Median Mean Min Max td Error 75%ile 90%ile Total PCBs (µg/l) 90 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 Page : 104 FIAL DRAFT: August 20

19 Table 1.3 ummary of physicochemical, metal/metalloid and organics data in biosolids during td Group Parameter (units) Period Median Mean Min Max Error 75%ile 90%ile Physicochemical Total solids (%w/w) Volatile solids (%w/w) Ammonium _Total (mg/l ) Grease total low range (mg/l) Fluoride (mg/l) Metals / Metalloids ilver-ag-aaurnace (µg/l) ilver Ag-ICP (µg/l) Arsenic As-vga (µg/l) Cadmium Cd-furnace (µg/l) Cadmium Cd-ICP (mg/l) Chromium Cr VI-furnace (µg/l Chromium Cr_VIi-furnace (µg/l ) Chromium Cr-furnace (µg/l) Chromium cr- ICP (µgll) Copper Cu-furnace (µg/l) Copper Cu-ICP (µg/l) Mercury Hg- VGA ug/l) Manganese Mn-furnace (µg/l) Page : 104 FIAL DRAFT: August 20

20 Manganese -ICP (mg/l) ickel i-furnace (µg/l) ickel i-icp (mg/l) Lead Pb-furnace (µg/l) Lead Pb ICP µg/l) elenium e-vga (µg/l)) Zinc Zn (mg/l) Zinc Zn-ICP (mg/l) Organics Aldrin (µg/l) α-bhc Bhc-a (µg/l) β-bhc-b (µg/l) α Chlordane (ug/l) Chlordane (ug/l) λ Chlordane- (µg/l) Chlorpyrifos (µg/l) DDT (uµ/l) DDD (µg/l) DDE (µg/l) Diazinon (ug/l) Dieldrin (µg/l) Endosulfan-s (µg/l) Page : 104 FIAL DRAFT: August 20

21 Endrin (µg/l) HCB (µg/l) Heptachlor-epoxide (µg/l) Heptachlor (µg/l) Lindane (µg/l) Malathion (µg/l) Methoxychlor (µg/l) Parathion (ug/l) Total PCBs (µg/l) Page : 104 FIAL DRAFT: August 20

22 1.1.4 Effluent and Biosolids Flow Data Effluent and biosolids flow data for the study period was obtained from the Burwood Beach WWTW. A summary of flow data for the period July 2011 to May 20 is provided in Table 1.4 and Figure 1.3. Table 1.4 Effluent and biosolids flow data for the study period (July May 20). Date Rainfall (mm) econdary Flow (ML) 1 By-Pass Flow (ML) 2 Total Flow (ML) WA (ML) 3 July Aug ep Oct ov Dec Jan Feb Mar Apr May Jun Jul Aug ep Oct ov Dec Jan Feb Mar Apr May ote 1. econdary Flow is total secondary treated flow through the plant (i.e. total volume of screened and degritted sewage into secondary plant over a 24 hour period from 12 midnight and discharged to ocean). ote 2. By-Pass Flow is total volume of screened and degritted sewage which bypasses the secondary plant over a 24 hour period from 12 midnight and is discharged to ocean. ote 3. WA is the Volume of Waste Activated ludge (biosolids) pumped from the clarifier underflow over a 24 hour period from 12 midnight and is discharged to ocean. Page : 104 FIAL DRAFT: August 20

23 Figure 1.3 Effluent and biosolids (WA) flow data for the study period (July May 20) Dilution Modelling / Dispersion Characteristics Consulting Environmental Engineers (CEE 2007) calculated a predicted initial dilution for the Burwood effluent outfall, assuming a discharge rate of 43 ML/d and all duckbill valves in operation. The model predicted a typical dilution of 219:1 for the effluent field. Allowing for the reduction in dilution due to the orientation of the diffuser ports parallel to the currents, initial dilution is expected to be in the range of 180:1 to 220:1. The Water Research Lab (WRL 2007) also carried out field tests of effluent dilution using rhodamine dye. The dilution of the surface field showed a typical dilution of 185:1. WRL (2007) reported that the average near-field dilution was 207:1 and the 95 th percentile minimum dilution was 78:1. CEE (2010) therefore considers it reasonable to base the environmental risk assessment of the effects of effluent discharge on an effluent plume near the ocean surface with an initial dilution in the range of 100:1 to 200:1. The dilution of a combined biosolids and effluent discharge through the biosolids diffuser was also calculated (CEE 2007). The CEE model predicted a typical dilution of 475:1 for discharged biosolids if they rose to the ocean surface, or about 250:1 if trapped by stratification at mid-depth (CEE 2007). The WRL hydrodynamic computer model showed a median dilution of 300:1, with a minimum dilution of 100:1 when strong stratification decreases the rise and dilution of the small biosolids plumes, and a maximum dilution at times of strong currents exceeding 1,000:1 (WRL 2007). The WRL model also showed the biosolids plume is often trapped well below the surface by the natural stratification of the ocean water column. WRL field tests of the biosolids plume, with dilution measured using rhodamine dye, showed a typical dilution of 841:1. WRL reported that the average near-field dilution of the biosolids plume was 268:1 and the 95 th percentile minimum dilution was 205:1, for a submerged plume (WRL 2007). Based Page : 104 FIAL DRAFT: August 20

24 on these results, it is considered reasonable to base the assessment of the effects of biosolids discharge on two conditions; surface plume with an initial dilution of 300:1 and submerged plume with an initial dilution of 200:1 (CEE 2010). WRL (1999) modelled the biosolids plume at 10 m depth and showed that the centre of the plume, at about 10 m depth, the dilution achieved is between 200:1 and 1,000:1. At a distance of 200 m from the diffuser, the dilution exceeds 1,000:1 and increases further with distance travelled. The diluted biosolids extends to the south of the diffuser, but would be indistinguishable except by the sensitive techniques used in the field studies. Based on the field tests and dilution modelling undertaken by WRL (1999, 2007) and CEE (2007), the following mixing zones (Table 1.5) were determined for reporting purposes only. Table 1.5 Classification of zones based on prior effluent dilution modelling. Distance from Diffuser Zones < 50 m outfall impact zone outfall impact > m nearfield mixing zone > m mixing zone midfield mixing zone > 200-2,000 m farfield mixing zone > 2,000 m reference zone reference Biosolids Deposition Previous diver inspections undertaken at the Burwood Beach outfall (i.e. by commercial divers inspecting the outfall infrastructure) reported that biosolids deposits at the seabed can vary significantly. In-situ diver observations have reported a biosolids thickness of 0 to 125 mm, with variation likely a result of weather conditions. Divers have noted biosolids being washed away after storms with no long-term accumulation on the seabed evident. More protected areas such as small caves have a greater depth of biosolids and a peak of 750 mm was recorded in 1994/96 (note that at this time effluent was not mixed with biosolids before discharge). ATO (1998) undertook a study of the movement of seabed sediments 1,100 m south east of the outfall using iridium-radiated glass beads. The beads were found to disperse over 100 m to the east and west and over 150 m to the north, providing an indication of the likely expected movement of sandy sediments on the seabed. It is expected that smaller biosolids particles would disperse at a greater rate and further than sand particles. Page : 104 FIAL DRAFT: August 20

25 1.2 Burwood Beach Marine Environmental Assessment Program A number of monitoring programs and studies have previously been undertaken to assess the impact of treated effluent and biosolids discharge on the marine environment at Burwood Beach (e.g. W Environment Protection Authority (EPA) 1994, 1996; The Ecology Lab 1996, 1998; Australian Water Technologies (AWT) 1996, 1998, 2000, 2003; inclair Knight Merz (KM) 1999, 2000; Ecotox ervices Australasia (EA) 2001, 2005; BioAnalysis 2006; Andrew-Priestley 2011; Andrew-Priestley et al. 2012). While providing a wealth of data on the marine environment here, it is considered that these previous studies have not effectively assessed the spatial extent and ecological significance of the outfalls impact (CEE 2010). The aim of the Burwood Beach Marine Environmental Assessment Program (MEAP) was to establish the impact footprint of the existing outfall, establish the gradient of impact with distance to the edge of the outfall and predict the potential footprint of future impacts. The current Burwood Beach Marine Infauna tudy aimed to address some of the knowledge gaps. This incorporated assessing both the spatial and temporal impact of the effluent and biosolids discharges on benthic marine infauna assemblages along the effluent dispersion pathway Initial Consultation Prior to commencement of the Burwood Beach MEAP, details of the proposed sampling program and survey methodology were discussed with Hunter Water, CEE and the W EPA (then the Office of Environment and Heritage (OEH) on 10 October This initial consultation was undertaken to ensure that the proposed MEAP was adequate in addressing the requirements of both the Client (Hunter Water) and the Regulator (W EPA). During this meeting, concerns with the proposed survey / sampling program were raised and where required the methodology was subsequently altered accordingly. 1.3 tudy Area Burwood Beach is located in ewcastle, on the Hunter Coast of W. It lies to the south of Merewether Beach and to the north of Dudley Beach (refer to Figure 1.1). The seabed in the vicinity of the outfall consists of small areas of low profile patchy rocky reef, which is subject to strong wave action and periodic sand movement, interspersed between large areas of soft sediment (sandy) habitat. These low profile reefs are emergent approximately 1 m above the sand. Water depth is approximately 22 m at the outfall diffuser (refer to Figure 1.2). Fine mobile sandy sediments occur in the gutters and low-lying seabed between reef patches. Extensive sandy beaches with intertidal rocky reef habitats occur along the shoreline adjacent to the outfall. Merewether Beach lies to the north and Dudley Beach to the south of Burwood Beach Page : 104 FIAL DRAFT: August 20

26 1.4 cope of Works / tudy Objectives While several studies have examined the macrobenthic sessile marine fauna living on the rocky reefs at Burwood Beach (e.g. The Ecology Lab 1996, 1997, 1998; AWT 2000, 2003; BioAnalysis 2006; Roberts and Murray 2006), there have been no studies undertaken to date that have assessed infauna within the marine sediments. An assessment of infauna assemblages, abundance and species richness at Burwood Beach, which incorporates spatial and temporal replication, was proposed to assist in determining potential impacts from the discharge of treated effluent and biosolids into the marine environment. The key objective of the Burwood Beach Marine Infauna tudy was to monitor changes in the distribution of marine infauna along the effluent and biosolids dispersion pathway, as a function of distance from the outfall. Changes in the abundance, richness and diversity of infauna and in the dominance of opportunistic species were monitored. The study also aimed to detect and characterise the following: Impacts on community structure of infauna communities. The extent or zone of impact. The gradient of any impact on biological indicators (species or groups) depending on distance from the outfall ull Hypothesis The null hypothesis of this study was: There is no significant difference between infauna diversity, abundance and richness at sampling sites close to the outfall compared to equivalent habitats with increasing distance from the outfall. There is no significant difference between the ratio of polychaetes to other taxa at sampling sites close to the outfall compared to equivalent habitats with increasing distance from the outfall. There is no significant difference between dominant infauna groups or between infauna assemblages close to the outfall compared to equivalent habitats with increasing distance from the outfall. Page : 104 FIAL DRAFT: August 20

27 1.5 Review of Previous tudies Impacts of ewage Discharges on Infauna Assemblages The release of sewage into the marine environment has been demonstrated to impact on marine biota at the cellular, individual and community levels (Underwood and Peterson 1988). The type and extent of impact varies and depends on the quantity and composition of sewage effluent. Impacts on marine biota have been reported as localised, in the immediate vicinity of the WWTW (Fairweather 1990) or wide ranging, such as kilometres from the WWTW source (Fry and Butman 1991; Zmarzly et al. 1994). Temporally, impacts may be pulse events or sustained press events (Underwood 1992, 1993). oft sediments provide habitat for a range of macroinvertebrate infauna (i.e. fauna living within the sediments) including crustaceans (amphipods, isopods and cumaceans), worms (polychaetes, nemerteans) and molluscs (bivalves and gastropods). Infauna may feed using filter feeding mechanisms (e.g. molluscs), active predation, or by gathering detritus from the sediments. Marine infauna assemblages have been used extensively to monitor the level of anthropogenic impacts on the marine environment. Infauna assemblages are useful as indicators due to their relatively sedentary lifestyle and as they live within the sediments. They are also relatively easy to quantitatively sample. Infauna communities have been established to respond to anthropogenic disturbance (Warwick 1993; Otway et al. 1996). Environmental changes, resulting from the discharge of treated sewage effluent into the marine environment, can include increased algal growth as a result of increased availability of nutrients (e.g. phosphorus and nitrogen), release of and potential exposure to organic and / or inorganic contaminants and pathogens (bacteria or fungi) from wastewater (Defeo et al. 2009). In turn, impacts on infauna communities can include changes in species abundance, species richness, the dominance of opportunistic species or the dominance of deposit feeders (Dauvin and Ruellet 2006; Dean 2008). Changes in infauna communities around the point of WWTW discharge may result from organic enrichment of bottom sediments (Pearson and Rosenberg 1976, 1978). Organic and inorganic contaminants in sewage can also bioaccumulate in soft-bottom organisms (Phillips 1977, 1978) causing alterations to infauna communities (Reish et al. 1987). One of the difficulties in using infauna assemblages to monitor impacts of WWTWs is their inherent spatial and temporal variability, making it difficult to attribute change to an impact rather than natural variation. Infauna communities are composed of a mosaic of successional patches, resulting from numerous interacting processes; also attributing to the significant spatial and temporal variation observed (Peterson 1977; Dayton and Oliver 1980). Infauna monitoring programs have been used to assess impacts from WWTWs in Australia and ew Zealand, both shoreline (where wastewater is discharged into the intertidal zone) and deep water (where outfall diffuser is extended out to sea and wastewater discharged into deeper waters). ome examples of infauna monitoring programs are provided in Table 1.6. ummaries of some of these studies are also provided. Page : 104 FIAL DRAFT: August 20

28 Table 1.6 Examples of infauna monitoring programs undertaken in Australia and ew Zealand. Outfall Monitoring period Hobart outfall present Blackmans Bay outfall 2007, 2010, 20 Anglesea outfall 2005, 2009 Altona outfall 2003, 2005, 2007, 2009, 2011, 20 Geelong outfall Latrobe Valley outfall Port Kembla outfall ydney outfalls 1976, 1995 Coffs Harbour outfall 2000, 2008 Kawana outfall 1990, 1996 Perth outfalls 2000, 2004 Werribee outfall 1982 Gisbourne outfall 2002 Marine infauna assemblages were assessed as part of the ydney Water W Environmental Monitoring Programme (EMP) which was undertaken during to assess impacts of ydney s deepwater outfalls, orth Head, Malabar and Bondi, on the marine environment (Otway et al. 1995, 1996). The EMP was required by the W EPA to assess impacts of the WWTWs on the receiving marine environments and also included studies on fish communities. The experimental design consisted of a before-after-control-impact (BACI) design with sampling undertaken before and after commissioning of the deepwater outfalls. In the receiving environment of each outfall, six sites (which consisted of three outfall sites and three reference sites) were sampled using a grab method, with three random sediment grabs per site. All sites were located in m of water. ediment was sieved through a 1 mm sieve to capture infauna. Polychaetes, crustaceans and molluscs were identified to family level while other taxa were identified to Phylum and Class. They found that the infauna communities were comprised of 54% polychaetes, 39% crustaceans, 3% molluscs and 4% miscellaneous taxa. They found that the abundance of infauna varied through time and there were significantly less individuals collected during winter. Overall, the abundance of organisms comprising these three communities fluctuated in time and space and no obvious patterns were evident. Analysis of polychaete families indicated that their populations fluctuated at varying spatial and temporal scales. Despite the variability reported by Otway et al. (1995, 1996) they were able to demonstrate impacts on infauna assemblages due to the commissioning of the three deepwater outfalls. Page : 104 FIAL DRAFT: August 20

29 Following commissioning of the Malabar outfall, they found that the combined number of Anthurid and Paranthurid isopods increased where the mean number of polychaete families decreased. At orth Head and Malabar, they reported that nereid polychaetes and crustaceans increased. They also found that the percentage of impacts were related to the flow data of effluent and suspended solids load; as the flow rate or suspended solid load increased, the impact on increased abundance of infauna around the WWTWs decreased (Otway et al. 1995, 1996). This may suggest a hormesis type impact, i.e. whereby at low concentrations the infauna abundance is stimulated by WWTW releases but at higher concentrations abundance is negatively impacted. uch a scenario would have negative implications on infauna abundance if flow rates or suspended solid loads increased in the future. Results of the ydney deepwater outfalls EMP were consistent with previous studies indicating spatial and temporal fluctuations in the abundance of soft-bottom infauna communities (e.g. Gray 1974; Pearson and Rosenberg 1978). The variability between results from the three outfalls was thought to relate to variable patterns in abundance and / or sediment grain size and structure (Otway 1995). Marine infauna assemblages were assessed as part of the replacement of Blackmans Bay Outfall from shoreline discharge to offshore (Kingsborough Council 2008). They analysed infauna communities north and south of the existing outfall at 200 m and 1000 m, as well as at the site of the proposed new outfall. There were no differences between the 200 m and 1000 m sites in the abundance or diversity of the infauna assemblages. However they found that the abundance and diversity of infauna was higher at the proposed site for the new outfall. It was speculated that impacts from the proposed outfall would be changes to the infauna composition and abundance of certain species. It was also noted that the spatial arrangement of sampling sites was not sufficient to quantify the variability of infauna assemblages. In the receiving environment of Black Rock outfall in Geelong, Victoria, marine infauna assemblages were monitored before and after the replacement of an outfall in The old outfall discharged into the intertidal zone and was replaced by an outfall which discharges into the subtidal zone, 1.2 km offshore at an average depth of 15 m. Analysis of the infauna assemblages around the subtidal zone of the new outfall showed that there were no outfall related impacts or changes following. The study found evidence that the polychaete population in the intertidal zone (where the old outfall had discharged) had decreased, but it was suggested that further monitoring was needed to confirm this. There is also other evidence of increased abundance and richness of marine infauna at sewage affected locations compared with control locations. Dauer and Conner (1980) reported that the total abundance, biomass and richness of polychaete populations were significantly greater at a location receiving sewage effluent (Tamba Bay, Mexico) in comparison to a control location. However, it should be noted that this particular study did not replicate at the location level and differences seen may be due to natural variability. In 2002, an assessment of the ecological effects of primary treated effluent, discharged into water depths of 18 m from the Gisborne wastewater outfall in ew Zealand was undertaken (Keeley et al. 2002). oft bottom benthic infauna samples were taken along two transects radiating away from the outfall in two directions (of the most likely effluent flow). Analysis of abundance and richness suggest an Page : 104 FIAL DRAFT: August 20

30 environmental gradient of enrichment which radiates away from the outfall including four approximate zones: tending towards abiotic at the outfall (i.e. 0 m), highly enriched 50 m from the outfall, a transitional area of detectable but diffuse enrichment out to 1,200 m and background levels beyond 1,200 m (Keeley et al. 2002). The species most responsible for the overall trend in abundance was the surface deposit feeding bristle worm (Prionospio sp.) which accounted for over half of all individuals collected. The majority of species found had surface-oriented feeding behaviours (e.g. scavenging, filter feeding, predation and omnivorous deposit feeding) (Keeley et al. 2002). Again, these situations are not comparable to Burwood Beach but do demonstrate that an increased infauna abundance or richness can be a potential response from sewage effluent release. In terms of infauna assessment, richness, abundance and diversity of infauna communities are the main variables used in the monitoring of environmental impacts. Polychaetes have been useful as monitors of environmental pollution and are known to respond to organic enrichment (Pearson and Rosenberg 1976; Gray and Pearson 1982), particularly in studies of monitoring of sewage outfalls (Reish 1957; Tsutsumi 1990; Weston 1990). Polycheates can respond to organic enrichment by the dominance of opportunistic polychaete species. In particular, polycheates from the Capitellidae family have been established as opportunistic (Dorsey 1982; Roper et al. 1989; Ward and Hutchings 1996). It is also known that there are opportunistic infauna species within the families of pionidae and ereidae (Dauvin and Ruellet 2006; Dean 2008). ome polychaete families can be sensitive and a lack of their presence, in ecosystems where they are known to occur, can also be an indication of an impact. An impact can also be shown through the overall dominance of polychaetes in comparison to other taxa. Intertidal infauna were assessed in sandy sediments adjacent to drains from the Werribee, a large outfall that discharges on the shoreline in Victoria and found that species diversity was low, abundance was high and the infauna assemblages were characterised by opportunistic species such as spionids, capitellids, nereid polychaetes and corophiid amphipods (Dorsey 1982). In summary, these studies indicate that richness, abundance and diversity are all important parameters in the assessment of potential anthropogenic impacts on marine infauna assemblages, with potential positive and negative impacts from the discharge of sewage effluent into marine environments Infauna Assessments at Burwood Beach o previous assessments of marine infauna have been undertaken at Burwood Beach. Page : 104 FIAL DRAFT: August 20

31 2 METHOD 2.1 Infauna ampling ites Infauna sampling for the Burwood Beach outfall was undertaken using a gradient sampling design. ites were positioned at increasing distances from the outfall at 10 m, 20 m, 50 m, 100 m, 200 m and 2,000 m (reference sites), along two radial axis (approximately north-east and south-west) (Figures 2.1 and 2.2) ( = 6 distances and 12 sites). GP co-ordinates and depths of each of the sampling sites are provided in Table 2.1. All sampling sites were located in areas of soft seabed and samples were taken along the same depth contour (~ 22 m), or as close to this depth as possible. Figure 2.1 Location of all infauna sampling sites. Page : 104 FIAL DRAFT: August 20

32 Figure 2.2 ampling sites near to the outfall. Page : 104 FIAL DRAFT: August 20

33 Table 2.1 GP co-ordinates and depths of infauna sampling sites. Location Distance ite Latitude () / Longitude (E) Depth (m) Outfall Impact Zone 10 m 10m ' / ' 23 10m ' / ' m 20m ' / ' 22 20m ' / ' 24 earfield Mixing Zone 50 m 50m ' / ' 21 50m ' / ' 24 Midfield Mixing Zone 100 m 100m ' / ' m ' / ' 25 Farfield Mixing Zone 200 m 200m ' / ' m ' / ' 25 Reference 2,000 m 2,000m ' / ' 22 2,000m ' / ' Temporal Assessment Four marine infauna surveys were undertaken over a two period. This included two cool water surveys during December 2011 and October 2012 and two warm water surveys during April 2012 and April Field ampling Methods Benthic infauna was collected using a diver operated core which was 22 cm deep and 16 cm in diameter. Three replicate cores were taken at each site and immediately transferred into individual sieve bags of size 1 mm (see Figure 2.3) ( = 3 replicates per site). At each site, the replicate cores were taken approximately 1-2 m apart. The sediment was sieved in-situ by the diver, tied off and all sample bags returned to the surface. On the boat, each sieve bag was transferred into a separate snap lock bag into which a 10% formalin solution was placed to cover the entire sample. Page : 104 FIAL DRAFT: August 20

34 Figure 2.3 Infauna sampling equipment. 2.4 Laboratory and Data Analysis Laboratory Analysis amples were sent to Aquen (Aquatic Environmental Consulting) for sorting and identification of infauna. amples were identified at least to family level and to species level where possible. Identification to family level has been established as adequate for the detection of impacts on infauna communities (Warwick 1988) Taxa Abundance, Richness and Diversity Taxa abundance, richness and diversity were calculated for the infauna data. A brief definition of each of these is provided below: Abundance: Relates to how common or rare taxa are relative to other taxa in a defined location or community. Richness: A measure related to the total number of different taxa present within a sample. Diversity: Taxa diversity accounts for the number of taxa and the evenness of taxa, giving a measure of the biodiversity and complexity of a population. Taxa diversity consists of two components, taxa richness and taxa evenness. Taxa richness is a simple count of taxa, whereas taxa evenness quantifies how equal the abundances of the taxa are. Page : 104 FIAL DRAFT: August 20

35 Taxa diversity was calculated using the hannon Weiner diversity index as follows; H = Σ - (Pi * ln Pi) i = 1 Where: H = the hannon diversity index Pi = fraction of the entire population made up of taxa i Σ = sum from taxa 1 to taxa (number of taxa encountered) Abundance was calculated as the mean proportion of total fauna and for individual phyla that were dominant in the dataset Polychaete Ratio Benthic indices have often been used to explore relationships between the relative abundance of sensitive taxa versus opportunistic taxa that may be indicators of organic enrichment (Dauvin and Ruellet 2006; Dean 2008). As polychaetes are well established indicators of environmental health their abundance was examined in relation to other taxa present using a polychaete ratio. The polychaete ratio was calculated by the division of combined polychaete abundance by the combined abundance of all other taxa. Σ Polychaete Abundance Σ Other Taxa Abundance (all taxa other than polychaetes) 2.5 ediment Characteristics It is evident that some factors are more important than others in determining the distribution of particular species. Particle size is perhaps the single most important ecological factor influencing the distribution of infaunal taxa such as polychaetes (Gray and Elliott 2010). The Burwood Beach ediment tudy, another component of the MEAP, was undertaken twice; during December 2011 and October Marine sediment sampling was undertaken at the same sites as the infauna study and sediments were analysed for metals, total organic carbon (TOC) and particle size distribution. It should be noted that sediment analyses were based on sediment samples of 2 cm depth (this was as per W EPA requirements to take just the top 2 cm of sediments for the Burwood Beach ediment tudy) compared to the 22 cm depth sediment cores used for infauna sampling. Page : 104 FIAL DRAFT: August 20

36 2.6 tatistical Analysis Univariate statistical analyses were performed using tatistica Version 7. Diversity, abundance and richness measures were examined for normality, using a normality plot and Levenes test for homogeneity of variance. Where p <0.05, the data was transformed via a log transformation ln (x +1) and the parameters transformed are indicated in the statistics results in ection ignificant differences (p < 0.05) between time, distance (fixed factors) and site (nested within distance) (random factor) along with significant interactions between time and distance were examined using a mixed model nested analyses of variance (AOVAs) under the General Linear Model (GLM) of tatistica. ote that the design was unbalanced due to missing sites during the December 2011 sampling event. Pairwise Tukey s post hoc tests were used to determine where differences occurred. Multi-dimensional scaling (MD) and cluster plots were generated in PRIMER 6, using infauna family abundance, to identify whether differences in infauna communities were evident between sites. Ordination of infauna family abundance was performed using MD scaling in PRIMER 6, based on ranked matrices of dissimilarities between samples, employing the square root transformation with Bray Curtis similarity. Goodness of fit (stress) was assessed using Kruskal s stress formula and compared to maximum values recommended by turrock and Rocha (2000). To identify which taxa had the highest contribution to the average similarity within each site, IMPER analysis was performed. ignificant differences in overall results of infauna assemblages between time and distance were analysed using a factorial nested Permutational Multivariate Analysis of Variance (PERMAOVA). Power analysis was undertaken on the first round of sampling data (refer to ection 3.1.8), and in combination with the statistical analysis, was intended to help design and modify, where applicable, future infauna studies. A Type I error rate of 5% (0.05) was adopted and a Type II error rate of 20% (0.2, power 80%) and an effect size of 50% was used. Page : 104 FIAL DRAFT: August 20

37 3 REULT 3.1 Univariate Analyses of Marine Infauna Average taxa abundance, richness and diversity of infauna are presented in Figures 3.1, 3.3 and 3.4. Images of infauna taxa which were in high abundance are provided in Figure 3.2. The ratio of polychaetes to all other taxa is presented in Figure 3.5. Abundance of polychaete taxa is presented in Figure 3.6 and abundance of all other taxa is presented in Figure 3.7. A summary of the raw data (i.e. infauna abundance at each site) for each sampling period is provided in Appendix 1. Mixed model nested AOVAs were undertaken for the measures of abundance, richness and diversity for all infauna and the ratio of polychaetes to all other taxa, which are all discussed separately in the following sections. A summary of all key statistical output is provided in Table 3.1. Differences in infauna measures were analysed to assess if there were significant differences for the main factors of time, distance and site (nested within distance) and for interactions between time by distance and time by site (distance). As some sites were missing (due to a lack of soft sediments to sample in predominately rocky reef areas) during December 2011 (i.e. 10m, 10m, 50m and 100m ) and October 2012 (i.e. 10m ), the model bases estimated effects on the distances or sites that were available. This means that the statistical analyses are comprised in terms of calculating temporal effects, between sampling events and seasons. During the first sampling round there were a number of sites that could not be sampled due to insufficient sediment available for sampling in areas where were dominated by reef habitat. These included 10m, 10m, 50m and 100m. During subsequent sampling events in April 2012, October 2012 and April 20 there were sufficient sediment at all sites, with the exception of 10m during October The fact that there is mobile sand offshore at Burwood Beach is an important factor that may influence the results of this study. Intermittent sand movement may influence the abundance, diversity and composition of the infauna communities Abundance The average abundance of various infauna taxa and total infauna taxa for each sampling period is detailed in the sections below. Figure 3.1 provides a graphical representation of the average total infauna abundance at each site (i.e. average of three sediment cores) for each survey event. Images of some abundant infauna taxa are provided in Figure 3.2. Overall, infauna abundance was higher during December 2011 and October 2012 due to high populations at several sites. The findings of the mixed model nested AOVA for abundance found a significant interaction between time and site (distance) (Table 3.1). A significant interaction demonstrates that the Page : 104 FIAL DRAFT: August 20

38 trends among sites are inconsistent over the four sampling events. Tukey s post hoc tests showed that this was due to higher total infauna abundance at the 20m, 50m and 200m s sites during December 2011 compared to April 2012, October 2012 and April 20. Total infauna abundance was also higher at 10m during October 2012 compared to April 2012 and April 20. DECEMBER 2011 During this sampling event a very low abundance of infauna was recorded at both of the reference sites (2,000m and 2,000m ) and at the 100m site, whereas other sites (e.g. 20m, 50m and 200m ) were all characterised by a high abundance of a single family. The most abundant taxon at reference site 2,000m were the gammarid amphipods, but mean abundance was very low with just one individual per sample. At the reference site 2,000m, Trochidae were most abundant, with a mean abundance of 18 individuals per sample. At the site 100m, the most abundant taxa were nematodes, with a mean abundance of six individuals per sample. Polygordiid polychaetes were present in highest abundance at the 200m and 20m sites, with respective site means of 66 and 209 individuals per sample. ematodes were also present in high abundance, particularly at sites 50m and 200m, with respective means of 225 and 69 individuals per sample. The most abundant taxa at 20m were spionid polychaetes, with a mean abundance of 30 individuals per sample. Importantly, across all sites the composition of families varied and no taxonomic group was consistently abundant. APRIL 2012 During the April 2012 sampling event infauna abundance was generally lower compared to the December 2011 sampling event, but for some sites it was similar (e.g. at sites 200m and 100m ). Overall, there was very low abundance across all distances and sites in April Gammarid amphipods were the most abundant taxon at the sites 10m, 50m, 100m, 200m and 200m (with respective site means of 12, nine, 16, 28 and 31 individuals per sample). ereid polychaetes were the most abundant taxon at 50m and 100m with respective means of 24 and 34 individuals per sample. Finally, Corophiidae (amphipods) were the most abundant family at sites 20m, 20m, 2,000m and 2,000m, with respective means of four, 16, four and 11 individuals per sample. ites closest to the outfall (the 10 m and 20 m distances) had total means of between 23 to 53 individuals per sample. Mean total abundance at the 50 m and 100 m distances were much higher and ranged from 49 to 120 individuals per sample. The 200 m distance also had high abundances of infauna in comparison to other distances, with mean total abundances of 91 and 5 individuals per sample for Page : 104 FIAL DRAFT: August 20

39 these sites. The 2,000 m reference sites had low abundances, with just 22 and 34 individuals per sample respectively. OCTOBER 2012 During the October 2012 survey event, with the exception of site 10m, infauna abundance was low at all sites. Abundance at 10m was at least four times that of other sites. For the 10m outfall site, Dorvilleidae, ereididae and Gammarid spp. were the most abundant taxa with respective means of 209, 86 and 77. For all other sites, gammarid amphipods were the most abundant taxon. Mean total abundance of Gammaridae was highest at 20m, 20m, 50m, 100m and > 2,000m with respective mean abundances of 20, 25, 22, and 17. The sites 50m, 100m, 200m, 200m and > 2,000m all had average mean abundances which were less than 10. Ostracods (seed shrimps) were the second most abundant taxon for the 20m, 20m, 50m and 200m sites with respective means of five, 18, 16 and two. At the 2,000m and 2,000m reference sites, Oligochaeta spp. and Gastropoda were the second most abundant taxa with means of 10 and four respectively. All other taxa had low abundances with an average mean of three or less. APRIL 20 During April 20, infauna abundance was similar at most sites with the exception of 10m and 50m which had elevated abundance levels in comparison to the other sites. Gammarid spp. were the most abundant taxon at sites 10m, 50m, and 100m, with respective mean abundances of 15, nine and six. Dorvilleidae was the most abundant family at 10m and 50m with respective mean abundances of 43 and 88. pionidae was the most abundant family at sites 20m and 100m with respective mean abundances of 11 and 12. ematodes were the most abundant at 20m, Paraonidae the most abundant at 200m and Polygordiidae at 200m, with respective mean abundances of 67, 12 and 30. At the > 2,000m and > 2,000m reference sites, Corophiidae was the most abundant family with respective mean abundances of and seven. Page : 104 FIAL DRAFT: August 20

40 December 2011 April 2012 October 2012 April 20 Figure 3.1 Abundance (mean ± E) of all infauna taxa surveyed. = 3 replicate sediment cores per site. sediment depth to sample. Colours indicate distance from the WWTW outfall. = /A due to insufficient Page : 104 FIAL DRAFT: August 20

41 Phylum: Annelida, Class: Polychaeta, uborder: incertae sedis, Family: Polygordiidae, pecies: Polygordius kiarama Phylum: Annelida, Class: Polychaeta, uborder: incertae sedis, Family: Polygordiidae, pecies: Polygordius kiarama Phylum: Annelida, Class: Oligochaeta, Family: undifferentiated, pecies: sp. a Figure 3.2 Infauna taxa in high abundance. Page : 104 FIAL DRAFT: August 20

42 Phylum: Annelida, Class: Oligochaeta, Family: undifferentiated, pecies: sp. b Phylum: ematoda, Class: undifferentiated, Family: undifferentiated, pecies: undifferentiated Phylum: Annelida, Class: Polychaeta, Family: pionidae Figure 3.2 (continued) Infauna taxa in high abundance. Page : 104 FIAL DRAFT: August 20

43 3.1.2 Richness Results for richness of infauna taxa are presented in Figure 3.3. Overall, there were no consistent trends in richness among sites or distances. During December 2011, there was little difference in richness between the available sites. In April 2012, there was a slight trend of increasing richness with distance from the outfall, out to about 200 m, followed by a decline at the reference distance. In October 2012, richness was similar among sites with the exception of at > 2,000 m, which was higher than all other sites. During April 20, richness was similar among all sites. The findings of the mixed model nested AOVA for richness showed a significant interaction between time and site (distance) (Table 3.1). This was due to significantly higher taxa richness during October 2012 at site 2000m in comparison to other sites, but not during December 2011, April 2012 and April 20, determined through the Tukey s post hoc analysis. There was a slight trend of increasing richness with distance up to 200 m during April 2012 and April 20. During December 2011 and October 2011, this was not consistent and there was higher richness at the 10 m and / or 20 m distances in comparison to 50 m, 100 m and 200 m. Page : 104 FIAL DRAFT: August 20

44 December 2011 April 2012 October 2012 April 20 Figure 3.3 Richness (number of taxa; mean ± E) of all infauna taxa surveyed. = 3 replicate sediment cores per site. insufficient sediment depth to sample. Colours indicate distance from the WWTW outfall. = /A due to Page : 104 FIAL DRAFT: August 20

45 3.1.3 Diversity Results for infauna taxa diversity at each sampling site are presented in Figure 3.4. Within each survey event there were variations in diversity among sites, however there was no consistent pattern across all four surveys. In December 2011, taxa diversity was higher at the 20m site while the other sites had similar levels. There was large variability at the > 2,000m, > 2,000m and 100m sites. In April 2012, there was similar diversity among sites but diversity was slightly elevated at the 100m and 50m sites. In October 2012 richness was lowest at the >2,000m site and highest at the > 2,000m site, and similar among all other sites. During April 20, diversity was similar among the 10 m, 20 m, 50 m and 200 m distances and the > 2,000m site. The 100 m distance and the > 2,000 m sites had higher taxa diversity in comparison. The mixed model nested AOVA found that there was a significant interaction between time and site (distance) (Table 3.1). This was due to significantly lower diversity at > 2,000 m sites in comparison to other sites during December 2011 only, determined through Tukey s post hoc analyses. There was also lower diversity at the > 2,000m site during October Page : 104 FIAL DRAFT: August 20

46 December 2011 April 2012 October 2012 April 20 Figure 3.4 Diversity (hannon wiener index) (mean ± E) of all infauna taxa surveyed. = 3 replicate sediment cores per site. due to insufficient sediment depth to sample. Colours indicate distance from the WWTW outfall. = /A Page : 104 FIAL DRAFT: August 20

47 3.1.4 Polychaete Ratio The ratio of polychaete families to all other taxa is presented in Figure 3.5. The polychaete ratio was consistently elevated at 10 m or 20 m sites. In December 2011, there was a significantly higher ratio of polychaetes to all other taxa at site 20m. In April 2012, the polychaete ratio was much lower compared to December 2011 and was similar among sites. In October 2012, the polychaete ratio was higher at 100m and 10m compared to all other sites. The mixed model nested AOVA found that there was a significant interaction between time and site (distance). Although this indicates that there are inconsistent trends among the sampling events, the Tukey s post hoc analyses demonstrate that during December 2011, October 2012 and April 20, this result was due to an elevated ratio at sites close to the outfall (i.e. < 20 m), compared to those at greater distances. For example, there was a significantly higher polychaete ratio during December 2011 at the site 20m, but not during other sampling events. There was also an elevated polychaete ratio at 10m during October 2012 and April 20 only. During April 2012, there was a similar polychaete ratio among all distances. Page : 104 FIAL DRAFT: August 20

48 December 2011 April 2012 October 2012 April 20 Figure 3.5 Ratio of polychaete abundance to all other taxa abundance (mean ± E). ote: there is a different scale for the December 2011 graph. = 3 replicate sediment cores per site. = /A due to insufficient sediment depth to sample. Colours indicate distance from the WWTW outfall. Page : 104 FIAL DRAFT: August 20

49 3.1.5 Polychaete Families The abundance of polychaete families is presented in Figure 3.6. As well as accounting for approximately half of all taxa surveyed in this study, some polychaete families are potential indicators of high organic loadings. Differences between the compositions of polychaete families occurred between the four surveys. During December 2011, the polychaete families were comprised of mainly Polygordiidae and pionidae. Polygordiidae had high abundance at 20m and 200m and pionidae had high abundance at 50m. In comparison, polychaetes in the April 2012 survey were mostly comprised of ereididae, Capitellidae and Dorvilleidae. These families occurred in higher abundances at the sites within 100 m of the outfall compared to the reference sites. Polychaete families at the reference sites were largely made up less abundant species that have been categorised as other. The group other was generally similar across all other sites. Polygordiidae were again present in the April 2012 survey, and with higher abundance when compared to December However, no Polygordiidae were detected at 20m during April 2012, where they had been previously abundant in December During October 2012 and April 20, the composition of polychaete families was quite different from December 2011 and April 2012 in terms of the taxa present and also in terms of distribution between sites. In general, Dorvilleidae, ereididae and pionidae were the families that occurred in the highest abundances. pionidae was also found to be among the most abundant polychaete families in December 2011 and April 2012, while Dorvilleidae were most abundant in April During October 2012, the 10m site had much higher abundance in comparison to all other sites and this was largely characterised by the Dorvilleidae family. During April 20, the 50m site had the highest abundance which was also dominated by the Dorvilleidae family. The Polygordiidae and Dorvilleidae families were analysed by mixed model nested AOVAs (Table 3.1). For both families it was found that there was a significant interaction between time and site (distance), indicating that the patterns in their abundance were different across the four surveys. For Polygordiidae, there was significantly higher abundance at 20m and 200m in comparison to other sites, but during December 2011 only. Dorvilleidae were significantly higher at 10m during October 2012 and at 10m and 50m during April 20. Page : 104 FIAL DRAFT: August 20

50 December 2011 October 2012 April 2012 April 20 Figure 3.6 Mean abundance of polychaete families surveyed. Families with low abundance (i.e. < 10 individuals across all sites) were grouped as other. = 3 replicate sediment cores per site. = /A due to insufficient sediment depth to sample. Page : 104 FIAL DRAFT: August 20

51 3.1.6 Other Infauna Taxa Abundance of dominant infauna families other than polychaetes is presented in Figure 3.7. During December 2011, there was a high abundance of ematoda at the 50m and 200m sites. At 200m there was a high abundance of Oligochaeta. The pattern of dominant families was different between the April 2012 and December 2011 surveys. Few nematodes were detected in April There was also a general pattern of increasing Gammarids and to a lesser extent of Ostracods, within 10 m to 200 m from the outfall. This pattern was not consistent for the reference sites and there was very low abundance of infauna families here compared to all other sites. During October 2012, the dominant families were similar to April 2012 (but different to December 2011). Gammarids and Ostracods were the most abundant taxa. However, in contrast to April 2012 there was a trend for decreasing abundance of Gammarids with distance from the outfall. During April 20, the abundance of other dominant taxa was highest at sites 10m, 20m and 50m. The 20m site was dominated by nematodes and the 50m site was dominated by Gammarids. ematodes and Gammarids were analysed by mixed model nested AOVAs, as these were taxa that had the highest abundance across the two surveys or demonstrated trends with distance from the outfall (Table 3.1). For Gammarids and nematodes, there was a significant interaction found between time and site (distance) indicating that their patterns of abundance were inconsistent across the four surveys. Page : 104 FIAL DRAFT: August 20

52 December 2011 April 2012 October 2012 April 20 Figure 3.7 Mean abundance of dominant infauna (other than polychaetes) surveyed. = 3 replicate sediment cores per site. due to insufficient sediment depth to sample. = /A Page : 104 FIAL DRAFT: August 20

53 3.1.7 ummary of AOVAs Table 3.1 provides a summary of mixed model nested AOVAs for selected dependent variables of infauna taxa during all infauna surveys. Table 3.1 ummary of mixed model nested AOVAs for selected dependent variables of infauna taxa. = 3 replicate sediment cores per site. ource Effect DF M F p M F p Infauna Abundance Infauna Richness Time Fixed * Distance Fixed Distance*Time Fixed ite(distance) Random * ite(distance)*time Random ** ** Error Infauna Diversity Polychaete Ratio Time Fixed ** Distance Fixed Distance*Time Fixed ite(distance) Random ite(distance)*time Random ** ** Error Polygordiidae Dorvilleidae Time Fixed * Distance Fixed Distance*Time Fixed ite(distance) Random ite(distance)*time Random ** ** Error Gammarid Amphipods ereid Worms Time Fixed ** Distance Fixed Distance*Time Fixed ** ite(distance) Random ite(distance)*time Random ** Error ** = significant, p < 0.01, * = significant, p < ote: all data was log transformed (ln x+ 1) to treat unequal variances. Page : 104 FIAL DRAFT: August 20

54 Table 3.1 (continued) ummary of mixed model nested AOVAs for selected dependent variables of infauna taxa. ource Effect DF M F p ematodes Time Fixed Distance Fixed Distance*Time Fixed ite(distance) Random ite (distance)*time Random ** Error ** = significant, p < 0.01, * = significant, p < 0.05, ns = not significant. Where Levenes test indicated unequal variances (p < 0.05), data was log transformed Power Analysis Power analyses were carried out on the December 2011 survey data (i.e. data from first sampling round). The first analysis was done to determine what replication would be required to detect significant differences among sites. A Type I error rate of 5% (0.05) was used, a Type II error rate of 20% (0.2, power 80%) was considered acceptable and a 50% effect size was used. The power analysis estimated the amount of replication required to detect a significant difference (p < 0.05) with a 50% effect size (Appendix 3). The amounts of estimated replicates per site were 115 for abundance, 12 for richness and four for diversity. The analysis indicates that the sampling size of three sediment cores per site and six sediment cores per distance was not sufficient replication to detect differences for abundance and richness. The second analysis was done to determine what power was achieved using the sample size used in the current study (i.e. n = 3 per site). A Type I error rate of 5% (0.05) was used, a 50% effect size was used and a sample size of 3 was used. The amount of power achieved was low with 5.2% for abundance, 12.3% for richness and.5% for diversity. It should be noted: The post hoc power analyses undertaken during the first sampling round suggested that much more replication would be required for abundance and richness, however, this is likely to also be due to the fact that low abundance was found at the 2,000 m distance during December 2011, which is used as the basis for the effect size. The very large estimate for replicates required to detect significant differences in abundance is due to the very low results for these measures at the reference sites and the high variability at all sites. Alternative reference sites with a more Page : 104 FIAL DRAFT: August 20

55 similar particle size distribution were found for the third and fourth sampling event (although similar infauna assemblages were still found at the new sites). Following the first and second sampling events, it was recommended that the replication should be increased. However, it was considered that the costs and logistics (i.e. diving and sampling days required) were too large. AOVAs undertaken on the first and second sampling events were able to detect differences between sites and distances (this is generally considered to be sufficient evidence that enough replication has been used). However, after incorporating the data from all four sampling events differences could not be detected. At the site level, results were also inconsistent among the four surveys and this is reflected in the analysis with significant interactions between time and site (distance). 3.2 Multivariate Analyses of Infauna December 2011 on-metric Multidimensional caling (MD) plots were used to compare patterns in the similarities of infauna assemblages surveyed during the December 2011 survey (Figure 3.8; full analysis in Appendix 2). Visual examination of the MD plot for the December 2011 survey indicates some grouping of sites (e.g. 50m, 100m, 200m and ) and distances (e.g. 100 m and 2,000 m). There is also some directional separation evident between southern and northern sites at specific distances from the outfall (e.g. 20 m and, 200 m and and 2,000 m and ). While all sites within 200 m of the outfall tend to lie on the right hand side of the plot, the reference sites all lie in the center and on the left, showing a degree of dissimilarity between them. Two-way global analysis of similarities (AOIM) indicated that there was a significant difference in infauna assemblages. For December 2011 (R = 0.412, p < 0.05), this was due to significant pairwise comparisons whereby the 20 m distance was different to 50 m and 2,000 m, the 50 m distance was different to 100 m and 2,000 m and the 200 m distance was different to 2,000 m. Page : 104 FIAL DRAFT: August 20

56 Figure 3.8 MD analysis (square root transformation with Bray Curtis measure of similarity) of infauna assemblages for December The IMPER analysis in Table 3.2 identifies and ranks families which are contributing the most to the average dissimilarity between sites and was used to identify which families primarily accounted for the observed assemblage differences (i.e. which taxa were unique) in December ote: IMPER ranking does not necessarily correspond to the most abundant taxa. Abundant taxa for each survey period are discussed in ection 3.1. There was high variability in the structure of infauna assemblages among distances. In particular, the >2,000 m distance had some distinctly ranked families such as ipunculidae, Lumbrineridae and Ophiuroidea spp. during December Page : 104 FIAL DRAFT: August 20