Marl update 2 survey sept 2017 final by Huon Aquaculture

MARINE AQUACULTURE RESEARCH LEASE PROVIDENCE BAY, NSW UPDATE 2 SURVEY Water column, sediment chemistry and biological characteristics

FINAL REPORT September 2017

Report to NSW DPI/HUON AQUACULTURE RESEARCH TEAM BY AQUACULTURE, MANAGEMENT & DEVELOPMENT PTY LTD

MARL Update 2 survey Final Report September 2017

Document Distribution Date

Name

Company

2/04/2018

Matt Whittle

Huon Aquaculture

4/04/2018

Matt Whittle

4/04/2018

Document

Version

Copies

Draft

Huon Aquaculture

Draft

Ian Lyall

Fisheries NSW

Draft

4/04/2018

Wayne O’Connor

Fisheries NSW

Draft

4/04/2018

Graeme Bowley

Fisheries NSW

Draft

24/04/2018

Troy Gaston

University of Newcastle

Draft

24/04/2018

Margaret Platell

University of Newcastle

Draft

24/04/2018

Matt Whittle

Huon Aquaculture

Draft

24/04/2018

Ian Lyall

Fisheries NSW

Draft

24/04/2018

Wayne O’Connor

Fisheries NSW

Draft

24/04/2018

Graeme Bowley

Fisheries NSW

Draft

Type

DISCLAIMER: The present report is provided for the exclusive use of NSW DPI/Huon Aquaculture Research Team as part of the requirements for the Water Quality and Benthic Environment Monitoring Plan as part of the Environmental Management Plan for the development of the Marine Aquaculture Research Lease in Providence Bay, NSW, in accordance with the State Significant Infrastructure (SSI-5118) consent conditions. Aquaculture, Management & Development P/L disclaims all liability for any adverse consequences arising from the use of information contained within this report by any third party. Dr. Dom O’Brien Managing Director. AMD 24th May 2018

MARL Update 2 survey Final Report September 2017

Contents 1

Summary ................................................................................................................................. 1

Operational Summary ............................................................................................................. 2

Location and Survey Map ........................................................................................................ 4

Water Column Characteristics ................................................................................................. 5

Visual Surveys ....................................................................................................................... 11 5.1 ROV Filming summary ..................................................................................................... 11 5.2 Observations from ROV filming ....................................................................................... 11 5.3 Sediment Cores - Photographs ........................................................................................ 17

Sediment Chemistry .............................................................................................................. 19 6.1 Redox Potential ............................................................................................................... 19 6.2 Sulphide Analysis ............................................................................................................ 20 6.3 Particle Size Analysis ....................................................................................................... 21 6.4 Organic Content .............................................................................................................. 23

Biological Analysis ................................................................................................................. 25

References ............................................................................................................................ 37

Appendices............................................................................................................................ 39

List of Figures Figure 1 - Location and Survey Maps â&#x20AC;&#x201C; MARL Providence Bay ................................................................................................4 Figure 2 - Oxygen and Temperature profiles for all sites ..........................................................................................................6 Figure 3 - Graphs of nutrients results for all sites split into seafloor and surface readings. ...................................................7 Figure 4 - Comparison of various nutrient results for the present survey against Huon internal monthly monitoring surveys ...............................................................................................................................................................................8 Figure 5 - Monthly Chlorophyll a levels at sites C1, C3, S1 and S2, compared to survey results for pen site S3/PB01. ........9 Figure 6 - Screenshots of ROV footage from all sites. .............................................................................................................13 Figure 7 - Sediment chemistry cores for all survey sites except S2 ........................................................................................17 Figure 8 - Redox potential at 30 mm depth in sediment cores .............................................................................................19 Figure 9 - Sulphide concentrations in sediment core samples ...............................................................................................20 Figure 10 - Particle size analyses of the top 100 mm of sediment. Mean percentage cumulative volume for size fractions at each site .......................................................................................................................................................................22 Figure 11 â&#x20AC;&#x201C; Mean Total Organic Content for all sites (includes standard error) ...................................................................23 Figure 12 - Comparison of organic content across all sites for the three surveys. ................................................................24 Figure 13 - K-dominance curves, using data pooled for each site, of the broad-scale benthic macroinvertebrates, obtained from four replicate grabs at each site during Update 2 Survey at Providence Bay. ....................................28 Figure 14 - Means (and SE) of the numbers of broad-scale (a) and family-level taxa (b) and for all benthic macroinvertebrates (c), obtained from four replicate grabs at each site during the Baseline (blue) and Update 2 (orange) surveys at Providence Bay. ..............................................................................................................................30 Figure 15 - Percentage difference for the numbers of all benthic macroinvertebrates obtained from four replicate grabs at each site, with reference to Baseline, for both Update 1 and Update 2 Surveys at Providence Bay. ....................30 Figure 16 - A schematic of the relationships between the various sites at (a) broad-scale, i.e. phylum/class/order, and (b) family-level for polychaetes, molluscs and decapods, obtained from four replicate grabs at each site during Update 2 Survey at Providence Bay. All sites were significantly different in each of the northern and southern MARL (separated by a dashed line), except where blue lines denote non-significance. ............................................32

MARL Update 2 survey Final Report September 2017 Figure 17 - nMDS ordinations using log10 transformed data on the numbers of the various benthic macroinvertebrate at both a (a) broad-scale, i.e. phylum/class/order, and (b) family-level for polychaetes, molluscs and decapods, obtained from four replicate grabs at each site during Update 2 Survey at Providence Bay. 2 denotes replicate grab C2-2..........................................................................................................................................................................33 Figure 18 - nMDS ordinations, based on centroids of the Bray-Curtis similarities calculated from log10 transformed data on the numbers of the various benthic macroinvertebrate at both a (a) broad-scale, i.e. phylum/class/order, and (b) family-level for polychaetes, molluscs and decapods, obtained from four replicate grabs at each site during Baseline, Update 1 and Update 2 surveys at Providence Bay. ......................................................................................34

List of Tables Table 1 - Chlorophyll a levels for all sites ...................................................................................................................................9 Table 2 - Nutrient and Chlorophyll a levels from control and compliance sites compared against ANZECC Trigger levels (2000) ...............................................................................................................................................................................10 Table 3 - Pen Bays ROV direction from edge of pen................................................................................................................11 Table 4 - Description of each ROV dive performed at the MARL site, Providence Bay. ........................................................14 Table 5 - Mean sediment grain size and %mud at all sites......................................................................................................21 Table 6 - Numbers of the benthic macroinvertebrates for each broad-scale taxa (bold values) and important families (non-bold values) for selected groups, recorded at nine sites during the Update 2 Survey at Providence Bay, and the total and percentage contribution of each to the overall fauna. ...........................................................................27 Table 7 - Results of two-way ANOVA of Survey and Site of the numbers of taxa (broad-scale and family-level) and the total abundance of benthic macroinvertebrates obtained from four replicate grabs at each site during the Baseline and Update 2 surveys at Providence Bay, and the total and percentage contribution of each to the overall fauna. ...................................................................................................................................................................29

List of Appendices Appendix 1 Survey coordinates for sediment (non ROV) sampling, based on the Mapping Grid of Australia Zone 56 (Datum GDA94) ...............................................................................................................................................................39 Appendix 2 Water Column dissolved oxygen and temperature Sonde results as averages for surface, seafloor and 5m depth increments in between ........................................................................................................................................40 Appendix 3 Water Column results for all sites â&#x20AC;&#x201C; Nutrients ....................................................................................................41 Appendix 4 Redox potential, measured in millivolts from 3cm depth in the sediment grabs or cores...............................42 Appendix 5 Sulphide analysis, measured in sediments at 3 cm from sediment surface ......................................................43 Appendix 6 Organic content â&#x20AC;&#x201C; raw data..................................................................................................................................44 Appendix 7 AST Labs nutrients analyses methodology ..........................................................................................................45 Appendix 8 Raw data for benthic macroinvertebrates, obtained from four replicate grabs at each site during the Update 2 Survey at Providence Bay. Note: Green text represents taxa that were recorded for the first time, while the shaded grey represents no animals recorded for that taxa in Update 2, but which were previously recorded in either Baseline or Update 1 Survey. ...............................................................................................................................46

MARL Update 2 survey Final Report September 2017

Summary

The Marine Aquaculture Research Lease (MARL) AL06/098 is located in Providence Bay, New South Wales (NSW). NSW DPI/Huon Aquaculture Research Team are using the MARL to assess the potential for the culture of Yellowtail Kingfish in these exposed NSW waters. The MARL has a consent for a standing biomass of 998 tonnes. In September 2016 DPI/Huon positioned the first of two grids on the lease which can carry up to six pens each. For the first year, two stocked pens were present in that grid. This report presents all of the water column and sediments data required under the Water Quality Monitoring, the Substrate Monitoring and the Benthic Macroinvertebrate Monitoring programs associated with a standing biomass of 130 tonnes, as required under the SSI5118 consent. Assessment of the water quality data at both the pen site and compliance sites suggests that the only observed increase vs. control sites was the slight (in terms of magnitude) increase in surface ammonia level observed at the pen site. All other nutrient, chlorophyll a and physical/chemical water data do not show any upward trends at the pen site or elsewhere around the farm. Water column monitoring results across all sites show that: both Nitrate (seafloor) and Total N (seafloor and surface) levels greatly exceed, and, Total P (seafloor and surface) levels slightly exceed the ANZECC trigger levels, confirming a need to establish or develop suitable local guideline levels. Filming of the seabed showed all sites shared the common features of medium to coarse rippled sand, some shell grit and old shells, with a depauperate fauna. Differences in the pen sites (PB01 & PB04) were restricted to the presence of dislodged shell debris below the pen, e.g., dead M. coccopoma barnacles. As for the baseline survey, drift alga was abundant at nearly all sites. All sites have mean sediment redox values above 240mV and sulphide concentration was below detection. TOC levels would appear to be slightly higher in the northern end of the survey area than the south but there would appear to be no significant increase in TOC levels at the pen site (S3) since Baseline. These observations suggest there to be no significant evidence of organic enrichment across all sites including the pen site. Overall patterns of particle size distribution were indicative of a sedimentary environment with moderate agitation of seabed sediments and associated low abundance of fine silt and clay fractions. The control site C2, was composed of coarse to very coarse sands and was therefore substantially different to all other sites. The number of benthic macroinvertebrates at the nine sites of Update 2 Survey were similar to that recorded in Baseline, and was far less than recorded in Update 1 Survey. This is most probably related to seasonal changes in small crustaceans. Comparisons of the community composition of the benthic macroinvertebrates from the Update 2 survey with those of both Baseline and Update 1 surveys (at both a broad and family scale), found overall significant differences between the three surveys. These differences were most marked between Update 2 vs the other two surveys, but also emphasised the substantial variation between sites in each of the survey periods. The faunal patterns (broad-scale and family-level comparisons) show that the pen site does not show an obvious difference when compared to the northern control and compliance sites. This is similar to Baseline. The difference between Baseline and Update 1 surveys was considered likely to reflect the increased organic input from farm operations. However, given the continued increase in stocking since that time then other factor(s) may be responsible for the present lack of difference. It is also possible that the conclusion re Update 1 Survey may have been confounded due to seasonal effects, i.e. spring vs autumn. As the broad-scale and family-level comparisons â&#x20AC;&#x153;toldâ&#x20AC;? different stories with respect to the faunal patterns in S3 and C2, it is suggested that the family-level identification continue in the future.

MARL Update 2 survey Final Report September 2017

Operational Summary

All samples for the Benthic Macroinvertebrate Monitoring Program, the Water Quality Monitoring Program and the Substrate Monitoring Program were collected by AMD and the University of Newcastle in September 2017. Reporting of Update 2 survey follows the same format as provided in the Baseline and Update 1 survey reports except for the following: 1) For the water quality data, S6 was omitted as there were already two sites in close proximity (S4 & S5) at the southern end of the lease without there being any farming activity (no southern grid as yet) in that part of the lease; 2) Some comparison to the Baseline and Update 1 survey results are provided where appropriate; and 3) core photographs and brief descriptions are again provided to gauge their utility as another visual methodology for assessing any changes in the sediments at such an exposed farm site. The present survey has been timed to occur one year after the Baseline survey to allow for a direct comparison of the results whilst eliminating any seasonal effects. At the time of the survey there was a standing biomass of approximately 130T of YTK on the MARL. Contractor:

AQUACULTURE, MANAGEMENT & DEVELOPMENT PTY LTD (AMD) ACN 079 618 385 Phone 0421 331797 e-mail: dompobrien@ gmail.com

Client:

NSW DPI/Huon Aquaculture Research Team Locked Bag 1, Nelson Bay, NSW, 23157 T: 02 4982 1232 | F: 02 4981 9074 Liaison: Wayne O’Connor; Matt Whittle

Field work: AMD Personnel: University Personnel: Huon Personnel: NSW DPI Personnel:

AMD Pty Ltd, University of Newcastle, Huon Aquaculture Company Dom O’Brien (21st-24th Sept) Margaret Platell (23rd Sept), Tom Ryan (13th Sept), Gerhardus Barnard (21st, 22nd Sept) Tim Smith (13th, 22nd Sept) Jason Clark (13th Sept), David Whyte (21st-24th Sept) Ian Lyall (23rd Sept)

Dates of fieldwork: 13th Sept – Infauna all sites 21st Sept – ROV for sites C1, C2, S1, S2, S3 22nd Sept – Seafloor TOC/S2-/Redox for C1.1, C1.2, C2, S1, S2, S3, S4, S5 23rd Sept – Water column - Nutrients, Chlor a, DO, Salinity, Temp. ROV for sites C3, S4, S5, S6. 24th Sept – Seafloor TOC/S2-/Redox for sites S6, C3 & C1.3.

Laboratory Analysis: Faunal analysis, Sediment Grain size, Chlorophyll a: University of Newcastle. Nutrients in water, Analytical State Laboratories (AST), Hobart, TAS. Organic content, Analytical Laboratory Services (ALS), Sydney, NSW. Filming for this assessment was carried out using a VideoRay Remotely Operated Inspection System using a colour video camera with 180 degrees of tilt range, high resolution and 0.004 lux of sensitivity, accompanied by two optimised LED arrays (3,600 lumens) adjustable lights. 2

MARL Update 2 survey Final Report September 2017

Seabed sampling was undertaken using a Van-Veen grab for infauna and Craib corer for sediment chemistry cores. Reporting: Section 7 and infauna parts for the Executive Summary, Dr Margaret Platell, University of Newcastle, NSW. The rest of the reporting is provided by AMD.

MARL Update 2 survey Final Report September 2017

Location and Survey Map

Figure 1 - Location and Survey Maps â&#x20AC;&#x201C; MARL Providence Bay Key to maps: S1, S2, S5 & S6 35m compliance (triplicate groups of ROV spot dives). S1.1, 2.1, 4.1, 5.1, 6.1 Sediment and water chemistry compliance sites C1, C2, C3 Control sites (also in triplicate). S4 Future pen site (also in triplicate). S3(PB01), PB04 Current pen sites, single ROV transects Red rectangle â&#x20AC;&#x201C; Lease boundary. NB: 2 pen locations are indicated for PB01, these indicate the position of the pen on the different sampling days. For sediment chemistry sample site coordinates please refer to Appendix 1.

MARL Update 2 survey Final Report September 2017

Water Column Characteristics

Methods Dissolved Oxygen (percentage saturation), pH and temperature data were measured using a YSI 600 series Sonde on 23rd September. The probe measured from the water surface down to within 2-5m of the seafloor at all sites except S6. Site S6 was not included in the present survey as 2 out of the 3 sites S4-S6 was deemed sufficient for a description of water column characteristics for that part of the lease. The full suite of nutrients was also collected on the 23rd September. Nutrients and Chlorophyll a were sampled at the surface and seafloor using a Niskin bottle with graduated cord to ensure that the deeper samples were taken just above (approximately 2m) the seafloor. The water samples were subsequently analysed for nutrients by AST laboratories Hobart. The methodology employed by the AST labs is provided in Appendix 7. Chlorophyll a samples were collected and analysed by the University of Newcastle. Seawater (0.5 â&#x20AC;&#x201C; 1.0L) was filtered through a 1.2um glass fibre filter (GF/C) under low vacuum for Chlorophyll a analysis (Dela Cruz et al., 2002). The filter paper was placed into a 15ml centrifuge tube and 10ml of 90% acetone added. The filter paper was macerated with a glass rod, centrifuge tube covered in foil and placed in the freezer (-20oC) for 1 hour. The sample was then centrifuged at 5000rpm for 5 minutes. The supernatant was decanted into a UV spectrophotometer and measured at 630, 647, 650 and 750nm. Calculations of Chlorophyll a were based on the American Public Health Association (APHA) Method 10200.

Results Dissolved oxygen (DO) as % saturation and temperature results are provided in Figure 2 and Appendix 2. DO ranged between 104-107% at the surface, decreasing down through the water column to 82-90% at the seafloor for most sites, except C1 (96%) and C3 this being the shallowest site at 98% saturation at 30m depth. These DO levels are more variable than those measured for the same sites in the Baseline survey with surface DO higher and seafloor DO lower than for Baseline. Water temperatures were also more variable than for the Baseline survey with temperatures ranging from 19.5o to 19.9o degrees at the surface for all sites to 17.2o to 18o for most sites at close to the seafloor. Again C1 (18.5o) and C3 (18.8o) were slightly higher than the other sites at the seafloor. Salinity was also highly consistent and >35 parts per thousand for all depths at all sites.

MARL Update 2 survey Final Report September 2017

Figure 2 â&#x20AC;&#x201C; Dissolved Oxygen and Temperature profiles for all sites

Water column nutrient results for surface and seafloor samples are provided in Figure 3 and Appendix 3. Please note that where the sample value is below the level of detection, then the figure used for the graphs and depicted in Appendix 3 in red is that taken from the AST lab report raw data (uncorrected for being below the limit of detection) and rounded. Nitrate+nitrite (NOx) and dissolved reactive phosphorus (DRP) levels were consistently higher at the seafloor compared to the surface, with all NOx readings being at or below the limit of reporting, i.e., 0.002mg/L at the surface, and <0.003-0.082mg/L at the seafloor. DRP ranged between 0.0050.008mg/L at the surface and 0.005-0.017mg/L at the seafloor. Kjeldahl Nitrogen, Total Nitrogen and Total Phosphorus readings generally showed little difference between the surface and the seafloor or across sites, with Kjeldahl N ranging from 0.29-0.37mg/L, Total N ranging from 0.290.41mg/L and Total P ranging from 0.03-0.05mg/L. Ammonia levels were also generally similar

MARL Update 2 survey Final Report September 2017

between the depths and across the sites (range of 0.004-0.013mg/L) except for the pen site (S3), where there was a spike in Ammonia of 0.024mg/L. These results are higher than for the Baseline survey except for: the low values for NOx at the surface, and, a small increase in ammonia (when compared to the other sites) registered at the surface for the S3/PB01 site. But all lie within the broadscale environmental range for the region (refer to Figure 4 below).

Figure 3 - Graphs of nutrient results for all sites split into seafloor and surface readings.

Huon Aquaculture is also undertaking in-house monthly samples for nutrient water quality across a representative range of sites (C1, C3, S1, S2) in order to establish or develop suitable local ANZECC guideline levels. The first 12 months of this data is presented in Figure 4. For Ammonia the Pen (S3/PB01) data for the Baseline, Update 1 and Update 2 surveys is also included for comparison. From the surface Ammonia graph, it is evident that levels in the Update 1 & 2 surveys are markedly greater at the pen than all other sites. However, given the Ammonia readings for C1/S3 in the previous month then these â&#x20AC;&#x2DC;elevatedâ&#x20AC;&#x2122; levels still fall within the broadscale environmental range for the region. As shown, seafloor Ammonia levels are well within the broadscale environmental range both within the surveys and on a seasonal basis. After 12 months of monthly surveys seafloor dissolved nutrient levels (NOx, DRP) are (during most months) markedly higher than at the surface across the Bay (Figure 4), with a possible apparent increase in these surface nutrients occurring earlier in 2017 than 2016. 7

MARL Update 2 survey Final Report September 2017

Figure 4 - Comparison of various nutrient results for the present survey against Huon internal monthly monitoring surveys

Chlorophyll a results for both surface and close to the seafloor samples taken on September 23rd are provided in Table 1. Across all sites, Chlorophyll a levels ranged from <0.2-0.65µg/L. Mean levels did not differ greatly between surface (0.37µg/L) and seafloor (0.40µg/L) sites. Monthly results (where available) for surface Chlorophyll a levels taken at a reduced range of monitoring sites are presented in Figure 5. These results show that Chlorophyll a levels throughout the survey area are generally below 2µg/L, but can on occasion become elevated across all sites (December 2016). As nutrients drive plankton productivity then it is reasonable to suppose that the high dissolved nutrient levels present in the December monthly survey may be part of the reason at least for the significant increase in Chlorophyll a levels at that time. The present survey and monthly 8

MARL Update 2 survey Final Report September 2017

results do not suggest that there is any strong trend across the survey sites, as might be expected in such an exposed area, and the pen site (S3) did not show any sign of an increase above background levels. Table 1 - Chlorophyll a levels for all sites

Figure 5 - Monthly Chlorophyll a levels at sites C1, C3, S1 and S2, compared to survey results for pen site S3/PB01.

MARL Update 2 survey Final Report September 2017

Interpretation – general comments on DO/nutrients/Chlorophyll a levels and trends. Water Quality monitoring is included as part of the survey programme primarily in order to; define the baseline concentrations for these variables on a seasonal basis for Providence Bay, and, to detect if there are any measurable changes/differences in these water quality variables around the fish farm, and if so at what distances from the farm these differences might be detected. Defining the Baseline characteristics and Seasonality of the water quality variables. This is being addressed through the monthly sampling surveys (with 12 months of data available), and from the Baseline, Update 1 and Update 2 surveys. From the data presented above and Table 2 below, the following broad trends are suggested: 1. Oxygen saturation levels can decrease in this region by as much as 20% from the surface to the seafloor at certain times of the year. 2. Dissolved nutrient levels for NOx & DRP (FRP - Table 2) differ markedly between the seafloor and the surface samples. For the other nutrient categories there was a slight increase in levels at the seafloor compared to surface values. 3. At control and compliance sites both NOx (seafloor) and Total N (seafloor and surface) levels greatly exceed, and Total P (seafloor and surface) and Chlorophyll a (surface) levels slightly exceed the ANZECC 2000 trigger levels, confirming a need to establish or develop suitable local guideline levels (as suggested by the Baseline survey results) for Providence Bay. 4. The monthly data suggests that there is an ingress of Nitrate rich waters into Providence Bay during the summer months, which may be linked to higher productivity as evidenced by the Chlorophyll a levels in December, 2016 (Figure 5). Results for the present survey suggest that this increase can start in the early Spring. Table 2 - Nutrient and Chlorophyll a levels from control and compliance sites compared against ANZECC Trigger levels (2000)

ALL SURVEYS (COMPLIANCE AND CONTROLS ONLY)

Parameter

Unit

NH4-N NOx-N Tot N FRP Tot P Chl a

µg/L µg/L µg/L µg/L µg/L µg/L

ANZECC Control sites Control sites Compliance Compliance Trigger Surface Seafloor sites Surface sites Seafloor level 2000 Mean Mean Mean Mean 20# 25# 120 10 25 1

7 6 267 5 28 1.58

9 42 301 10 30 0.62

6 6 267 5 27 1.31

8 49 297 11 35 0.61

# - From ANZECC - NSW elevated levels due to frequent upwelling events

Farm observations The present survey has been timed to coincide with a standing biomass of approximately 130T of YTK on the MARL, with the additional expectation that this would happen approximately 12 months after the Baseline survey, thereby eliminating seasonal effects in any comparison between the Baseline and present Update 2 surveys. From the present survey, considering some of the seasonal variation suggested by the monthly data, then the only observed increase at the pen site is the slight (in terms of magnitude) increase in surface ammonia level, noting that compliance sites remained at the same levels or lower than the control sites. All other nutrient, Chlorophyll a and physical/chemical water data do not yet show any upwards trend at the pen site or elsewhere around the lease. 10

MARL Update 2 survey Final Report September 2017

Visual Surveys 5.1

ROV Filming summary

The appearance of the seabed in the vicinity of the MARL was recorded by filming spot dives of the sea floor using a VideoRay Remote Observation Vehicle (ROV). The spot dive locations were: -

Compliance sites - at 35m outside the lease boundary (S1, S2, S5, S6). Previously unoccupied pen bay - consisting of triplicate samples in the vicinity of the prospective pen bay in the future southern grid within the lease area (S4). Presently used (stocked) pen bays - consisting of single ROV transect locations under the occupied pen bays within the lease area (S3/PB01 and PB04). Control sites at least 500 m from the lease boundary (C1, C2, C3).

A map of these survey sites is provided in Figure 1 and their coordinates listed in Table 4. The positions of all dives were located or marked by DGPS using a Getac mobile GIS unit with attached Novatel Smart Antenna Differential GPS. Digital recording of all control, compliance and internal lease dive sites for the MARL has been forwarded to NSW Department of Planning & Environment, and screenshots of each site provided in Figure 6. 5.2

Observations from ROV filming Interpretation – general comments on spot dive locations

Table 4 provides a list of seabed sediment characteristics and fauna observations from the 26 ROV spot dives undertaken at the control, compliance and pen sites. All sites shared the common features of medium to coarse rippled sand (e.g., screenshots, Figure 6), some shell grit and old shells, with a depauperate fauna. There were very occasional Pennatulaceans (Cnidaria), juvenile Flathead and a Stingaree, brittle stars (ophiuroid), hermit crabs and ribbon worms; observed at only one or two sites. There was no further evidence for the dark grey rounded ‘globules’ present at the S1.1 site in the Baseline survey. The present survey again shows that light coloured amorphous (possibly sand) accretions/globules are widely distributed with evidence for them at a number of sites (e.g., S4, S5, S6 & C3). The pen sites (PB01 & PB04) showed some slight differences to the rest of the sites as might be expected. These differences were restricted to the presence of dislodged shell debris below the pen, e.g., dead Megabalanus coccopoma barnacles (shells only). Drift algae was abundant at nearly all sites, except C3, where only a few very sparse and old pieces of plant material were observed. There appeared to be a density gradient of drift algae in general from the northern (most dense) to the southern sites as observed during the Baseline survey. Table 3 - Pen Bays ROV direction from edge of pen

Grid Position

Fish Pen

Direction to centre of pen (degrees)

PB01 PB04

1601 1602

40 40

MARL Update 2 survey Final Report September 2017

The only introduced species identified from the survey footage was Megabalanus coccopoma. This was present as dislodged (possibly through wave action on the pens or by in-situ cleaning of the nets) clumps of shell debris underneath both occupied pen bays.

MARL Update 2 survey Final Report September 2017

Figure 6 - Screenshots of ROV footage from all sites.

MARL Update 2 survey Final Report September 2017 Table 4 - Description of each ROV dive performed at the MARL site, Providence Bay. Easting (GDA94 MGA55)

Northing (GDA94 MGA55)

Date (DD-MMYYYY)

Time

Depth (m)

Dive Type

C1.1

433769

6388153

21-Sep-17

10.04.36 am

control

C1.2

433805

6388169

21-Sep-17

10.20.28 am

control

C1.3

433732

6388106

21-Sep-17

10.35.15 am

37.5

control

C2.1

433294

6388514

21-Sep-17

10.49.45 am

37.0

control

C2.2

433329

6388588

21-Sep-17

11.07.43 am

37.5

control

C2.3

433308

6388556

21-Sep-17

11.26.34 am

37.5

control

1.1

432791

6387474

21-Sep-17

11.59.48 am

38.5

35m

1.2

432761

6387497

21-Sep-17

1.00.48 pm

35m

1.3

432826

6387439

21-Sep-17

1.14.35 pm

38.5

35m

2.1

432425

6387434

21-Sep-17

2.21.27 pm

37.5

35m

Site

Comments Seabed = Rippled sand, occasional old shell. Fauna = None observed. Flora = A lot of loose drift algae, mostly reds. Seabed = Rippled sand, occasional old shell, some shellgrit. Fauna = None observed. Flora = A lot of loose drift algae, mostly reds. Seabed = Rippled sand, occasional old shell, some shellgrit. Fauna = None observed. Flora = A lot of loose drift algae, mostly reds. Seabed = Rippled sand, occasional old shell, some shellgrit. Fauna = Suspected Ribbon worm and small hermit crab. Flora = A lot of loose drift algae, mostly reds. Seabed = Rippled sands, quite coarse. Moderate shell debris and shellgrit. Fauna = None observed. Flora = A lot of drift algae quite consolidated, mostly reds. Seabed = Rippled sands, quite coarse at start of filming, finer later. Moderate shell debris and shellgrit. Fauna = None observed. Flora = A lot of drift algae quite consolidated, mostly greens/reds. Seabed = Rippled sand, occasional old shell, some shellgrit Fauna = None observed. Flora = A lot of loose drift algae, mostly reds. Seabed = Rippled sand, occasional old shell, some shellgrit Fauna = Suspected brittle star. Flora = A lot of loose drift algae, mostly reds. Seabed = Rippled sand, occasional old shell, some shellgrit Fauna = Hermit crab. Flora = A lot of loose drift algae, mostly reds. Seabed = Rippled sand, occasional old shell, some shellgrit Fauna = Flathead, Stingaree. Flora = A lot of loose drift algae, mostly reds.

MARL Update 2 survey Final Report September 2017 Easting (GDA94 MGA55)

Northing (GDA94 MGA55)

Date (DD-MMYYYY)

Time

Depth (m)

Dive Type

2.2

432458

6387469

21-Sep-17

2.41.26 pm

37.5

35m

2.3

432388

6387396

21-Sep-17

3.05.10 pm

37.5

35m

4.1

432358

6386760

23-Sep-17

10.19.16 am

40.5

internal

4.2

432361

6386777

23-Sep-17

10.29.43 am

40.5

internal

4.3

432344

6386771

23-Sep-17

10.40.43 am

40.5

internal

5.1

432519

6386589

23-Sep-17

9.50.13 am

35m

5.2

432548

6386634

23-Sep-17

9.59.25 am

35m

5.3

432484

6386554

23-Sep-17

10.09.23 am

35m

6.1

432159

6386559

23-Sep-17

9.20.23 am

40.5

35m

Site

Comments Seabed = Rippled sand, occasional old shell, some shellgrit. Fauna = None observed. Flora = Moderate amount of loose drift algae, mostly reds. Seabed = Rippled sand, occasional old shell, some shellgrit. Fauna = Urchin test and suspected Flathead. Flora = Moderate amount of loose drift algae, mostly reds. Seabed = Rippled sand, amorphous small lumps on surface, occasional old shell, some shellgrit. Fauna = None observed. Flora = A lot of loose drift algae, mostly reds. Seabed = Rippled sand, amorphous small lumps on surface, occasional old shell, some shellgrit. Fauna = Brittle star. Suspected ribbon worm. Flora = A lot of loose drift algae, mostly reds. Seabed = Rippled sand, amorphous small lumps on surface, occasional old shell, some shellgrit. Fauna = None observed. Flora = A lot of loose drift algae, mostly reds. Seabed = Rippled sand, amorphous small lumps on surface, occasional old shell, some shellgrit. Fauna = None observed. Flora = A lot of loose drift algae, mostly reds. Seabed = Rippled sand, amorphous small lumps on surface, occasional old shell, some shellgrit. Fauna = None observed. Flora = A lot of loose drift algae, mostly reds. Seabed = Rippled sand, amorphous small lumps on surface, occasional old shell, some shellgrit. Fauna = Suspected small crustacean. Flora = A lot of loose drift algae, mostly reds. Seabed = Rippled sand, amorphous small lumps on surface, occasional old shell, some shellgrit. Fauna = None observed. Flora = A lot of loose drift algae, mostly reds.

MARL Update 2 survey Final Report September 2017 Site

Easting (GDA94 MGA55)

Northing (GDA94 MGA55)

Date (DD-MMYYYY)

Time

35m

Depth (m)

Dive Type

6.2

432126

6386597

23-Sep-17

9.29.52 am

6.3

432201

6386523

23-Sep-17

9.40.18 am

35m

C3.1

430275

6384853

23-Sep-17

7.47.35 am

36.5

control

C3.2

430272

6384806

23-Sep-17

8.00.17 am

36.5

control

C3.3

430221

6384856

23-Sep-17

8.11.23 am

36.5

control

PB01 (3.3)

432568

6387284

21-Sep-17

2.06.02 pm

38.5

Stocked pen

PB04

432491

6387218

21-Sep-17

1.46.15 pm

38.5

Stocked pen

Comments Seabed = Rippled sand, amorphous small lumps on surface, occasional old shell, some shellgrit. Fauna = Suspected Maldanid polychaetes. Flora = A lot of loose drift algae, mostly reds. Seabed = Rippled sand, amorphous small lumps on surface, occasional old shell, some shellgrit. Fauna = None observed. Flora = A lot of loose drift algae, mostly reds. Seabed = Rippled sand, amorphous small lumps on surface, occasional old shell, some shellgrit. Fauna = Suspected Sea pen. Flora = Very small amount of old loose drift algae. Seabed = Rippled sand, amorphous small lumps on surface, occasional old shell, some shellgrit. Fauna = Suspected Sea pen. Flora = Small amount of old loose drift algae. Seabed = Rippled sand, amorphous small lumps on surface, occasional old shell, some shellgrit. Fauna = None observed. Flora = Very small amount of old loose drift algae. Seabed = Rippled sand, occasional old shell, some shellgrit. Fauna = Unidentified fish. Megabalanus shells (debris) probably not live. Flora = A lot of loose drift algae, mostly reds. Seabed = Rippled sand, occasional old shell, some shellgrit. Fauna = Flathead and unidentified skate or ray. Megabalanus shells (debris) probably not live. Flora = A lot of loose drift algae, mostly reds.

MARL Update 2 survey Final Report September 2017

5.3

Sediment Cores - Photographs C1

S3(PB01)

Figure 7 - Sediment chemistry cores for all survey sites except S2

Interpretation â&#x20AC;&#x201C; general observations on cores Photographs of all sediment chemistry cores except for S2 (photographs accidentally deleted) are presented in Figure 7 above. General observations are as follows: 1) There would appear to be a slight general trend from fine to coarser grained sand from north to south across the groups of sites (C1/C2 to S1/S2/S3 to S4/S5/S6 to C3). With the notable difference that two of the C2 cores were predominantly coarse to very coarse-grained sand and therefore substantially different to all other cores.

MARL Update 2 survey Final Report September 2017

2) All cores are uniform in colour throughout (light brown sand colour) showing well-oxygenated sands in all cases. The only signs of black deposits were at approximately 5cm depth in one C1 and one C2 core (Figure 7). The presence of these faint black deposits at both control sites is not reflected in the sediment chemistry which is measured at 3cm depth. As for Update Survey 1, there were no visual signs of organic matter at the pen site (S3/PB01).

MARL Update 2 survey Final Report September 2017

Sediment Chemistry

Triplicate samples were taken at each of the nine sample sites for all of the variables tested in this section, except for sediment grain size for which one sample only was taken per site. Results for each site are generally presented below as an average (and associated variability) of the triplicate samples. 6.1

Redox Potential

Methods Redox potential was measured in millivolts at 30mm below the sediment surface using a TPS intermediate junction Redox (ORP) Sensor attached to a TPS pH 80 meter. Calibration and functionality of the meter were checked before each test using Zobells Standard Solution (229mV at 25Â°C). Pre measurement calibration provided a reading of 240mV at 19Â°C. Measurements were made within 3 hours of the samples being collected. Corrected Redox potential values were calculated by adding the standard potential of the reference cell to the measured redox potential and are reported in millivolts. Results and interpretation All sites have mean sediment redox values above 240mV (Figure 8). The observed high redox values are indicative of well oxygenated, unimpacted sediments (Macleod & Forbes, 2004) and it would appear that whatever impacted S1 since the baseline survey and S3 in the previous survey has now dissipated or was highly localised. Raw data is presented in Appendix 4.

Figure 8 - Redox potential at 30 mm depth in sediment cores

MARL Update 2 survey Final Report September 2017

6.2

Sulphide Analysis

Methods Sediment sulphide was measured in broad accordance with the protocols outlined in Macleod and Forbes (2004) and modified for a TPS intermediate junction series (IJ-Ag2S) silver/sulphide ion selective electrode and TPS WP-90 Specific Ion-pH-mV meter. Measurements were made using a modified syringe, 2mL of sediment was removed at 30mm depth from the core or grab and mixed with 2mL of reagent (sulphide anti-oxidant buffer, SAOB) in a small beaker. The sediment/SAOB mixture was carefully stirred with the probe for 15-20 seconds, until the reading stabilised. The accuracy and functionality of the meter and probe was assessed prior to analysis commencing, using standards of known concentration. The meter and probe have a self-calibration facility (mV to uM sulphide) that allows for the meter to provide sulphide concentration directly without the need for post processing conversion. Results and interpretation Sulphide concentration in sediments was well below detection at all sites (Figure 9) and it would appear that whatever impacted S1 since the baseline survey and S3 in the previous survey has now dissipated or was highly localised. The observed sulphide concentrations presently, suggest there to be no significant evidence of organic enrichment (Macleod & Forbes, 2004). A table of the raw data is presented in Appendix 5.

Sulphides 50 45 40

Sulphides (uM)

35 30 25

20 15 10 5 0 S1

S5 Site

Figure 9 - Sulphide concentrations in sediment core samples

MARL Update 2 survey Final Report September 2017

6.3

Particle Size Analysis

Methods Surface sediments were taken from the grab samples provided for the faunal analysis. These samples were homogenised and then sieved for particle size determination. Results and interpretation Apart from C2, all sediments sampled across the area were dominated by medium sand fractions with the great majority of sediments (>50% at each site) being in the 0.25 and 0.5mm size classes. The sediments were clean with a very low proportion of mud fractions (i.e. < 0.063mm). The mean grain size across all of these sites was within a narrow range with a trend of increasing mean grain size from north to south across the survey area as was also identified during the Baseline and Update 1 surveys, and there were no site specific differences identified since Baseline survey. At C2, the sample was composed of coarse to very coarse sands (0.5-2mm in size) and was therefore substantially different to all other sites. This result was also different to the results for the previous surveys, and suggests that there may be some form of mosaic of coarser sand interspersed with the fine to medium sands at this site possibly due to more sorting/agitation of the sediments. Detailed results are presented in Figure 10, with the proportion of muds provided in Table 5. The patterns of particle size distribution for all sites were indicative of a sedimentary environment with moderate agitation of seabed sediments and associated low abundance of fine silt and clay fractions. These patterns are considered typical of sediments in deep (i.e. >20m) and exposed locations. The overall similarity in particle size distribution between the majority of sites implies similar depositional environments. Table 5 - Mean sediment grain size and %mud at all sites

Site C1 C2 C3 S1 S2 S3 S4 S5 S6

Mean Grain Size (um) Mean % Mud 249.60 0.063 1097.30 0.038 441.93 0.058 267.62 0.143 412.38 0.061 371.90 0.050 446.47 0.049 435.08 0.036 407.46 0.079

MARL Update 2 survey Final Report September 2017

Figure 10 - Particle size analyses of the top 100 mm of sediment. Mean percentage cumulative volume for size fractions at each site .

MARL Update 2 survey Final Report September 2017

6.4

Organic Content

Methods A single undisturbed sediment core sample taken using a perspex core with an internal diameter of at least 50mm at each sample site specified in the survey for the purposes of organic content analysis. The top 3cm of each was oven dried at 60Â°C prior to analysis of total organic carbon. Total organic carbon (TOC) was measured by loss on ignition (450Â°C in a muffle furnace for 4 hours) by AST. Results and interpretation Results from the organic content analysis are presented in Figure 11. As for both previous surveys, the organic content was very low for all samples, ranging from <0.04% to 0.18% (refer to Appendix 6), with an average of 0.07% across all samples. TOC levels would appear to be slightly higher in the northern end of the survey area than the south as shown by higher levels at C1, C2 and S1. There would appear to be no significant increase in TOC levels at the pen site (S3) since Baseline. A comparison of the results from all three surveys (Figure 12) shows the relative decrease in organic content at sites C1 and S1 between this and the previous surveys.

Figure 11 â&#x20AC;&#x201C; Mean Total Organic Content for all sites (includes standard error)

MARL Update 2 survey Final Report September 2017

Figure 12 - Comparison of organic content across all sites for the three surveys.

MARL Update 2 survey Final Report September 2017

Biological Analysis

Methods Collection: Macroinvertebrates were collected at each of the nine sampling sites in and around the MARL using a Van Veen grab that sampled a 0.07 m2 area of seabed on the 13th of September in 2017. Four replicate grab samples were collected at each of the control (C1, C2, and C3), pen (S3 and S4) and compliance (S1, S2, S5 and S6) sites, resulting in a total of 36 grab samples. Grab samples were placed in plastic bags containing 5-10% buffered formalin, and stored for 4 weeks, ensuring adequate fixation of organisms. Each sample was sieved, within a fume hood, through 1 mm mesh, rinsed thoroughly and stored separately in jars with 70% ethanol. Laboratory analysis: Samples were processed in the laboratory by firstly rinsing the sample with fresh water through a very fine mesh sieve (125 um), and then examining for the lighter/floating organisms (typically crustaceans and polychaetes) using a dissecting microscope. The remaining shell grit was microscopically examined for heavier/denser organisms (such as molluscs and echinoderms). This process is termed picking. Using a dissecting microscope, each organism was then identified to a higher taxonomic level, i.e. phylum, class or order, and, in the case of the polychaetes, molluscs and decapods, further identified to family level where possible using Fauchald (1977), Beasley et al. (1998), Underwood and Hoskin (1999), Jansen (2000) and Poore (2004), as well as online keys â&#x20AC;&#x201C; Grove and de Little (2017) and Wilson et al. (2017). This process is called sorting. Statistical analyses: The relationship between the rank of each benthic macroinvertebrate taxon and its percentage contribution for each sites was described using K-dominance curves. Summaries of the main taxa were tabulated (Table 6) and raw data provided (Appendix 8). The number of benthic macroinvertebrate taxa, both at a broad scale, i.e. phylum, class or order, as appropriate, and family level (within the polychaetes, molluscs and decapods) and the abundance of all benthic macroinvertebrates were each analysed using two-way ANOVA, with Survey (Baseline vs Update 2) and Site as the two factors. Leveneâ&#x20AC;&#x2122;s test showed that the numbers of broad-scale taxa were not required to be transformed (Broad-scale, df = 6,15, F = 1.25, P = 0.368). However, there was no transformation for either family-level taxa or the total abundance that appropriately reduced the significance of Leveneâ&#x20AC;&#x2122;s test and the data was therefore not transformed, i.e. Familylevel, df =6,15, F = 2.07, P = 0.026; Total abundance df = 6,15, F = 2.73, P = 0.003). Means and standard error plots are presented for the number of taxa, both at a broad scale and family level, and the total abundance of all benthic macroinvertebrates (Figure 14 a-c). Multivariate analyses of the abundance of each of the benthic macroinvertebrate taxa were carried out, treating both broad-scale and family-level taxa separately, using various subroutines of the PRIMER 7 package (Clarke et al., 2014). The total numbers of each of the various taxa in each replicate grab were log10 transformed and the Bray-Curtis measure used to create a similarity matrix. One-way analyses of Similarities (ANOSIM) was used to determine whether there were any significant differences between samples at the different sites, using the magnitude of the R-statistic to ascertain the extent of any such difference, and the benthic faunal similarities between the various sites were visualised using non-metric multidimensional-scaling (nMDS) ordination. RELATE was used to assess the correspondence between the broad-scale and family-level matrices. 25

MARL Update 2 survey Final Report September 2017

Comparisons were made of the numbers of benthic macroinvertebrates at both the broad-scale and family-level taxa obtained in this survey (Update 2) and both Baseline and Update 1. Thus, centroids of the samples for the different Sites in the different surveys were calculated from the Bray-Curtis similarity matrix using the add-on PERMANOVA routine (Anderson et al. 2008) within PRIMER7, and visualised using nMDS ordination. Two-way analyses of Similarities (ANOSIM) was used to determine whether there were any significant differences between Survey and Site, focussing on any differences between Survey. Results and interpretation Comments re processing, sediment characteristics and taxonomic identifications: Following sieving of the material from eight of the nine sites through a 1 mm mesh, approximately 100-250 ml of a mixture containing sediment, shell grit and animals remained, which meant relatively short picking times of 30 min to 1 h. However, in the case of C2, nearly all of the sample material was retained on the sieve for three of the four samples (except for C2-2) with many small individuals present in those samples. This meant that picking times ranged between 4 and 8 hours for those three samples. However, sorting times ranged from 20 min to 60 min for all samples. All samples contained a mixture of shell grit and oceanic sand, except for those three samples at C2 which contained large amounts of rounded lithic granules (> 1 mm in diameter). It is noted that this sediment type was present at the nearby S1 in Update 1 Survey (AMD, 2017). For each of the samples from S3 (the active pen site), there were small weathered fragments of M. coccopoma and no anoxic sediments were observed. This is in contrast to Update 1 - with large fragments of M. coccopoma observed in all samples and anoxic sediments found in two of the four samples (S32 and S3-3, AMD, 2017). It is also noted that there were a few small and weathered fragments of M. coccopoma present at C1 in this Update 2 survey. General description: A total of 3210 benthic macroinvertebrates were recorded, representing a total of 32 broad-scale taxa (including 11 phyla) and, collectively within the polychaetes, molluscs and decapod crustacean over 20 families (Table 6 and Appendix 8). Some of the families caught in previous surveys were not obtained (those greyed out in Appendix 8), and these were typically represented by low numbers of individuals in other surveys (except for the opheliid polychaetes). Of the newly-sampled families, these included single individuals of a polychaete (cf. Heterospionidae) and one crustacean (Penaeidae). Thirty seven small dorvilleid polychaetes were recorded, with most of these being represented at C2 (Table 6), possibly due to the unusual sediments present at that site during this survey (see earlier). For comparison, 19 dorvilleids were obtained over all sites in the Update 1 Survey, while just one was recorded in the Baseline Survey (AMD, 2016; 2017). As for Baseline, there were very few capitellid polychaetes (Appendix 8). Caprellid amphipods were found again in low numbers, i.e. 1 or 2 at each of the sites (Appendix 8), but which was greater than the total of 3 found in Update 1 Survey (AMD, 2017). The Phylum Chordata was also represented, but not included in subsequent analyses, with three small teleosts being obtained at two sites, i.e. an ophichthyid (sand eel) at C2, and a pleuronectid (flounder) and platycephalid (flathead) at S3.

MARL Update 2 survey Final Report September 2017 Table 6 - Numbers of the benthic macroinvertebrates for each broad-scale taxa (bold values) and important families (non-bold values) for selected groups, recorded at nine sites during the Update 2 Survey at Providence Bay, and the total and percentage contribution of each to the overall fauna. BENTHIC MACROINVERTEBRATES

3 4 3 3 -

4 6 15 8

5 12 -

6 24 3 6 -

3 6 5 2 -

10 80 16 3

10 28 0 12 -

9 17 1 4 -

23 4 1 3 -

126 17 25 22 17 15

17 5

25 15 2 3 1 -

56 15 9 6 8 3

77 8 23 19 5 8

24 5 5 4 1

15 24 10 3 7 3 11 1 10 197 8 155 2 1 9

26 2 2 2 136 8 73 19 1 5

12 16 7 7 3 6 5 1 338 17 254 22 1 19 1

4 14 4 3 1 1 9 2 7 265 5 134 22 5 5

15 24 12 5 7 1 11 4 7 395 19 252 10 4 6 8

14 14 10 10 4 1 3 383 4 158 52 20 13

15 7

10 20

9 15

85 6

91 5

Decapoda: Diogenidae Decapoda: Pasiphaeidae

5 1

18 -

7 5

2 -

Decapoda: 5 other families Decapoda: megalopa & nisto

1 -

2 -

3 -

22 22

14 14

279

Cnidaria Bryozoa Porifera Other invertebrate phyla (4) Annelida: Oligochaeta Annelida: Polychaeta Polychaeta: Spionidae Polychaeta: Syllidae Polychaeta: Onuphidae Polychaeta: Dorvilleidae Polychaeta: Hesionidae Polychaeta: 15 other families Mollusca (Total) Mollusca: Bivalvia Bivalvia: Tellinidae Bivalvia: 8 other families Mollusca: Scaphopoda Mollusca: Gastropoda Gastropoda: Marginellidae Gastropoda: 8 other families Crustacea (Total) Crustacea: Ostracoda Crustacea: Amphipoda Crustacea: Isopoda Crustacea: Leptostraca Crustacea: Mysidacea Crustacea: Cumacea Crustacea: Tanaidacea Crustacea: Decapoda

Echinodermata (Total) Echinodermata: Ophiuroidea Echinodermata: 2 other classes TOTAL

Total

9 11 1 6 2

44 105 111 64 13

1.4 3.3 3.5 2.0 0.4

45 12 5 12 2 1

26 10 2 4 1 2

419 86 71 71 37 35

13.1 2.7 2.2 2.2 1.2 1.1

9 9 8 1 7 1 1 241 3 112 27 22 6

13 9 8 1 7 1 150 64 20 9 6

7 11 11 3 8 147 2 94 17 4 7

115 123 72 16 56 9 42 13 29 2252 66 1296 191 6 86 60

3.6 3.8 2.2 0.5 1.7 0.3 1.3 0.4 0.9 70.2 2.1 40.4 6.0 0.2 2.7 1.9

116 20

63 8

46 5

15 8

450 94

14.0 2.9

18 2

4 1

5 -

2 2

61 11

1.9 0.3

4 4

5 2

3 -

4 2

22 8

0.7 0.2

19 19

4 3

3 3

5 5

2 2

5 5

79 78

2.5 2.4

<0.1

311

407

347

494

588

329

237

218

3210

In terms of abundance, the benthic macroinvertebrate fauna was again dominated by crustaceans (70%), especially amphipods (40%) and tanaids (14%), with polychaetes (13%, esp. spionids) and molluscs (nearly 4%, esp. tellinid bivalves and marginellid gastropods) making smaller contributions to the overall numbers (Table 6). It is noted that tanaids, and particularly the very small, tubedwellers were far less abundant than in the Update 1 Survey (AMD, 2017). Of the larger decapod 27

MARL Update 2 survey Final Report September 2017

crustaceans, diogenids (hermit crabs) and pasaipheids (shrimps) were the most abundant in Update 2 (Table 6). Interestingly, the empty gastropods that provide homes for hermit crabs were observed to provide protection for some amphipods and tanaids (that also secrete a tube that projects from the aperture) and even for two onuphid polychaetes. For S3 (the current pen site), one small (< 20 mm total length) platycephalid (flathead) and one small (12 mm TL) pleuronectid (flounder) were obtained, which is the first time that such fish have been recorded at the MARL (Table 6). It is also noted that, as for Baseline (AMD, 2016), no buccinid gastropods were recorded at S3 or elsewhere (Appendix 8), which contrasts with the 19 individuals recorded at this site in Update 1 Survey (AMD, 2017) Ostracod abundance was greatest at S3 (19 of 66 over the entire MARL), similar to S1 (17 found, Table 6), but was far less than the 171 ostracods recorded at S3 in Update 1 Survey (AMD, 2017). Observed patterns in the k-dominance plots show that there are relatively diverse communities and low to moderate levels of single taxon dominance in all nine sites (Figure 13). This reflects the contribution of amphipods being greatest at eight of the nine sites (26.8 to 62.4%) followed by either polychaetes at C1 (8.2%), isopods at S1 and C3 (5.4-7.9%) or tanaids at S2, S3, S4, S5 and S6 (18.427.4%). At the ninth site (C2) polychaetes contributed 40.5% followed by amphipods (27.4%) (Figure 13).

Figure 13 - K-dominance curves, using data pooled for each site, of the broad-scale benthic macroinvertebrates, obtained from four replicate grabs at each site during Update 2 Survey at Providence Bay.

Univariate analyses: For Update 2, the numbers of broad-scale taxa ranged between 9 and 16 (similar for Baseline), and the number of family-level taxa of polychaetes, molluscs and decapods ranged between 4 and 14 (again similar to Baseline). The number of individuals per replicate grab ranged between 26 and 200 (this being similar to the 23 to 167 in Baseline) and the overall mean (and SE) abundance was 89.3 (6.4) and thus similar to Baseline (AMD, 2016). ANOVA showed that (1) the number of broad-scale taxa differed only with Survey, (2) the number of families of polychaetes, molluscs and decapods showed a significant interaction between Survey and Site and (3) the total abundances of benthic macroinvertebrates differed with Site and there was an interaction between Survey and Site (Table 7). For the two-way interactions (see below), 28

MARL Update 2 survey Final Report September 2017

post-hoc comparisons to ascertain where the differences resided were restricted to Update 2, as results for Baseline were reported in AMD (2017). Table 7 - Results of two-way ANOVA of Survey and Site of the numbers of taxa (broad-scale and family-level) and the total abundance of benthic macroinvertebrates obtained from four replicate grabs at each site during the Baseline and Update 2 surveys at Providence Bay, and the total and percentage contribution of each to the overall fauna. Source Broad-scale taxa Survey Site Survey x Site Residual Family-level taxa Survey Site Survey x Site Residual Total abundance Survey Site Survey x Site Residual

P (%)

1 8 7 51

14.06 3.84 4.35 2.88

4.90 1.34 1.51

0.031 0.246 0.184

1 8 7 51

17.02 13.14 22.41 7.235

2.35 1.82 3.10

0.131 0.096 0.008

1 8 7 51

1434.52 2887.77 3690.98 964.73

1.49 2.99 3.826

0.228 0.008 0.002

For the broad-scale taxa, the significant effect with Survey reflected the slightly greater mean number of taxa in Update 2 compared to Baseline, with a mean (SE) of 12.1 (2.9) and 11.1 (0.3), respectively (see also Figure 14). Tukeyâ&#x20AC;&#x2122;s test for site within survey did not detect a significant difference for either Update 2 or Baseline. The mean (SE) number of family-level taxa ranged from 6 (1.1) for S2 to 11.25 (1.1) for S3. The mean (SE) total abundance of macroinvertebrates were greatest at S4, 147 (20.9), being significantly greater than sites C3, 54.5 (1.4) and S6, 59.3 (13.1) (see also Figure 14).

MARL Update 2 survey Final Report September 2017

Figure 14 - Means (and SE) of the numbers of broad-scale (a) and family-level taxa (b) and for all benthic macroinvertebrates (c), obtained from four replicate grabs at each site during the Baseline (blue) and Update 2 (orange) surveys at Providence Bay.

The percentage difference in the abundance of all macroinvertebrates in comparison to Baseline were calculated for each of Update 1 and Update 2 surveys, with the values for C3 using Update 2 survey as the â&#x20AC;&#x153;baselineâ&#x20AC;?. This shows that the northern sites showed inconsistent and small differences between surveys, while the percentage increases were particularly high in the southern sites during Update 1 survey (Figure 15).

Figure 15 - Percentage difference for the numbers of all benthic macroinvertebrates obtained from four replicate grabs at each site, with reference to Baseline, for both Update 1 and Update 2 Surveys at Providence Bay.

Multivariate analyses: When the Bray-Curtis similarity matrix, based on log 10-transformed abundances of the broad-scale benthic macroinvertebrates for the nine sites, was subjected to ANOSIM, there was a significant overall difference among those sites (R = 0.66, P = 0.1%). This 30

MARL Update 2 survey Final Report September 2017

magnitude of the R-statistic value is similar to that for Update 1 (R = 0.65) but greater than that of the Baseline (R = 0.39) (AMD, 2016; 2017). For the northern sites (in which the pen was located), all were highly significantly different from each other, i.e. R = 0.73-0.86, P = 2.9%, except for S3 vs both C1 and S2 (R = 0.54, P = 5.7% and R = 0.20, P = 11.4%, respectively), with the former being of marginal significance and the latter not significant. This new lack of difference between one of the northern compliance sites (S2) and the stocked pen site (S3) suggests that there may be an influence from the stocking of fish, while the lack of difference between S3 and C1 is consistent with the possible influence of M. coccopoma, with small weathered fragments being observed for the first time at this northern control site. However, unlike the other northern sites, S2 was also shown now to not differ from two of the more southern sites (S5: R = 0.13, P = 20.0% and S6: R = 0.15, P = 22.9%), perhaps reflecting other influences within the MARL. ANOSIM also showed that there was a significant difference for broad-scale taxa between all control sites, with this difference being least for C2 vs C3 (R = 0.54, P = 2.9%) and far greater for C1 vs C2 and C3 (R = 0.87 and R = 0.91, resp., P = 2.9%). For the southern part of the MARL, there were significant differences between all sites (R = 0.31 â&#x20AC;&#x201C; 0.91, P = 2.9%) except for S6 vs both S5 and C3 (P = 17.1-20.0%). When these analyses were repeated for the family-level taxa, in which the polychaetes, molluscs and decapod crustaceans were identified to family, ANOSIM showed overall significance (P = 0.1%), with the R-statistic value being less than for broad-scale, i.e. 0.50 vs 0.66. This R-statistic value was less than for both Baseline (0.71) and for Update 1 (0.56) (AMD, 2016; 2017). In contrast to the broad-scale taxa, however, both C1 and S2 are now significantly different from S3 at P = 2.9% (C1: R = 0.88 and S1: R = 0.32). Furthermore, S2 vs both C1 (R = 0.48, P = 5.7%) and C2 were not significantly different (R = 0.52, P = 5.7%). The family-level taxa for the northern sites was again shown to differ from those of the southern sites (P=2.9%), with R-statistic values ranging between 0.68 and 0.89, with the exception of C2 vs all southern sites (R = -0.08 to 0.33, P = 5.7 to 38.0%) except for C3 (R = 0.49, P = 8.6%). This probably reflects the coarser grain size at C2 (see earlier), which appears unusual for the northern sites. ANOSIM also showed that there was a significant difference for C1 vs both C2 and C3 at P = 2.9 % (R = 0.72 and 0.69, respectively). The family-level taxa at each of the southern sites was not shown to be significantly different from each other (R = -0.12 to 0.18, P = 5.7 to 68.6%), except for C3 vs both S4 and S6 (R=0.45 and 0.42, resp., P=2.9%). In summary, ANOSIM of the broad-scale data for the pen site (S3) vs the two compliance sites (S1 and S2) showed that significant differences were detected for S1 (R = 0.79, P = 2.9%) but not for S2 (R = 0.20, P = 11.4). For the family-level comparisons, however, those for S1 and S2 both differed from those of S3 (R = 0.98 and 0.34, resp., P = 2.9%). The overall pattern for the site differences, as shown by ANOSIM, in each of the northern and southern part of the MARL is also summarised in Figure 16.

MARL Update 2 survey Final Report September 2017

Figure 16 - A schematic of the relationships between the various sites at (a) broad-scale, i.e. phylum/class/order, and (b) family-level for polychaetes, molluscs and decapods, obtained from four replicate grabs at each site during Update 2 Survey at Providence Bay. All sites were significantly different in each of the northern and southern MARL (separated by a dashed line), except where blue lines denote non-significance.

RELATE showed that the two matrices, i.e. broad scale vs family level, had a significant correlation with each other (Rho = 0.32, P = 0.1%), but this was lower than has been previously recorded, i.e. 0.61 in Update 1 Survey (AMD, 2017) and 0.60 in Baseline Survey (AMD, 2016). nMDS ordination of the broad-scale benthic macroinvertebrates showed that, as before for Baseline and Update 1 surveys, the samples for C1 and C2 were distinct from those of most other sites (Figure 17a). The samples for the southern control (C3) lay beneath C1 and close to those of the remaining southern sites (S4-S6), while those for S1 tended to be located beneath C1 and just above the pen site (S3). Again, S2 lay closer to the southern sites (Figure 17a). Like Baseline (but unlike Update 1 Survey), the samples for S3 showed no obvious tendency to be discrete from the other sampling sites, although they lay on the edge of the cloud of samples (Figure 17a). nMDS ordination of the family-level taxa of polychaete, molluscs and decapods showed a more dispersed pattern to that of the broad-scale taxa (Figure 17a,b), with the samples for the different sites falling in a particular part of the plot but being relatively more interspersed with those of the other sites. This probably reflects the increased sensitivity of the family-level to variations within the sedimentary chemistry at each site and/or the relative sparseness of the representation of those families, i.e. 1-2 individuals. This is exemplified for C2, in which the point for sample C2-2 (a sample which contained no lithic granules) lay at the centre top of the plot, while those for the other three samples lay close together at the bottom right of the plot. SIMPER and the raw data (Appendix 8) showed that C2-2 contained no syllid, hesionid or onuphid polychaetes (unlike the other three replicate grabs) and one individual each of a leucosiid and majid crab (while crabs were found in no other replicates). The samples for S3 lay again at the edge of the cloud of the other samples and lay relatively close together (Figure 17b).

MARL Update 2 survey Final Report September 2017

Figure 17 - nMDS ordinations using log10 transformed data on the numbers of the various benthic macroinvertebrate at both a (a) broad-scale, i.e. phylum/class/order, and (b) family-level for polychaetes, molluscs and decapods, obtained from four replicate grabs at each site during Update 2 Survey at Providence Bay. 2 denotes replicate grab C2-2.

For comparisons between the benthic macroinvertebrate composition at Baseline, Update 1 and Update 2 surveys, a centroid nMDS plot (i.e. depicts each Site/Survey as a single point, collapsed from the four replicate grabs in that survey), was constructed for both broad-scale and family-level taxa (Figure 17a,b) and interpreted via two-way ANOSIM, focussing on any differences between surveys but taking into account any site differences. From the broad-scale taxa, two-way ANOSIM showed that there were significant differences overall with Survey (R = 0.59, P = 0.1%) and between each survey at P = 0.1%, with the R-statistic value least between Baseline and Update 2 survey (0.44) and higher for Update 1 vs both Baseline and Update 2 (R = 0.70 and 0.63, resp.). This is supported by the points for Update 1 Survey lying to the right/beneath of most other samples from Baseline and Update 2 surveys (Figure 18a). For S3 (the current pen site), the points for both Baseline and Update 2 are close together in the cloud of samples, while that for Update 1 lies well to the right (Figure 18a). Interestingly, the R-statistic value for Site was also high (R = 0.57, P = 0.1%), and all sites were significantly different from each other, except for S5 and S6 (R = 0.15, P = 7.6%).

MARL Update 2 survey Final Report September 2017

Figure 18 - nMDS ordinations, based on centroids of the Bray-Curtis similarities calculated from log10 transformed data on the numbers of the various benthic macroinvertebrate at both a (a) broad-scale, i.e. phylum/class/order, and (b) family-level for polychaetes, molluscs and decapods, obtained from four replicate grabs at each site during Baseline, Update 1 and Update 2 surveys at Providence Bay.

For the family-level taxa, ANOSIM also showed that there were significant differences overall with Survey (R = 0.58, P = 0.1%) and between each survey at P=0.1%, with the R-statistic value being similar for all comparisons, i.e. ranging between 0.51 and 0.66. This is supported by the points for the surveys tending to be distributed in different parts of the plot, i.e. Baseline at the uppermost part, Update 1 in the centre and Update 2 towards the bottom right part of the plot (Figure 18b). There is now more separation between the points for S3 (current pen site) for three surveys than for broad-scale taxa, with the point for Baseline lying to the right and above that of Update 1, while that of Update 2 lies at the lowermost part of the plot (Figure 18b). Concluding Remarks Based on the above faunal patterns for Update 2 Survey, it is evident that S3, the pen site, does not show an obvious difference when compared to the northern control and compliance sites, when taking into account both broad-scale and family-level comparisons. This is similar to that in Baseline, i.e. before the fish were introduced into the pens (AMD, 2016), but contrasts with that found in Update 1, carried out in April of 2017 (AMD, 2017). In this latter survey, the benthic macroinvertebrate composition at S3 was showing differences, which reflected increased numbers overall and of particular taxa such as ostracods and gastropods (esp. buccinids) at the pen site. At the time, this difference was considered likely to reflect the increased organic input (fish food and 34

MARL Update 2 survey Final Report September 2017

faeces) from farm operations. However, given now that the pens have been stocked for a further five months, with these fish having being fed continually since that time, and presumably with more faecal and feed deposition, it is likely that some other factor is responsible for the present lack of obvious difference. It is also possible that the conclusion re Update 1 Survey in AMD (2017) may have been confounded with the Baseline and Update 1 surveys having occurred at contrasting times of year, i.e. spring vs autumn. It is worth noting that, when comparing the macroinvertebrate composition at the family level for the three surveys together, the centroid for S3 in Update 2 Survey does not fall within the large group of samples from other surveys and sites, implying that there is at least some level of change at this site in the Update 2 Survey. From the results of Update 2 Survey, it is now considered likely that either the heavy wave action or recent net cleaning operations, which acted to remove fouling communities from the stocked sea pen to the seabed below and thus contributed to the difference between S3 and other sites in Update Survey 1, has not occurred in the present survey. Recall in Update 1 that large fragments of M. coccopoma colonies were observed, with one individual at least still being alive (AMD, 2017 and video footage from that survey) and anoxic sand, presumably from decomposition of those fragments, was also observed. In this Update 2 Survey, no anoxic sand and only small remnants of M. coccopoma colonies were obtained at S3 (also supported by results of ROV in the present study), and there were very small and weathered remnants at 2 of the sites at nearby C1. This conclusion that wave action/net cleaning resulting in fouling community deposition to the seafloor and thus influences the environment is consistent with work elsewhere, see e.g. Edgar et al. (2010). It is recommended that information around the extent of wave action/processes and timing of net cleaning operations be obtained and used to investigate further these apparent effects on the seabed. In the present survey, M. coccopoma fragments were detected for the first time in C1, which lies close to S3, and which indicates that dynamic processes are redistributing these fragments away from the stocked sea pen. These remnants were small and sparsely distributed, and the faunal composition of C1 did not show any notable differences. However, the dynamic processes that are present in the study area, which clearly involve sediment movement around the broader area, could also imply that effects from the stocked sea pens may be relatively diffuse and thus possibly underestimated using a straightforward sea pen vs compliance vs control site comparison. As the broad-scale and family-level comparisons â&#x20AC;&#x153;toldâ&#x20AC;? different stories with respect to the faunal patterns in S3 and C2, it is recommended that the family-level identification of polychaetes, molluscs and decapods continue in the future. This concurs with the recommendation in Update 1 Survey, and which would enable the quantification of any future increases in sensitive polychaete taxa, such as capitellids and dorvilleids or any concomitant declines in bivalves or ophiuroids (Macleod & Forbes, 2004; Edgar et al., 2010). It is noted that the numbers of dorvilleids was the highest overall since the MARL commenced (i.e. 37) while the number of capitellids was still low (4) and the number of bivalves and ophiuroids (72 and 78, respectively) was less than in Baseline (100 and 130, which did not sample C3, AMD (2016)). The lower than expected correlation between the broad-scale and family-level matrices also points towards these taxonomic approaches being complementary, rather than directly corresponding to each other. Caprellid amphipods were found in low numbers, i.e. 1 or 2, at most sites in the present survey, where these amphipods were seldom recorded previously (AMD, 2016; 2017). As these crustaceans are often found in algal assemblages (Jones & Morgan, 1994), most probably feeding on the associated detritus (Guerra-Garcia, 2009), this is considered likely to reflect the recorded extent of drift algae throughout the MARL (this report). 35

MARL Update 2 survey Final Report September 2017

Three small teleost individuals were obtained using the grab sampler, including an early juvenile of both a flounder and flathead at the pen site (S3). Further fish data is also being collected through the ROV surveys and a separate study being undertaken by the University of Newcastle using Baited Remote Underwater Video Stations (BRUVS). The choice of control sites continues to be enhanced by the addition of the third control site (C3) in 2017, as it is similar in taxonomic composition to the other southern sites and C1 and C2 are not considered “ideal” controls within the MARL as both contain relatively finer sediments. It was recommended in the Update 1 Survey that both sites be retained for sampling in the future, owing to their finer sediments and a relatively short picking time. However, in Update 2 Survey, the relatively dynamic environment of the MARL has meant that the finer sediment (typically present) at C2 was “swamped” by deposition of large lithic granules which were not removed by sieving through 1 mm mesh, meaning extremely long picking times for those samples. It is therefore recommended that sampling be continued for both C1 and C2 but, if this extreme dominance by lithic granules occurs again, considerations be made as to comparisons between sites with such material, given the obvious influence on the community composition and the very long picking times. More efficient picking methods should also be investigated. The number of benthic macroinvertebrates at the nine sites of Update 2 Survey (3213) were similar to that recorded in Baseline, which did not include sampling for C3 (2704), and was far less than the 7631 recorded five months previously in Update 1 Survey (AMD, 2017). This is most probably related to the seasonal increases in tanaids and other crustaceans during autumn 2017 (Update 1 Survey) in comparison to spring in 2016 and 2017 (Baseline and Update 2 Survey). These marked changes in overall numbers of invertebrates and their community compositions between time of year also raises the question as to the best timing for future comparisons of possible effects of the MARL. It is recommended that sampling be again undertaken in the autumn of 2018, as well as that which is already planned for spring in 2018. Comparisons of the community composition of the benthic macroinvertebrates from the Update 2 survey with those of both Baseline and Update 1 surveys at both a broad and family scale, found overall significant differences between the three surveys. These differences were most marked between Update 2 vs the other two surveys, but also emphasised the substantial variation between sites in each of the survey periods.

MARL Update 2 survey Final Report September 2017

References

ANZECC & ARMCANZ (2000). Australian and New Zealand Guidelines for Fresh and Marine Water Quality, Australian and New Zealand Environment and Conservation Council & Agriculture and Resource Management Council of Australia and New Zealand, Canberra Aquaculture, Management & Development Pty Ltd (2016). Marine Aquaculture Research Lease – Providence Bay Baseline Survey Environmental Assessment. Report to Huon Aquaculture Company Pty Ltd. Aquaculture, Management & Development Pty Ltd (2017). Marine Aquaculture Research Lease – Providence Bay Update 2 Survey Environmental Assessment. Report to Huon Aquaculture Company Pty Ltd. Anderson, M.J., Gorley, R.N. & Clarke, K.R. (2008). PERMANOVA+ for PRIMER: Guide to Software and Statistical Methods. Plymouth, UK: PRIMER-E. Plymouth Marine Laboratory. Beasley, P.L., Ross, G.J.B. & Wells, A. (eds) (1998). Mollusca: The Southern Synthesis. Fauna of Australia. Vol. 5. CSIRO Publishing, Melbourne. Part A xvi 563 pp. Part B Viii 565-1234 pp. Clarke, K. R., Gorley, R. N., Somerfield, P. J. & Warwick, R. M. (2014). Change in Marine Communities: An Approach to Statistical Analysis and Interpretation. PRIMER-E, Plymouth, 260 pp. Dela Cruz J, Ajani P, Lee R, Pritchard T & Suthers I (2002). Temporal abundance patterns of the red tide dinoflagellate Noctiluca scintillans along the southeast coast of Australia. Marine Ecology Progress Series 236, 75-88. Edgar, G.J., Davey, A. & Shepherd, C. (2010). Application of biotic and abiotic indicators for detecting benthic impacts of marine salmonid farming among coast regions of Tasmania. Aquaculture 307, 212-218. Fauchald, K. (1977). The Polychaete Worms: Definitions and Keys to the Orders, Families and Genera. Natural History Museum of Los Angeles County & The Allen Hancock Foundation, University of Southern California, 188 pp. Guerra-Garcia, J.M. (2009). What do caprellids (Crustacea, Amphipoda) feed on? Marine Biology, 156, 1881-1890. Grove, S. & de Little, R. (2017). A guide to the seashells and other marine molluscs of Tasmania. http://www.molluscsoftasmania.net/ Jansen, J. (2000). Seashells of South-East Australia. Capricornica Publications, Lindfield, 18 pp. Macleod, C.K. & Forbes, S. (2004). Guide to the Assessment of Sediment Condition at Marine Finfish Farms in Tasmania. Tasmanian Aquaculture and Fisheries Institute – University of Tasmania, Hobart, Australia, 65 pp. Poore, G.C.B. (2004). Marine Decapod Crustacea of Southern Australia. A Guide to Identification. Museum of Victoria & CSIRO Publishing, Collingwood, 574 pp. Underwood, A.J. & Hoskin, M.G. (1999). Ecological Sampling for Assessment of Potential Environmental Impacts of the Trial Snapper Farm at Port Stephens, NSW (Pisces Marine Aquaculture Pty. Ltd.). Draft Report, February 1999. Centre for Research on Ecological Impacts of Coastal Cities, 15 pp.

MARL Update 2 survey Final Report September 2017

Wilson, R.S., Hutchings, P.A. & Glasby, C.J. (2017). Polychaetes: An interactive Identification Guide. Retrieved from http://researchdata.museum.vic.gov.au/polychaetes

MARL Update 2 survey Final Report September 2017

Appendices

Appendix 1 Survey coordinates for sediment (non ROV) sampling, based on the Mapping Grid of Australia Zone 56 (Datum GDA94)

SITE c1.1 c2.1 s1.1 s2.1 s3.1 s4.1 s5.1 s6.1 c3.1

MGA56_Easting MGA56_Northing 433769 433294 432791 432425 432573 432358 432519 432159 430275

6388153 6388514 6387474 6387434 6387294 6386760 6386589 6386559 6384853

MARL Update 2 survey Final Report September 2017

Appendix 2 Water Column dissolved oxygen and temperature Sonde results as averages for surface, seafloor and 5m depth increments in between

Dissolved Oxygen (% Saturation) Depth 1 5 10 15 20 25 30 35 Seafloor Temperature (degrees C) Depth 1 5 10 15 20 25 30 35 Seafloor

SITE C1 107 106 106 105 105 104 99

C2 106 107 106 105 105 104 98

C3 106 105 104 103 102 100 99

S1 107 106 106 105 104 91 84

S2 107 106 106 105 99 97 92

S3 104 106 106 106 105 103 99

S4 107 107 106 101 95 92 85

S5 107 106 106 104 101 98 89 87 84

SITE C1 19.9 19.4 19.4 19.3 19.3 19.3 18.8

C2 19.9 19.5 19.5 19.4 19.3 19.2 18.7

C3 19.5 19.4 19.2 19.1 19.1 19 18.9

S1 19.7 19.5 19.4 19.3 19.1 17.9 17.3

S2 19.7 19.4 19.4 19.2 18.7 18.4 17.8

S3 19.5 19.4 19.3 19.3 19.3 19.2 18.8

S4 19.6 19.5 19.2 18.7 18.3 18 17.4

18.5

17.9

18.8

17.2

17.6

17.9

17.2

S5 19.5 19.5 19.3 19.2 18.8 18.6 17.8 17.7 17.4

MARL Update 2 survey Final Report September 2017

Appendix 3 Water Column results for all sites â&#x20AC;&#x201C; Nutrients

SITE UNIT LOR C1 Surface C2 Surface S1 Surface S2 Surface S3 Surface S4 Surface S5 Surface C3 Surface C1 Seafloor C2 Seafloor S1 Seafloor S2 Seafloor S3 Seafloor S4 Seafloor S5 Seafloor C3 Seafloor Overall Mean Overall Standard Deviation Surface Mean Surface Standard Deviation Seafloor Mean Seafloor Standard Deviation

Ammonia

NOx

Kjeldahl Nitrogen

Total Nitrogen

Total Phosphorus

Reactive Phosphorus

mg-N/L 0.005

mg-N/L 0.002

mg-N/L 0.1

mg-P/L 0.01

mg-P/L 0.003

0.009 0.008 0.006 0.008 0.024 0.007 0.01 0.008 0.013 0.008 0.012 0.005 0.006 0.004 0.005 0.009 0.009 0.005 0.010 0.006 0.008 0.003

0.002 0.002 0.001 0.001 0.002 0.001 0.002 0.003 0.045 0.003 0.081 0.077 0.05 0.082 0.063 0.027 0.028 0.033 0.002 0.001 0.054 0.028

0.3 0.31 0.33 0.31 0.3 0.32 0.3 0.29 0.37 0.3 0.32 0.29 0.29 0.33 0.29 0.3 0.309 0.021 0.308 0.013 0.311 0.028

0.3 0.31 0.33 0.31 0.31 0.32 0.3 0.29 0.41 0.3 0.4 0.36 0.35 0.41 0.36 0.32 0.336 0.041 0.309 0.012 0.364 0.041

0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.03 0.04 0.04 0.05 0.05 0.04 0.05 0.05 0.04 0.042 0.005 0.039 0.004 0.045 0.005

0.006 0.005 0.005 0.006 0.006 0.005 0.008 0.005 0.011 0.005 0.017 0.014 0.011 0.015 0.012 0.009 0.009 0.004 0.006 0.001 0.012 0.004

NB: figures in red indicate where the lab result was actually below the LoR (Limit or Reporting) and the figure substituted is the actual reading provided by the lab below that limit and rounded to the third decimal place.

MARL Update 2 survey Final Report September 2017 Appendix 4 Redox potential, measured in millivolts from 3cm depth in the sediment grabs or cores

Core or Grab No S1.1 S1.2 S1.3 S2.1 S2.2 S2.3 S3.1 S3.2 S3.3 S4.1 S4.2 S4.3 S5.1 S5.2 S5.3 S6.1 S6.2 S6.3 C1.1 C1.2 C1.3 C2.1 C2.2 C2.3 C3.1 C3.2 C3.3

Redox at 3cm mV 242 221 263 272 279 280 249 214 261 269 269 269 271 274 267 269 265 273 250 257 245 270 259 275 231 279 272

MARL Update 2 survey Final Report September 2017 Appendix 5 Sulphide analysis, measured in sediments at 3cm from sediment surface

Core or Grab Sulphide 3cm No (uM) S1.1 0.00 S1.2 0.00 S1.3 0.00 S2.1 0.00 S2.2 0.00 S2.3 0.00 S3.1 0.00 S3.2 0.00 S3.3 0.00 S4.1 0.00 S4.2 0.00 S4.3 0.00 S5.1 0.00 S5.2 0.00 S5.3 0.00 S6.1 0.00 S6.2 0.00 S6.3 0.00 C1.1 0.00 C1.2 0.00 C1.3 0.00 C2.1 0.00 C2.2 0.00 C2.3 0.00 C3.1 0.00 C3.2 0.00 C3.3 0.00

MARL Update 2 survey Final Report September 2017 Appendix 6 Organic content â&#x20AC;&#x201C; raw data

Sample No C1.1 C1.2 C1.3 C2.1 C2.2 C2.3 S1.1 S1.2 S1.3 S2.1 S2.2 S2.3 S3.1 S3.2 S3.3 S4.1 S4.2 S4.3 S5.1 S5.2 S5.3 S6.1 S6.2 S6.3 C3.1 C3.2 C3.3

LOI % 0.15 0.16 0.12 0.18 0.07 0.1 0.13 0.11 0.12 0.07 0.12 0.06 0.07 0.06 0.07 0.04 0.1 0.09 0.06 0.07 0.1 0.1 0.06 0.06 0.04 0.04 0.06

MARL Update 2 survey Final Report September 2017 Appendix 7 AST Labs nutrients analyses methodology Ammonia, in-house method 1205 Analysis was performed using a Lachat Flow Injection analyser. This method is based on APHA Standard methods (2005) 4500NH3 H. The ammonia determination is based on the Berthelot reaction. Ammonia reacts in alkaline solution with hypochlorite to form monochloramine at a pH between 8 and 11.5 which, in the presence of phenol, catalytic amounts of nitroprusside (nitroferricyanide) and excess hypochlorite, forms indophenol blue absorbing at 630 nm. Results are reported as mg/L as N, with a minimum reporting limit of 0.005 mg/L.

Nitrate and Nitrite, in-house method 1205 Analysis was performed using a Lachat Flow Injection analyser. This method is based on APHA Standard methods (2005) 4500NO3- I. The nitrate and nitrite determination involves the nitrate being quantitatively reduced to nitrite by passage of the sample throµgh a copperised cadmium column at a pH of 8. The NOx (reduced nitrate plus original nitrite), is then determined by diazotization with sulphanilamide under acidic conditions to form a diazonium ion which is coupled with N-(1-Napthyl) Ethylenediamine Dihydrochloride, (NEDD). The resulting pink dye absorbs at 520 nm. Results are reported as mg/L as N, with a minimum reporting limit of 0.002 mg/L. Dissolved Reactive Phosphorus, in-house method 1205 Analysis was performed using a Lachat Flow Injection analyser. This method is based on APHA Standard methods (2005) 4500-P G. The phosphate determination involves the orthophosphate ion (PO43-) reacting with ammonium molybdate and antimony potassium tartrate under acidic conditions to form a complex. This complex is reduced with ascorbic acid to form a blue complex which absorbs light at 880 nm. Results are reported as mg/L as P, with a minimum reporting limit of 0.003 mg/L.

Total Nitrogen in water Analysis was performed using a Lachat Flow Injection analyser. This method is based on APHA Standard methods 4500-Norg D. The Total Kjeldahl Nitrogen (TKN) present in the sample is determined by converting the nitrogen to ammonium sulphate in a sulphuric acid potassium digestion procedure. Ammonium–nitrogen is subsequently determined by colorimetric auto-analyser method. TKN represents the ammoniacal and organic nitrogen present in the sample. Total Nitrogen is calculated by adding oxides of nitrogen (determined from the nitrate and nitrite analysis), to the TKN value. Results are reported as mg/L as N, with a minimum reporting limit of 0.1 mg/L. Total Phosphorus in water Phosphorus compounds are converted in orthophosphate in the Kjeldahl digest, the resulting solution is analysed by Flow Injection analysis. This method was developed by Lachat Instruments and is a combination of “Quikchem” Method 10 - 115 - 01 - 1 - C and Method 10 - 107 - 06 - 2 – E. The chemistry used is based on APHA Standard methods 4500-P G. Results are reported as mg/L as P, with a minimum reporting limit of 0.01 mg/L.

MARL Update 2 survey Final Report September 2017

Appendix 8 Raw data for benthic macroinvertebrates, obtained from four replicate grabs at each site during the Update 2 Survey at Providence Bay. Note: Green text represents taxa that were recorded for the first time, while the shaded grey represents no animals recorded for that taxa in Update 2, but which were previously recorded in either Baseline or Update 1 Survey. INVERTEBRATES Cnidaria: Anthozoa: Actinaria Cnidaria: Scleractinia Bryozoa Porifera Nemertea Sipuncula Phoronida Chaetognatha Annelida: Oligochaeta Annelida: Polychaeta TOTAL Polychaeta: Orbiniidae Polychaeta: Spionidae Polychaeta: cf Heterospionidae Polychaeta: Magelonidae Polychaeta: Trochochaetidae Polychaeta: Cirratulidae Polychaeta: Arenicolidae Polychaeta: Capitellidae Polychaeta: Maldanidae Polychaeta: Opheliidae Polychaeta: Phyllodocidae Polychaeta: Polynoidae Polychaeta: Sigalionidae Polychaeta: Hesionidae Polychaeta: Syllidae Polychaeta: Pisionidae Polychaeta: Nereididae Polychaeta: Glyceridae Polychaeta: Nephtyidae Polychaeta: Onuphidae Polychaeta: Lumbrineridae Polychaeta: Eunicidae Polychaeta: Dorvilleidae Polychaeta: Sabellidae Polychaeta: Chaetopteridae Polychaeta: Oweniidae Polychaeta: Sabellongidae Polychaeta: Sternapsidae Polychaeta: Flabelligeridae

C1-1 C1-2 C1-3 C1-4 C2-1 C2-2 C2-3 C2-4 S1-1 S1-2 S1-3 S1-4 S2-1 S2-2 S2-3 S2-4 S3-1 S3-2 S3-3 S3-4 S4-1 S4-2 S4-3 S4-4 S5-1 S5-2 S5-3 S5-4 S6-1 S6-2 S6-3 S6-4 C3-1 C3-2 C3-3 C3-4 2 1 1 -

1 1 -

4 2 1 1 -

1 2 -

3 1 2 -

2 3 -

7 5 1 1 -

4 7 1 -

9 3 2 1 3 -

1 1

57 10 1 8 10 1 10 3 14 -

2 2 2

11 6 1 2 1 1 -

4 1 3 2

33 1 4 11 6 5 3 -

3 5 3

25 3 4 4 1 7 3 -

2 -

4 1 2 -

1 2 -

4 1 1 -

1 3 -

7 1 5 1 -

3 -

2 1 -

6 3 2 2 -

4 2 1 1 -

6 2 1 2 1 - -

2 11 -

4 4 1 1 -

6 4 2 -

1 1 -

9 7 1 1 -

2 1 -

12 6 1 2 3 -

2 -

10 4 1 3 1 1 -

1 4 2 1 -

15 2 1 1 3 4 2 2 -

6 40 2 -

19 3 1 2 3 1 3 4 2 -

4 12 2 3 4 2

31 1 1 5 15 2 1 1 3 2 -

13 4 1 -

16 4 1 2 1 7 1 -

15 1

21 3 1 1 2 6 4 -

4 12 2 -

9 5 2 -

1 2 1 5 -

5 1 1 1 1 1 -

2 9 1 -

7 2 3 1 1 -

3 5 3 -

11 2 1 1 3 2 -

1 1 -

3 6 1 1 -

7 3 1 2 1 -

3 2 2 -

12 4 2 4 -

2 9 -

15 3 1 1 2 2 2 -

1 1 1 -

11 2 1 1 4 1 -

5 1 2

9 5 1 2 1 -

1 2 3 -

8 2 3 1 1 1 -

2 8 1 1 -

3 1 1 -

6 1 2 2 1 -

MARL Update 2 survey Final Report September 2017

Polychaeta: Ampharetiidae Polychaeta: Pectinariidae Polychaeta: Sabellariidae Polychaeta: Terebellidae sensu lato Polychaeta: Terebellidae Polychaeta: Telothelepidinae Polychaeta: Trichobranchidae Polychaeta: Polycirridae Polychaeta: Unidentified ter. sl. Polychaeta: Unidentified errant Mollusca TOTAL Mollusca: Bivalvia TOTAL Bivalvia: Nuculanidae Bivalvia: Mactridae Bivalvia: Mesodesmatidae Bivalvia: Myochamidae Bivalvia: Tellinidae Bivalvia: Veneridae Bivalvia: Galeommatidae Bivalvia: Cardiidae Bivalvia: Carditidae Bivalvia: Corbulidae Bivalvia: Psammobiidae Bivalvia: Mytilidae Bivalvia: Hiatellidae Bivalvia: Pectinidae Bivalvia: Pholalidae Bivalvia: Unidentified Mollusca: Scaphopoda TOTAL Scaphopoda: Gadiludae Scaphopoda: Dentaliidae Mollusca: Gastropoda TOTAL Gastropoda: Trochidae Gastropoda: Turbinidae Gastropoda: Acteonidae Gastropoda: Architectonidae Gastropoda: Terebridae Gastropoda: Turritellidae Gastropoda: Scaphandridae Gastropoda: Pyramidellidae Gastropoda: Rissoellidae Gastropoda: Naticidae Gastropoda: Muricidae Gastropoda: Buccinidae Gastropoda: Turridae Gastropoda: Fascolariidae Gastropoda: Marginellidae

7 3 1 1 1 1 1 3 2 1 -

4 1 1 1 1 2 1 -

6 3 1 2 1 1 2 1 1

7 3 3 4 3 -

1 1 1 -

2 1 2 2 1 2 1 3 1 7 1 1 5 1 1 1 3 1 1 1 1 - 1 1 1 - 1 1 1

1 1 5 1 1 4 4

1 1 1 -

4 1 1 3 1 1 1

1 1 -

8 2 2 1 1 5 3 1 1

6 1 1 5 2 2

7 2 1 1 1 1 4 1 1 1

3 3 3 -

8 6 2 1 2 1 2 1

2 2 1 1 -

9 7 2 1 3 1 2 1 -

4 4 2 2 1 1

2 2 1 1 1 -

2 2 1 1 -

1 1 1 -

2 2 4 3 1 1 1 1 1 -

2 2 1 1 -

2 2 4 4 2 2 -

4 4 2 1 1 1 1 -

2 2 3 3 1 1 1 -

- 4 2 4 2 1 1 1 1 1 1 -

1 1 2 2 1 1 -

3 3 1 1 1 -

MARL Update 2 survey Final Report September 2017

Gastropoda: Columbellidae Gastropoda: Olivellidae Gastropoda: Olividae Gastropoda: Pseudolividae Gastropoda: Mitridae Gastropoda: Aplysiidae Gastropoda: Unidentified Crustacea TOTAL Crustacea: Ostracoda Crustacea: Amphipoda TOTAL Crustacea: Amphipoda: Gammaridea Crustacea: Amphipoda: Caprellidea Crustacea: Isopoda TOTAL Crustacea: Isopoda: Anthuridea Crustacea: Isopoda: Cymothoida: Gnathidae Crustacea: Isopoda: Valvifera Crustacea: Isopoda: Flabellifera: Sphaeromatidae Crustacea: Isopoda: Flabellifera- Serolidae Crustacea: Leptostraca Crustacea: Mysidacea Crustacea: Cumacea Crustacea: Tanaidacea Crustacea: Decapoda TOTAL Decapoda: Leucosiidae Decapoda: Majiidae Decapoda: Calappidae Decapoda: Hexapodidae Decapoda: Pasiphaeidae Decapoda: Callianassidae Decapoda: Crangonidae Decapoda: ?Pandalidae Decapoda: ?Penaidae Decapoda: Palinuridae Decapoda: Diogenidae Decapoda: Crab Megalopa Decapoda: Scyllarid Nisto Crustacea: Stomatopoda Arthropoda: Pycnogonida Echinodermata TOTAL Echinodermata: Asteroidea Echinodermata: Clypeasteroida Echinodermata: Echinoidea Echinodermata: Ophiuroidea Chordata: Teleostei Fish remains (otoliths, bones) M. coccopoma remains

17 3 12 12 1 1 1 4 4 x

1 50 41 1 42 37 42 37 1 1 4 2 3 1 1 2 1 4 8 4 8 x -

1 89 4 64 63 1 1 1 1 2 15 2 2 6 6 x x

37 33 3 29 18 29 18 3 9 2 9 1 1 1 2 4 1 1 2 2 - 3 10 3 10 1 x -

49 5 22 21 1 3 2 1 2 6 11 11 -

17 4 4 4 3 1 2 4 3 3 1 1 -

1 64 2 50 50 3 2 1 3 1 4 1 1 3 3 -

73 6 50 48 2 8 8 1 4 1 3 1 1 1 5 5 -

82 2 68 68 3 3 3 3 3 2 1 6 6 -

119 7 86 86 8 8 9 1 8 3 5 5 5 x -

21 1 6 6 2 1 1 1 8 3 1 1 -

75 1 39 38 1 4 1 1 1 1 1 2 28 1 1 -

1 113 54 54 14 1 6 7 4 1 34 6 2 4 1 1 -

56 3 35 34 1 2 2 1 15 1 1 -

1 85 1 55 55 4 1 1 2 1 4 18 2 1 1 0 x x

1 80 2 42 42 1 1 2 2 31 2 2 x

## 3 88 88 2 1 1 2 2 27 3 1 2 1 x

1 ## 13 67 67 3 2 1 1 4 15 1 1 1 x x

115 1 41 41 13 2 2 7 2 7 6 34 13 13 2 2 -

1 90 2 24 24 19 1 2 16 7 2 34 2 1 1 2 2 -

## 50 49 1 15 1 14 5 2 32 1 1 1 1 -

73 1 43 43 5 1 1 2 1 1 3 16 4 4 -

48 20 20 10 10 7 3 8 1 1 -

60 1 34 34 4 2 2 5 1 14 1 1 1 1 -

67 1 32 32 7 2 3 2 3 1 20 3 1 2 1 1 -

66 1 26 26 6 3 3 7 1 21 4 2 2 2 2 -

16 31 3 18 3 17 1 2 2 1 1 2 1 2 1 7 8 1 2 1 2 - 1 1 -

65 27 27 9 1 4 4 6 2 19 2 2 1 1 -

38 16 16 7 3 1 3 2 1 12 -

35 2 25 25 3 3 2 1 1 1 1 1 1 -

33 20 20 3 1 2 1 1 6 2 1 1 2 2 -

44 28 28 7 2 5 4 4 1 1 -

35 21 20 1 4 1 3 1 1 4 4 1 1 1 1 2 2 -