Marl update 1 survey apr 2017 final

MARINE AQUACULTURE RESEARCH LEASE PROVIDENCE BAY, NSW UPDATE 1 SURVEY Water column, sediment chemistry and biological characteristics

FINAL REPORT (corrected) April 2017

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

MARL Update 1 survey Final Report April 2017

Document Distribution Date

Name

Company

01/07/17

David Whyte

Huon Aquaculture

1/09/17

David Whyte

1/09/17

Document

Version

Copies

Draft

V1.1

1

Huon Aquaculture

Draft

V3

1

Ian Lyall

Fisheries NSW

Draft

V3

1

1/09/17

Troy Gaston

University of Newcastle

Draft

V3

1

1/09/17

Margaret Platell

University of Newcastle

Draft

V3

1

1/09/17

Wayne O’Connor

Fisheries NSW

Draft

V3

1

1/09/17

Graeme Bowley

Fisheries NSW

Draft

V3

1

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. 13th September 2017

MARL Update 1 survey Final Report April 2017

Contents 1

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

2

Operational Summary ............................................................................................................. 4

3

Location and Survey Map ........................................................................................................ 5

4

Water Column Characteristics ................................................................................................. 6

5

Visual Surveys ....................................................................................................................... 13 5.1 ROV Filming summary ..................................................................................................... 13 5.2 Observations from ROV filming ....................................................................................... 13 5.3 Sediment Cores - Photographs ........................................................................................ 19

6

Sediment Chemistry .............................................................................................................. 21 6.1 Redox Potential ............................................................................................................... 21 6.2 Sulphide Analysis ............................................................................................................ 22 6.3 Particle Size Analysis ....................................................................................................... 23 6.4 Organic Content .............................................................................................................. 24

7

Biological Analysis ................................................................................................................. 26

8

References ............................................................................................................................ 35

List of Figures Figure 1 - Location and Survey Maps – MARL Providence Bay ................................................................................................5 Figure 2 - Oxygen and Temperature profiles for all sites ..........................................................................................................7 Figure 3 - Graphs of nutrients results for all sites split into seafloor and surface readings ....................................................8 Figure 4 - Comparison of various nutrient results for the present survey against Huon internal monthly monitoring surveys ...............................................................................................................................................................................9 Figure 5 - Monthly Chlorophyll a levels at sites C1, C3, S1 and S2 .........................................................................................11 Figure 6 - Screenshots of ROV footage from all sites. .............................................................................................................15 Figure 7 - Sediment chemistry cores for all survey sites .........................................................................................................19 Figure 8 - Redox potential at 30 mm depth in sediment cores .............................................................................................21 Figure 9 - Sulphide concentrations in sediment core samples ...............................................................................................22 Figure 10 - Particle size analyses of the top 100 mm of sediment. Mean percentage cumulative volume for size fractions at each site. ......................................................................................................................................................23 Figure 11 - Total Organic Content for all sites (includes standard error) ...............................................................................24 Figure 12 - Total Organic Content comparison between Baseline survey (orange bars) and Update 1 survey (blue bars) 25 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 1 survey at Providence Bay. ....................................29 Figure 14 - Means (and SE) of the numbers of broad-scale (a,d) and family-level taxa (b,e) and for all benthic macroinvertebrates (c,f), obtained from four replicate grabs at each site during the Baseline and Update 1 surveys at Providence Bay. ...........................................................................................................................................................31 Figure 15 - nMDS ordinations using data on the benthic macroinvertebrates 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 1 survey at Providence Bay. ...................................................................................................................33

MARL Update 1 survey Final Report April 2017

List of Tables Table 1 - Chlorophyll a levels for all sites .................................................................................................................................10 Table 2 - Nutrient and chlorophyll a levels from all sites compared against ANZECC Trigger levels (2000) ........................12 Table 3 - ROV List of spot dives undertaken ...........................................................................................................................13 Table 4 - Pen Bays direction of dives across seafloor ..............................................................................................................14 Table 5 - Description of each ROV dive performed at the MARL site, Providence Bay. ......................................................16 Table 6 - Mean sediment grain size and %mud at all sites ........................................................Error! Bookmark not defined.

Table 7 - of the benthic macroinvertebrates for each broad-scale taxa and important families for selected groups, recorded at nine sites during Update 1 survey at Providence Bay, and the total and percentage contribution of each to the overall fauna ................................................................................................................................................28 Table 8 - Results of one-way ANOVA of 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 Update 1 survey at Providence Bay. ...........................................................................................................................................................30 Table 9 - Results of one-way ANOVA of Site of the numbers of taxa (broad-scale and family-level for polychaetes, molluscs and decapods) and the total abundance of benthic macroinvertebrates obtained from four replicate grabs at each site during the Baseline survey at Providence Bay. ................................................................................31

List of Appendices Appendix 1 Survey coordinates for sediment sampling, based on the Mapping Grid of Australia Zone 56 (Datum GDA94) ..........................................................................................................................................................................................37 Appendix 2 Water Column results for all sites - Probe Data ..................................................................................................38 Appendix 3 Water Column results for all sites – Nutrients ....................................................................................................39 Appendix 4 Redox potential, measured in millivolts from 3cm depth in the sediment grabs or cores...............................40 Appendix 5 Sulphide analysis, measured in sediments at 3 cm from sediment surface ......................................................41 Appendix 6 Organic content – raw data..................................................................................................................................42 Appendix 7 AST Labs nutrients analyses methodology.……………………………………………………………….………………………………..43 Appendix 8 Raw data for benthic macroinvertebrates, obtained from four replicate grabs at each site during the baseline survey at Providence Bay………………………………………………………………………………………………………………………………….44

MARL Update 1 survey Final Report April 2017

1

Summary

The Marine Aquaculture Research Lease (MARL) AL06/098 is located in Providence Bay in New South Wales (NSW). The MARL is positioned in a marine location that is exposed to the prevailing easterly winds and swell but is afforded some small protection from the north by Broughton Island and from the west through the NSW coastline. However, as the MARL is situated 5kms from this low lying coastline, the lease can also be relatively exposed to strong westerlies.

NSW DPI/Huon Aquaculture Research Team (NSW DPI/Huon) intend to use the MARL to assess the potential for the culture of Yellowtail Kingfish (YTK) in these exposed NSW waters. NSW DPI/Huon will position two grids on the lease which can carry up to six pens each. The first grid was installed in September 2016. For the first year all pens will be located on the northern grid within the MARL, and during the present survey two pens were located in that northern grid. The MARL has a consent for a standing biomass of 998t.

In accordance with the State Significant Infrastructure (SSI-5118) consent conditions, NSW DPI/Huon prepared a Water Quality and Benthic Environment Monitoring Plan which was approved by the Department of Planning & Environment., This report presents all of the water column and sediments data required under the Water Quality Monitoring Program, the Substrate Monitoring Program and the Benthic Macroinvertebrate Monitoring Program associated with the 50T standing biomass survey (present survey – Update 1 survey), as required under the SSI-5118 consent prior to commencement of aquaculture operations.

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 April 2017.

Reporting of Update 1 survey follows the same format as provided in the Baseline survey report (identified as the Baseline survey below), except for the following modifications: 1) The northern pen site S3 is now also called pen bay 1 (PB01) for the purposes of the ROV transects. This reflects the fact that it is the intention of the survey programme to include at least three pen bays in each grid (once they have been stocked) for all subsequent ROV surveys. The two presently occupied pen bays, PB01 and PB04 were included in the survey. 2) A control site to the south of the MARL has been included in the present survey and will be included in subsequent surveys to improve coverage across Providence Bay. 3) Photographs of sediment cores have been added to the sediment reporting structure as they may provide a simple but effective means for assessing sediment health. 4) Some comparison to the Baseline survey results are provided where appropriate.

The present survey has been timed to coincide with a standing biomass of approximately 50T of YTK on the MARL lease, with the additional expectation that this would happen approximately 6 months after Baseline Survey, thereby also providing a seasonal (spring/autumn) comparison.

Water column monitoring results from the present survey and internal (DPI/Huon) monthly sampling results are provided in part to assess whether the default trigger limits for physical and chemical stressors for coastal waters (ANZECC guidelines, 2000) are appropriate for Providence Bay. It is again evident as it was in the Baseline survey 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.

Further the data provided for both the present survey and monthly sampling has identified that: i) oxygen saturation levels decreased by 5-10% in the top 20m of the water column for all sites during 1

MARL Update 1 survey Final Report April 2017

the survey with sites in the southern end of the MARL lease having lower DO values at 15-20m depth, ii) dissolved nutrient levels (NOx, DRP) differed markedly between the seafloor and the surface samples, however there were no other discernible trends for nutrients both between sites and between seafloor and surface values, and, iii) the monthly data suggests that there is an ingress of Nitrate rich waters into Providence Bay during the summer months.

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 as yet show any upward trends at the pen site or elsewhere around the farm.

Filming of the seabed was conducted with a Remote Observation Vehicle (ROV). The seabed was generally uniform at all compliance, internal habitat and control sites. All sites shared the common features of medium to coarse rippled sand, some shell grit and old shells, with a depauperate fauna consisting in the main of polychaete tubes. There were occasional Pennatulaceans (Cnidaria), juvenile Flathead and Flounder at several sites, the occasional brittle star (ophiuroid), hermit crab and ribbon worm. In the Baseline survey, S1.1 showed evidence for dark grey rounded ‘globules’ suggested to be deposits of fine organic matter which was also present in grab and core samples and which also appeared to significantly affect the chemical nature of the site (e.g., redox/sulphide).

The present survey shows that amorphous globules (generally lighter in colour) may be more widely distributed as there was evidence for them at a number of sites. The pen sites showed some slight differences to the rest of the sites as might be expected, with the presence of dislodged fouling organisms of the introduced barnacle species Megabalanus coccopoma, and what appeared to be a small increase in general wild fish numbers. The only algae observed were small pieces of green drift algae at a few of the sites.

All sites apart from S1 have mean sediment redox values above 190mV. At S1 all three samples were lower compared to the results from all other sites. Sulphide concentration in sediments was below the detection limit at all sites except for S1 & S3. Only one of the triplicate samples at S3 was above detection at 5uM, whereas all three S1 samples registered a positive value for sulphides ranging from 0.01 – 2uM. The reading for S3 suggests that there may be some very slight effect from fish stocking now being picked up at this pen site. Generally the observed redox and sulphide values were indicative of well oxygenated, unimpacted sediments. The more anoxic values for the samples at S1 suggest that the source of organic matter identified at this site in the Baseline survey is still present.

Similar to the Baseline survey, sediments across the Bay 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 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 survey. 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. There were no site specific differences identified since the Baseline survey.

As for the Baseline survey the organic content was very low at all sites, ranging from <0.02% to 0.75%, with an average of 0.07% across all sites with one of the triplicate samples at C1 being higher than the other samples. As shown in the comparison between the two surveys, C1 and S1 appear to have consistently higher TOC levels, albeit that the overall levels are very small in all cases. There would appear to be no increase in TOC levels at the pen site (S3) between the two surveys.

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MARL Update 1 survey Final Report April 2017

A total of 7631 benthic macroinvertebrates was recorded, comprising 22 broad-scale taxa and, collectively within the polychaetes, molluscs and decapod crustaceans, over 65 families. The greatest numbers of broad-scale taxa (mean(SE) = 13.5(0.3)) and macroinvertebrates, i.e.

280.0(35.4), were recorded at S3 (the pen site), with ANOVA detecting some differences from the northern control and compliance sites. This contrasts with that recorded in the Baseline survey, with no significant differences present among any of the eight sites (with C3 not included). The evidence of difference with S3 is supported by nMDS ordination and ANOSIM showing that the overall community composition at this pen site (with both broad-scale and family-level taxa), was significantly different to all other sites, with this difference being more marked at the level of broadscale taxa.

Based on the above faunal patterns, and when comparing with the Baseline survey, it is evident that S3 (the pen site) is now showing evidence of difference with respect to the northern control and compliance sites, reflecting increased numbers of benthic macroinvertebrates overall and of particular taxa such as ostracods and buccinid gastropods. These differences are likely to reflect the increased organic input (fish food and faeces) from farm operations. The analyses also demonstrate that these faunal differences are not present at either of the compliance sites (S1 and S2). In this survey, the broad-scale taxa analyses of the benthic macroinvertebrates were sufficient to detect differences among sites.

Although the number of broad scale and family level taxa were similar between Baseline and Update 1 surveys, the number of individuals in the Update 1 survey was more than two and a half times than that recorded for the Baseline survey. This is related to the addition of the further control site (C3 â&#x20AC;&#x201C; 1312 individuals from four replicate grabs) and likely to reflect seasonal increases in tanaids and other crustaceans, as these surveys occurred in different seasons, i.e. Spring (September 2016) for Baseline and Autumn (April 2017) for Update 1.

It is recommended that family-level identification occur in subsequent sampling events as the lease is still developing, with two stocked pens, i.e. PB01 and PB04,currently located close to the pen site (S3). For example, buccinid gastropods were recorded at S3, which are known scavengers (Aguzzi et al., 2012). It is also important to be able to quantify either any future changes in polychaete taxa, such as increases in capitellids and dorvilleids, or in the abundances of particular bivalve families, some of which may decline (Macleod & Forbes, 2004; Edgar et al., 2010).

Remnants of the colonies of the barnacle M. coccopoma, which attach and grow on the fish nets and that are dislodged during wave action and routine net cleaning operations, were found only at S3, while the caprellid amphipods which occur on M. coccopoma (Platell, unpub. data) were essentially not recorded at this pen site. Thus, these barnacles do not apparently act as vectors for caprellids in this survey, however, they may be providing additional organic input, and possibly a food source for the observed buccinid gastropods.

This survey introduced a third control site (C3), which is similar in taxonomic composition to the other southern sites (S4, S5 and S6), and it is therefore recommended that sampling be continued at this site. Although C1 and C2 may not represent ideal â&#x20AC;&#x153;controlâ&#x20AC;? sites (see AMD, 2016) owing to their initial difference from the northern compliance sites (S1 and S2) and pen site (S3) and their typically finer grain size (this report and AMD, 2016), it is recommended that sampling at both C1 and C2 continue, as they represented only a small investment in processing time for benthic macroinvertebrates.

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MARL Update 1 survey Final Report April 2017

2

Operational Summary

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; David Whyte

Field work: AMD Personnel: University Personnel:

Huon Technical Personnel:

AMD Pty Ltd, University of Newcastle, Huon Aquaculture Company Dom O’Brien (18-20th April) Margaret Platell (17th April), Vincent Raoult (19th Sept), Tom Ryan (17-19th April), Gerhardus Barnard (17th April) Tim Smith (19 th -20th April) Elisha Lovell (All days)

Dates of fieldwork: 17th April – Infauna 18th April – ROV 19th April – Water column - Nutrients, Chlor a, DO, Salinity, Temp.

Seafloor TOC/S2-/Redox for C3, S6.1, S6.2.

20th April – Seafloor TOC/S2-/Redox for the rest of the sites.

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.

A Getac mobile GIS unit with attached Novatel Smart Antenna Differential GPS was used to locate all sites for both ROV and seabed sampling.

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.

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MARL Update 1 survey Final Report April 2017

3

Location and Survey Map

Figure 1 - Location and Survey Maps â&#x20AC;&#x201C; MARL Providence Bay Key to maps: S1, S2, S5 & S6 C1, C2, C3 S4 S3(PB01), PB04 Red rectangle â&#x20AC;&#x201C;

35 m compliance (triplicate groups of ROV spot dives).

Control sites (also in triplicate).

Future pen site (also in triplicate).

Current pen sites, single ROV transects Lease boundary.

For sample site coordinates please refer to Appendix 1.

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MARL Update 1 survey Final Report April 2017

4

Water Column Characteristics

Methods Dissolved Oxygen (percentage saturation), pH and temperature data through the water column were measured using a YSI Pro ODO Digital Optical Dissolved Oxygen Meter on 19th April 2017. The probe measured from the water surface down to a depth of 20m only.

The full suite of nutrients was collected on the 19th April. 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 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, 6650 and 750nm. Calculations of Chlorophyll a were based on the American Public Health Association (APHA) Method 10200.

Results Percentage saturation oxygen and temperature results are provided in Figure 2 and Appendix 2.

These demonstrate that the concentration gradients for all variables are generally similar down through the water column for all control and S1-S3 sites.

Dissolved oxygen as % saturation (%sat) ranged between 101-102% at the surface, decreasing down through the water column to 97-99% at 20m depth for the control and S1-S3 sites. The %sat levels at S4-S6 sites were slightly lower at depth (15m, 20m) with a minimum of 93%.

Water temperatures were highly consistent at all sites and depths at 21.8oC to 22.0oC.

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MARL Update 1 survey Final Report April 2017

Figure 2 - Oxygen and Temperature profiles for all sites

Water column nutrients results for surface and seafloor samples are provided in Figure 3 and Appendix 3.

Ammonia, nitrate+nitrite (NOx) and dissolved reactive phosphorus (DRP) levels were consistently higher at the seafloor compared to the surface, with ammonia levels ranging from 0.007-0.010mg/L at the seafloor and 0.005-0.060mg/Lat the surface, NOx ranging from 0.004-0.059mg/L at the seafloor and <0.002-0.004mg/Lat the surface, and DRP ranging from 0.005-0.012mg/L at the seafloor and 0.004-0.005mg/Lat the surface (Figure 3). These results are generally similar to the Baseline survey, with the exception of a relatively small increase in ammonia (when compared to the other sites) registered at the surface for the S3/PB01 site. Kjeldahl Nitrogen (KN), Total Nitrogen (TN) and Total Phosphorus (TP) did not show any particular trends or differences between the surface and the seafloor or across sites, again as for the Baseline survey, with Kjeldahl N ranging from 0.21-0.3mg/L, Total N ranging from 0.21-0.34mg/L and Total P ranging from 0.01-0.04mg/L.

7

MARL Update 1 survey Final Report April 2017 NOx-N (mg/L)

Ammonia-N (mg/L) 0.014

0.07

0.012

0.06

0.01

0.05

0.008

0.04

0.006

0.03

0.004

0.02

0.002

0.01 0

0

C1

C2

S1

S2

S3

S4

S5

S6

C1

C3

S1

C2

S2

S3

S4

S5

S6

C3

S5

S6

C3

S6

C3

Seafloor Nox

Surface Nox

Seafloor Ammonia

Surface Ammonia

Total N-N (mg/L)

Kjeldahl N-N (mg/L) 0.35

0.4

0.3

0.35 0.3

0.25

0.25

0.2

0.2

0.15

0.15

0.1

0.1

0.05

0.05 0

0 C1

C2

S1

S2

S3

S4

S5

S6

C1

C3

S1

C2

S2

S3

S4

Seafloor Total Nitrogen

Surface Total Nitrogen

Seafloor Kjeldahl Nitrogen

Surface Kjeldahl Nitrogen

Total P-P (mg/L)

Reactive P-P (mg/L) 0.045

0.014

0.04

0.012

0.035 0.01

0.03

0.008

0.025 0.02

0.006

0.015

0.004

0.01 0.002

0.005 0

0 C1

C2

S1

S2

Surface Reactive Phosphorus

S3

S4

S5

S6

Seafloor Reactive Phosphorus

C3

C1

C2

S1

S2

Surface Total Phosphorus

S3

S4

S5

Seafloor Total Phosphorus

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

Huon Aquaculture is presently also undertaking in-house monthly samples for nutrient water quality across a reduced range of sites (C1, C3, S1, S2) in order to establish or develop suitable local guideline levels. When compared to the monthly results, surface ammonia levels for S3 are also slightly elevated above the range demonstrated by those control and compliance sites. Seafloor nitrate levels in the current survey are shown to be well within the range experienced through the monthly monitoring programme.

After 6 months of monthly surveys seafloor dissolved nutrient levels (NOx, DRP) are consistently and significantly higher than at the surface across the Bay (Figure 4).

8

MARL Update 1 survey Final Report April 2017

NOx mgN/L

0.16

Monthly Nitrate + Nitrate (NOx) at control and compliance sites

0.14

Nitrate + Nitrite - C1 SEAFLOOR

0.12

Nitrate + Nitrite - C1 SURFACE

0.1 Nitrate + Nitrite - C3 SEAFLOOR 0.08 Nitrate + Nitrite - C3 SURFACE

0.06 0.04

Nitrate + Nitrite - S1 SEAFLOOR

0.02

Nitrate + Nitrite - S1 SURFACE

0.025

24/05/2017

19/04/2017

24/03/2017

22/02/2017

17/01/2017

14/12/2016

16/11/2016

16/10/2016

0

Nitrate + Nitrite - S2 SEAFLOOR Nitrate + Nitrite - S2 SURFACE

Monthly Dissolved Reactive Phophorus (DRP) at control and compliance sites Phosphorus, Dissolved Reactive - C1 SEAFLOOR

DRP mgP/L

0.02

Phosphorus, Dissolved Reactive - C1 SURFACE Phosphorus, Dissolved Reactive - C3 SEAFLOOR

0.015

Phosphorus, Dissolved Reactive - C3 SURFACE

0.01

Phosphorus, Dissolved Reactive - S1 SEAFLOOR

0.005

Phosphorus, Dissolved Reactive - S1 SURFACE 24/05/2017

19/04/2017

24/03/2017

22/02/2017

17/01/2017

14/12/2016

16/11/2016

16/10/2016

0

Phosphorus, Dissolved Reactive - S2 SEAFLOOR Phosphorus, Dissolved Reactive - S2 SURFACE

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 April 19th are provided in Table 1.

Across all sites, Chlorophyll a levels ranged from <0.1-1.04µg/L. Mean levels did not differ significantly between surface (0.36µg/L) and seafloor (0.38µg/L) sites. The spread of values (as shown by the SD values) at the surface sites was greater than for the seafloor sites (Table 1).

Monthly results (where available) for surface Chlorophyll a levels taken at the monthly 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 16’). 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 9

MARL Update 1 survey Final Report April 2017

significant increase in Chlorophyll a levels at that time. The present survey and monthly 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

NB: For the purposes of calculating means, below the limit of detection values (prefixed by <) have been treated as 50% of the limit of detection.

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MARL Update 1 survey Final Report April 2017

Chlor a ug/L

Chlor a across monthly sample sites 10 9 8 7 6 5 4 3 2 1 0

C1 C3 S1 S2

2/09/2016 16/10/2016 14/12/2016 15/01/2017 24/03/2017 19/04/2017

Date

Figure 5 - Monthly Chlorophyll a levels at sites C1, C3, S1 and S2

Interpretation â&#x20AC;&#x201C; 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. From the present survey and the first six months of monthly data the following trends are suggested (also refer to Table 2 below): 1. Oxygen saturation levels decreased by 5-10% in the top 20m of the water column for all sites during the survey, with sites in the southern end of the MARL lease having lower DO values at depth (for this Baseline Survey5-20m depth). This is despite the fact that oxygen saturation values at the Northern and Southern control sites were the same.

2. As for the Baseline survey the dissolved nutrient levels (NOx, DRP) differed markedly between the seafloor and the surface samples. There were no other discernible trends for nutrients both; between sites, and, between seafloor and surface values.

3. Both Nitrate (seafloor) and Total N (seafloor and surface) levels greatly exceed, and Total P (seafloor and 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 (Figure 5).

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MARL Update 1 survey Final Report April 2017 Table 2 - Nutrient and chlorophyll a levels from all sites compared against ANZECC Trigger levels (2000) SURVEY 1

Parameter

Unit

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

µg/L µg/L µg/L µg/L µg/L µg/L

SURVEY 2

Mean Mean Mean Mean Mean Mean ANZECC Mean Mean Mean Mean Surface Seafloor Surface Seafloor Surface Seafloor Trigger Surface Seafloor Surface Seafloor Control sites Control sites Compliance Compliance Pen site S3 Pen Site S3 level 2000 Baseline Baseline Baseline Baseline only only sites only sites only only only 15 8 13 6 9 9 9 6 10 13 8 5 4 9 2 27 3 38 2 14 2 4 120 280 290 251 280 263 293 235 260 260 300 25 30 30 21 24 27 23 15 20 20 30 10 5 7 4 8 5 9 5 6 5 5 1 0.68 1.62 0.36 0.38 0.5 0.43 0.39 0.39 0.05 0.12

Farm observations The present survey has been timed to coincide with a standing biomass of approximately 50T of YTK on the MARL lease, with the additional expectation that this would happen approximately 6 months after the Baseline survey, thereby also providing a seasonal (spring/autumn) comparison.

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 farm.

Of interest for the future management of the farm may be the observation that oxygen saturation levels can vary slightly in the midwater depth between the southern and northern end of the lease.

12

MARL Update 1 survey Final Report April 2017

5

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 3. 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 Table 3 - ROV List of spot dives undertaken DATE SITE EASTING NORTHING

20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418 20170418

c1.1 c1.2 c1.3 c2.1 c2.2 c2.3 PB01/s3 PB04 s1.1 s1.2 s1.3 s2.1 s2.2 s2.3 s4.1 s4.2 s4.3 s5.1 s5.2 s5.3 s6.1 s6.2 s6.3 c3.3 c3.2 c3.1

433768.9 433804.9 433732 433294.3 433328.9 433307.9 432567.6 432490.7 432791.1 432760.8 432825.6 432425.1 432458.1 432387.6 432358.1 432361 432344.3 432519 432547.6 432484.4 432159 432126.3 432201.4 430221.3 430271.5 430275.2

6388153 6388169 6388106 6388514 6388588 6388556 6387284 6387218 6387474 6387497 6387439 6387434 6387469 6387396 6386760 6386777 6386771 6386589 6386634 6386554 6386559 6386597 6386523 6384856 6384806 6384853

TIME

11:23:20 AM 11:36:02 AM 11:50:17 AM 12:04:12 PM 12:14:59 PM 12:26:18 PM 12:45:32 PM 1:03:23 PM 1:22:23 PM 1:32:18 PM 1:45:36 PM 2:00:34 PM 2:13:35 PM 2:26:37 PM 2:44:20 PM 2:55:18 PM 3:04:43 PM 3:24:05 PM 3:35:29 PM 3:45:31 PM 3:58:02 PM 4:07:00 PM 4:21:13 PM 4:38:18 PM 4:47:50 PM 4:57:19 PM

Note: all times indicated above are approximately 1 hour in advance of real time as the Getac unit had not compensated for (and could not be adjusted on the day) the change in daylight saving time.

13

MARL Update 1 survey Final Report April 2017

Interpretation â&#x20AC;&#x201C; general comments on spot dive locations Table 5 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 consisting in the main of polychaete tubes. There were occasional Pennatulaceans (Cnidaria), juvenile Flathead and Flounder, brittle stars (ophiuroid), hermit crabs and ribbon worms at several sites. In the Baseline survey, S1.1 showed evidence for dark grey rounded â&#x20AC;&#x2DC;globulesâ&#x20AC;&#x2122; suggested to be deposits of fine organic matter which was also present in grab and core samples and which also appeared to significantly affect the chemical nature of the site (e.g., redox/sulphide). The present survey shows that amorphous globules although generally lighter in colour in the present survey may be more widely distributed as there was evidence for them at a number of sites (e.g., S5.1, S6.1, C3.1 screenshots in Figure 6).

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 fouling organisms below the pen restricted to Megabalanus coccopoma and what appeared to be a small increase in general wild fish numbers.

The only algae observed was small pieces of green drift algae at sites S1, S2, PB 1(S3) and C3.

Table 4 - Pen Bays direction of dives across seafloor

Grid Position

PB01 PB04

Fish Pen

1601 1602

Direction to centre of pen (degrees)

Standing Biomass

110 110

34.8 16.4

(T)

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 the organism underneath both occupied pen bays.

14

MARL Update 1 survey Final Report April 2017

Figure 6 - Screenshots of ROV footage from all sites.

15

MARL Update 1 survey Final Report April 2017 Table 5 - Description of each ROV dive performed at the MARL site, Providence Bay. Note: dates and times refer to that shown on the ROV screen. For the actual time and date add 14 hours for all sites dated 17/4/17, as ROV was still on USA time. Correct date/time indicated for sites dated 18/4/17. C1.2 also erroneously labelled on footage as C1.1.

Easting Northing Date Depth Dive Site (GDA94 (GDA94 (DD-MMTime Comments (m) Type MGA55) MGA55) YYYY) Seabed = Rippled sands.

8.18.51 C1.1 433769 6388153 17-Apr-17 37 control Fauna = Suspected occasional worm tube. Brittle stars. Small crab. pm Flora = None.

Seabed = Rippled sands.

8.33.16 C1.2 433805 6388169 17-Apr-17 37 control Fauna = Suspected occasional worm tubes. pm Flora = None.

Seabed = Rippled sands.

8.46.07 C1.3 433732 6388106 17-Apr-17 37.5 control Fauna = Suspected occasional worm tubes. pm Flora = None.

Seabed = Rippled sands.

8.57.40 C2.1 433294 6388514 17-Apr-17 36.5 control Fauna = Suspected brittle star. Suspected occasional worm tubes. pm Flora = None.

Seabed = Rippled sands, quite coarse. Moderate amount of shell debris. Amorphous small 9.08.35 globules at surface.

C2.2 433329 6388588 17-Apr-17 37.5 control pm Fauna = Unidentified fish. Worm tubes encrusting some globules.

Flora = None.

Seabed = Rippled sands, quite coarse. Moderate amount of shell debris. Amorphous small 9.19.35 globules at surface.

C2.3 433308 6388556 17-Apr-17 37.5 control pm Fauna = Suspected Nassarid or hermit crab. Small Flathead.

Flora = None Seabed = Rippled sand. Some shell debris.

10.16.18 1.1 432791 6387474 17-Apr-17 39 35m Fauna = A few mysids. Occasional stalk, Turritellid shell. pm Flora = Some green drift algae.

Seabed = Rippled sand. Some shell debris. Very occasional black amorphous 'globule'.

10.25.45 1.2 432761 6387497 17-Apr-17 38.5 35m Fauna = A few suspected hermit crabs. Flathead.. pm Flora = None.

Seabed = Rippled sand. Some shell debris.

10.39.10 1.3 432826 6387439 17-Apr-17 38.5 35m Fauna = Suspected mysids. Flounder. Cuttlefish. Suspected worm tube (occasional). pm Flora = None.

Seabed = Rippled sand. Some shell debris.

10.54.25 2.1 432425 6387434 17-Apr-17 38.5 35m Fauna = Ribbon worm. Suspected worm tube pm Flora = None.

16

MARL Update 1 survey Final Report April 2017 Site

Easting (GDA94 MGA55)

Northing (GDA94 MGA55)

Date (DD-MMYYYY)

Time

38

35m

Depth (m)

Dive Type

2.2

432458

6387469

17-Apr-17

11.07.20 pm

2.3

432388

6387396

17-Apr-17

11.22.12 pm

38

35m

4.1

432358

6386760

17-Apr-17

11.37.58 pm

40

internal

4.2

432361

6386777

17-Apr-17

11.49.19 pm

40

internal

4.3

432344

6386771

18-Apr-17

2.05.06 pm

40

internal

5.1

432519

6386589

18-Apr-17

2.17.35 pm

41

35m

5.2

432548

6386634

18-Apr-17

2.29.23 pm

41.5

35m

5.3

432484

6386554

18-Apr-17

2.39.36 pm

41.5

35m

6.1

432159

6386559

18-Apr-17

2.51.36 pm

40

35m

6.2

432126

6386597

18-Apr-17

3.04.59 pm

40

35m

6.3

432201

6386523

18-Apr-17

3.14.56 pm

40.5

35m

Comments Seabed = Rippled sands. A few broken shells and shellgrit. V.occasional black amorphous lump/globule.

Fauna = Occasional worm tubes. Flounder. Suspected anemone. Suspected hermit crab.

Flora = Some drift green algae.

Seabed = Rippled sand. Some shell debris.

Fauna = Suspected occasional worm tube.

Flora = Some drift green algae.

Seabed = Rippled sand. Shell debris. Amorphous small lumps on surface.

Fauna = Fanworm (Serpulid?). Fine and larger worm tubes. Flounder, Flathead. Suspect tunicate.

Flora = None.

Seabed = Rippled sand. Shell debris. Amorphous small lumps on surface.

Fauna = Fine and larger worm tubes. Flathead. Suspected tunicate.

Flora = None.

Seabed = Rippled sand. Shell debris. Amorphous small lumps on surface.

Fauna = Fine and larger worm tubes. Stingaree, Flathead. Brittlestars common.

Flora = None.

Seabed = Rippled sand. Shell debris. Amorphous small lumps on surface.

Fauna = Fine and larger worm tubes. Octopus. Suspected Nassarid. Brittle stars.

Flora = None..

Seabed = Rippled sand. Shell debris. Amorphous small lumps on surface.

Fauna = Many fine and larger worm tubes. Brittle stars. Sea pen. Maldanid polychaete (Bamboo worm), Flathead, Gurnard. Ribbon worm. Suspected anemone.

Flora = None.

Seabed = Rippled sand. Shell debris. Amorphous small lumps on surface.

Fauna = Many fine and larger worm tubes. Brittlestars common Flora = None.

Seabed = Rippled sand. Some shell debris. Amorphous small lumps on surface.

Fauna = Fine and larger worm tubes. Brittlestars. Flounder, Flathead.

Flora = None Seabed = Rippled sand. Some shell debris. Amorphous small lumps on surface.

Fauna = Fine and larger worm tubes. Ribbon worm. Brittlestars. Suspected Gurnard.

Flora = None Seabed = Rippled sand. Some shell debris. Amorphous small lumps on surface.

Fauna = Fine and larger worm tubes. Ribbon worm. Brittlestars. Flathead.

Flora = None

17

MARL Update 1 survey Final Report April 2017 Easting (GDA94 MGA55)

Northing (GDA94 MGA55)

Date (DD-MMYYYY)

Time

Depth (m)

Dive Type

C3.1

430275

6384853

18-Apr-17

3.55.10 pm

36

control

C3.2

430272

6384806

18-Apr-17

3.41.01 pm

36.5

control

C3.3

430221

6384856

18-Apr-17

3.31.38 pm

36.5

control

PB01 (3.3)

432568

6387284

17-Apr-17

9.41.02 pm

39.5

Stocked pen

PB04

432491

6387218

17-Apr-17

9.58.19 pm

39

Stocked pen

Site

Comments Seabed = Rippled sand. Some shell debris. Amorphous small lumps on surface.

Fauna = Fine and larger worm tubes. Ribbon worm. Brittlestars. Suspected goby, Flathead.

Flora = None.

Seabed = Rippled sand. Some shell debris. Amorphous small lumps on surface.

Fauna = Fine and larger worm tubes. Brittlestar. Flathead.

Flora = Piece of drift green algae.

Seabed = Rippled sand. Some shell debris. Amorphous small lumps on surface.

Fauna = Fine and larger worm tubes. Brittlestars. Flathead, suspected small Ray. Flora = None Seabed = Rippled sands. Some shell debris.

Fauna = Megabalanus barnacles. A few small flathead. Occasional worm tubes. Suspected mysids and v.small hermit crabs. Ribbon worm.

Flora = Small amount of drift green (mostly) algae.

Seabed = Rippled sands. Some shell debris..

Fauna = Megabalanus barnacles (debris). A few flounder & flathead, a ray or numbfish.

Suspected v.small hermit crabs. Ribbon worms.

Flora = None.

18

MARL Update 1 survey Final Report April 2017

5.3

Sediment Cores - Photographs C1

C2

S1

S2

S5

C3

S3

S6.1,6.2

S4

S6.3

Figure 7 - Sediment chemistry cores for all survey sites

Interpretation â&#x20AC;&#x201C; general observations on cores Photographs of all sediment chemistry cores are presented in Figure 7 above. General observations are as follows: 1) There is a general trend from fine to coarser grained sand from north to south across the groups of sites (C1/C2, S1/S2/S3, S4/S5/S6, C3).

2) The finer and more consolidated sands at C1/C2, restrict the penetration of the core into the sediment at those sites, making for shorter cores.

19

MARL Update 1 survey Final Report April 2017

3) All cores are uniform throughout showing well-oxygenated sands in all cases; except the C2 and S1 cores which both show patches (2-3cm2) of fine black organic material, at 5cm depth in C2 and 3cm depth in S1. The observation of black deposits at S1 reflects the general sediment chemistry results obtained for that site and the previous video and sediment chemistry results provided in the Baseline survey. The presence of the black deposit at C2 is not reflected in the sediment chemistry.

It is possibly also noteworthy that there were no visual signs of organic matter at the pen site (S3/PB01).

20

MARL Update 1 survey Final Report April 2017

6

Sediment Chemistry

Triplicate samples were taken at each of the nine sample sites for all of the variables tested in this section. 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 25°C). Pre measurement calibration provided a reading of 227mV at 24.5°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 apart from S1 have mean sediment redox values above 190mV (Figure 8). At S1 all the triplicate samples were lower in comparison to the results from all other sites. The observed high redox values are indicative of well oxygenated, unimpacted sediments (Macleod & Forbes, 2004).

The more anoxic value for the samples at S1 suggests that the source of organic matter identified at this site in Baseline Survey is still present. Raw data is presented in Appendix 4.

Redox 300

Redox Potential (mV)

250

200

150

100

50

0

-50

-100 S1

S2

S3

S4

S5

S6

C1

C2

C3

Site

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

21

MARL Update 1 survey Final Report April 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 below detection at all sites except for S1 & S3 (Figure 9).

Only one of the triplicate samples at S3 was above detection at 5uM, whereas all three S1 samples registered a positive value for sulphides ranging from 0.01 â&#x20AC;&#x201C; 2uM. It would appear that whatever has impacted S1 (first observed during the Baseline Survey) is still present at that site. The reading for S3 suggests that there may be some very slight effect now being picked up at this pen site. These readings though are all extremely low and overall the observed sulphide concentrations therefore showed negligible significant evidence of organic enrichment (Macleod & Forbes, 2004). Raw data is presented in Appendix 5.

Figure 9 - Sulphide concentrations in sediment core samples

22

MARL Update 1 survey Final Report April 2017

6.3

Particle Size Analysis

Methods The top 100mm of each sediment core was homogenised and then sieved for particle size determination.

Results and interpretation Sediments across the area sampled 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 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 Baseline Survey. Detailed results are presented in Figure 10, with the proportion of muds provided in Table 6. There were no site specific differences identified since Baseline survey.

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 sites implies similar depositional environments.

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

23

MARL Update 1 survey Final Report April 2017 Table 6 - Mean sediment grain size and %mud at all sites

Site C1 C2 C3 S1 S2 S3 S4 S5 S6

6.4

Mean % Mud 0.00023 0.00057 0.00020 0.00050 0.00010 0.00038 0.00036 0.00060 0.00032

Mean Grain Size (um) 227.81 248.83 423.25 252.45 412.50 393.26 471.90 456.65 470.04

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 60°C prior to analysis of total organic carbon. Total organic carbon was measured by loss on ignition (450°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 Baseline survey the organic content was very low at all sites, ranging from <0.02% to 0.75%, with an average of 0.07% across all sites. One of the triplicate samples at C1 appeared higher than the other samples (refer to Appendix 6), and as shown in the comparison between the two surveys (Figure 12), C1 and S1 appear to have consistently higher TOC levels, albeit that the overall levels are very small in all cases.

There would appear to be no increase in TOC levels at the pen site (S3) between the two surveys.

0.80

0.70

Mean % Loss on Ignition

0.60 0.50 0.40 0.30 0.20 0.10 0.00 -0.10

C1

C2

S1

S2

S3

S4

S5

S6

C3

-0.20 Figure 11 - Total Organic Content for all sites (includes standard error)

24

MARL Update 1 survey Final Report April 2017 0.50 0.45

Mean % Loss on Ignition

0.40

0.35 0.30 0.25 0.20

0.15 0.10 0.05 0.00 C1

C2

S1

S2

S3

S4

S5

S6

C3

Figure 12 - Total Organic Content comparison between Baseline survey (orange bars) and Update 1 survey (blue bars)

25

MARL Update 1 survey Final Report April 2017

7

Biological Analysis

Methods Collection: Macroinvertebrates were collected at each of the eight sampling sites as for Baseline, and a further sampling site (C3) to the south, using a Van Veen grab which sampled a 0.07 m 2 area of seabed. Four replicate grab samples were collected at each of the control (C1 and C2, and the new C3), pen (S3 and S4) and compliance (S1, S2, S5 and S6) sites, with a total of 36 grab samples collected. 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 rinsing the sample with 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). 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 & Hoskin (1999), Jansen (2000) and Poore (2004), as well as online keys – Grove & de Little (2017) and Wilson et al. (2017). Identification of polychaetes was enhanced by examination of specimens by Dr Pat Hutchings at the Australian Museum, while, for particularly the bivalves and gastropods, the collection of greater numbers of larger/less damaged individuals further enhanced their separation into different taxa when considered with samples from the Baseline survey. Isopods and amphipods were separated into sub-orders (not done in the Baseline survey analysis) and reported in Appendix 8, following the observation that (1) there was considerable diversity in the isopods and (2) caprellid amphipods were particularly abundant on the barnacles (Megabalanus coccopoma) that grow on the seacage netting (Platell, unpub data).

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 7) and raw data provided in Appendix 8.

The abundance of all benthic macroinvertebrates and the number of taxa, both at a broad scale, i.e. phylum, class or order, as appropriate, and family level (within the polychaetes, molluscs and decapods) were each analysed using one-way ANOVA, with site as a factor. Levene’s test showed that the numbers of broad-scale taxa were not required to be transformed (Broad-scale, df=8, F=0.96, P=0.487), while family-level taxa and the total abundances required log 10 transformation, with non-significance reported following such transformation, i.e. Family-level, df=8, F=1.95, P=0.093; Total abundance df=8, F=2.17, P=0.074).

Following the analyses for this report, it was found that during the Baseline survey analysis, the numbers of broad-scale taxa and family-level (for polychaetes, molluscs and decapods) taxa had been overestimated. Consequently, these analyses have been re-conducted for this report using the corrected data. Unlike the previously reported broad-scale taxa data (Aquaculture, Management & Development Pty Ltd (AMD), 2016), Levene’s test showed that no transformation or von Bonferroni correction was needed for the broad-scale taxa in these analyses (df=7, F=1.56, P=0.195). However, as before, there were no transformations that reduced the value of Levene’s F-value for the family26

MARL Update 1 survey Final Report April 2017

level taxa and analyses were therefore carried out on the non-transformed data and using the von Bonferroni correction (df=7, F=2.88, P=0.025).

Means and standard error plots are presented for the abundance of all benthic macroinvertebrates and the number of taxa, both at a broad scale and family level, and for both the Baseline and present surveys (Figure 14a-f).

Multivariate analyses of the numbers 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). Thus, the total numbers of each of the taxa in each replicate grab were log10 transformed and the Bray-Curtis measure used to create a similarity matrix.

Analyses of Similarities (ANOSIM) was used to determine whether there were any significant differences between samples at the different sites 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.

Results and interpretation Comments re processing, sediment characteristics and taxonomic identifications: It is noted that the time to sieve, pick and sort each of the samples varied between sites, with those for the more northern sites, and most particularly C1 and C2, taking a relatively short time (e.g. 1hr 30 min). In contrast, those of the southern sites, including C3, had relatively large amounts of sediment retained and many small crustaceans, meaning that processing took up to 6 hours for some of those samples.

Most samples contained a mixture of shell grit and oceanic sand, but for some samples at S1, large amounts of rounded lithic granules (> 1 mm) were observed. At S3, there were large fragments of M. coccopoma colonies present in all samples, ranging from 2 (Sample 3) to over 50 individuals (Sample 4), and anoxic material (black sand) was observed in both Sample 2 and 3. Smaller fragments of M. coccopoma within the sieved sand were recorded at only S3.

General description: A total of 7631 benthic macroinvertebrates were recorded, comprising 22 broad-scale taxa and, collectively within the polychaetes, molluscs and decapod crustaceans, over 65 families (Table 7 and Appendix 8). Of the newly-sampled families, buccinid gastropods were only recorded at S3, while low numbers of dorvilleid polychaetes were found at S1, S3 and all southern sites (Table 7). Although dorvilleids were not recorded in the Baseline survey (AMD, 2016), the collection of 19 very small specimens in the Update 1 survey enabled a positive identification by Pat Hutchings and a re-examination of the taxonomically-similar polychaetes in Baseline showed that a total of three individuals were present (1 in S4 and 2 in S6). As for Baseline, there were very few capitellid polychaetes (Appendix 8). Very low numbers of caprellid amphipods were recorded, i.e. 2 at C2 and 1 at S3 (Appendix 8).

27

MARL Update 1 survey Final Report April 2017 Table 7 - of the benthic macroinvertebrates for each broad-scale taxa and important families for selected groups, recorded at nine sites during Update 1 survey at Providence Bay, and the total and percentage contribution of each to the overall fauna BENTHIC MACROINVERTEBRATES

C1

C2

S1

S2

S3

S4

S5

S6

Cnidaria Bryozoa Other invertebrate phyla Annelida: Oligochaeta Annelida: Polychaeta Polychaeta: Spionidae Polychaeta: Nereididae Polychaeta: Onuphidae Polychaeta: 27 other families Mollusca (total) Mollusca: Bivalvia Bivalvia: Nuculanidae

1 1 2 0 19 5 0 0 14 36 26 9

1 0 16 3 0 0 13 74 2 0

2 9 69 13 4 12 40 13 0 0

5 5 2 0 46 29 0 6 11 19 10 0

11 1 7 0 210 52 123 7 28 37 12 0

56 6 0 113 41 13 32 27 16 11 4

14 44 9 0 126 65 13 13 35 13 6 2

10 3 4 0 137 68 10 35 24 13 4 1

2 22 6 0 137 15 46 28 48 55 20 5

43 132 39 9 873 291 209 133 240 276 91 21

0.6 1.8 0.6 0.1 11.9 4.0 2.8 1.8 2.6 3.7 1.2 0.3

Bivalvia: Mesodesmatidae Bivalvia: 11 other families

0 17

0 2

0 0

2 8

0 12

5 2

0 4

1 2

10 5

18 52

0.2 0.6

1 9

2 70

2 11

1 8

2 23

3 2

1 6

0 9

6 29

18 167

0.2 2.3

0 2 0 7 114 4

61 1 0 8 289 3

0 3 0 8 104 3

1 1 0 6 299 1

0 1 19 3 766 171

0 1 0 1 1177 2

0 4 0 2 1369 8

0 1 0 8 746 2

2 13 0 14 1078 11

64 27 19 57 5942 205

0.9 0.4 0.3 0.6 80.7 2.8

Crustacea: Amphipoda Crustacea: Isopoda

86 0

184 4

66 3

164 29

383 23

275 37

311 37

252 31

324 53

2045 217

21.8 2.3

Crustacea: Leptostraca Crustacea: Mysidacea

1 9

1 4

0 6

0 7

6 5

0 33

1 40

0 20

0 6

9 130

0.1 1.8

Crustacea: Cumacea Crustacea: Tanaidacea

4 2

8 79

6 14

4 93

9 144

30 791

8 954

9 421

15 661

93 3159

1.3 42.9

Crustacea: Decapoda Decapoda: Pasiphaeidae

8 6

6 3

6 1

1 0

25 7

9 0

9 6

11 3

8 1

83 27

0.9 0.4

Decapoda: Leucosiidae Decapoda: Diogenidae

2 0

0 0

0 5

0 0

14 3

3 1

0 0

0 5

2 1

21 15

0.3 0.2

0 0 0 10 8 2 183

2 1 0 4 4 0 384

0 0 0 1 1 0 198

1 0 0 3 3 0 379

0 1 0 0 0 0 1032

2 3 0 6 6 0 1374

1 2 1 6 6 0 1581

1 2 0 5 5 0 918

4 0 0 12 12 0 1312

11 9 1 47 45 2 7631

0.1 0.1 <0.1 0.6 0.6 <0.1

Mollusca: Scaphopoda Mollusca: Gastropoda Gastropoda: Trochidae Gastropoda: Architectonidae Gastropoda: Buccinidae Gastropoda: 10 other families Crustacea (total) Crustacea: Ostracoda

Decapoda: 5 other families Crab megalopa & scyllarid nisto Crustacea: Stomatopoda Echinodermata (total) Echinodermata: Ophiuroidea Echinodermata: 2 other classes TOTAL

C3

Total

%

In terms of abundance, the benthic fauna was dominated by crustaceans (80.7%, esp. tanaids and amphipods), with polychaetes (11.9%, esp. spionids) and molluscs (7.0%, esp. nuculanid and mesodesmatid bivalves, trochid gastropods and architectonid gastropods) making smaller 28

MARL Update 1 survey Final Report April 2017

contributions (Table 7, Appendix 8). Sixty of the 64 trochids (and of the 167 gastropods overall) were found in one replicate grab at one site (C2). Of the decapods, pasaipheids (shrimps), leucosiids (crabs) and diogenids (hermit crabs) were the most abundant (Table 7). Furthermore, four small individuals of palinurids (true lobsters) and five scyllarid nisto (slipper lobsters) were present.

For S3 (the current pen site), buccinid gastropods were recorded at only this site, large numbers of ostracods were recorded and ophiuroids were not present, but were present at other sites in low numbers (Table 7).

Observed patterns in the k-dominance plots show that there are relatively diverse communities and low to moderate levels of single taxon dominance in particularly the southern sites (tanaids: S4 and S5 (57 - 60%), C3 (50%)), with the next most abundant taxa (amphipods) contributing 20-25% to the fauna at these three sites (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 1 survey at Providence Bay.

Univariate analyses: For Update 1 survey, the numbers of broad-scale taxa ranged between 7 and 15 (same as for Baseline), and the number of family-level taxa of polychaetes, molluscs and decapods ranged between 6 and 24 (higher than Baseline) for each of the replicate grab samples (see the amended values later in this section). The number of individuals per replicate grab ranged between 24 and 705 (this upper value being far higher than Baseline) and the overall mean (and SE) abundance was 204.5(27.8), being much greater than the 84.1 (6.8) individuals at Baseline.

ANOVA showed that, for Update 1 survey, there was a significant difference for each of the three variables, i.e. the number of broad-scale benthic macroinvertebrate taxa, families of polychaetes, molluscs and decapods and the total abundances at the different sites (Table 8). These were then followed up by the use of post-hoc tests to determine where the differences resided (see below).

29

MARL Update 1 survey Final Report April 2017 Table 8 - Results of one-way ANOVA of 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 Update 1 survey at Providence Bay. Source Broad-scale taxa Site Residual Family-level taxa Site Residual Total abundance Site Residual

df

MS

F

P (%)

8 27

11.50 1.85

6.210

0.000

8 27

0.07 0.02

4.634

0.001

8 27

0.52 0.04

15.19

0.000

Tukey’s test showed that the number of broad-scale taxa was significantly greater at S3 (mean(SE) = 13.5(0.3)) than from both of the northern control sites (C1 and C2) and one northern compliance site (S1), with means ranging between 8.75 and 10 for those three sites (Figure 14d). There was no significant difference between the number of broad-scale taxa at S3 and the other northern compliance site (S2), in which the mean (SE) was 11.5(0.3). There were no significant differences among any of the southern sites, with means (SE) of between 12(0.7) for S6 and 13.5(0.6) for C3, and none of which were significantly different from S3 (Figure 14d).

For the families of polychaetes, molluscs and decapods, Tukey’s test showed significant differences between C3 vs the other control sites, with values being much greater at C3, i.e. 19.3(2.6), vs C1 or C2, 9.0(0.8) and 8.0(1.4), respectively (Figure 14e). There were no significant differences between S3 vs any other sites or between the southern sites.

For the total abundances, Tukey’s test showed that the total abundances at S3 were significantly greater (P = 0.000) than those at C1 and S1 but not C2 (P = 0.059) or S2 (P = 0.056), see Figure 14f.

Thus, greater overall abundances were recorded at S3, i.e. mean (SE) of 258.0(35.4), than at C1 (45.8(7.4) or S1 (49.5(11.3)). There were no significant differences for the total abundances between the southern sites, which ranged between 229.5(30.2) at S6 to 395.3(103.3) at S5 (Figure 14f).

Univariate analyses revisited for Baseline: The numbers of broad-scale taxa ranged between 7 and 15, and the number of family-level taxa of polychaetes, molluscs and decapods ranged between 2 and 17, for each of the replicate grab samples, each of which were less than originally reported (AMD, 2016). ANOVA of the Baseline numbers of broad-scale benthic macroinvertebrate taxa showed that they did not differ overall between sites (

30

MARL Update 1 survey Final Report April 2017

Table 9), as was previously found, with an overall mean (SE) of 10.8(0.3) (Figure 14a). However, there was nearly a significant difference (at P=0.01) between the numbers of families of polychaetes, molluscs and decapods at the various sites, reflecting the mean values being lowest at C1, i.e. 6.00(1.35), and greatest at S5, i.e. 13.75(1.97) (Figure 14b). The means and SE for the total abundances, which were also previously shown not to be significantly different (AMD, 2016), are shown in Figure 14c.

31

MARL Update 1 survey Final Report April 2017 Table 9 - Results of one-way ANOVA of Site of the numbers of taxa (broad-scale and family-level for polychaetes, molluscs and decapods) and the total abundance of benthic macroinvertebrates obtained from four replicate grabs at each site during the Baseline survey at Providence Bay. Source Broad-scale taxa Site Residual Family-level taxa Site Residual

df

MS

F

P (%)

7 24

4.50 2.81

1.60

0.182

7 24

26.46 9.18

2.86

0.026

Figure 14 - Means (and SE) of the numbers of broad-scale (a,d) and family-level taxa (b,e) and for all benthic macroinvertebrates (c,f), obtained from four replicate grabs at each site during the Baseline and Update 1 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=0.65, P=0.1%). For the northern sites (in which the pen was located), all were significantly different from both each other, i.e. R=0.40-1.0, P=2.9%, except for C2 vs S1 (R=0.25, P=17.9%), and from all southern sites. These results were similar to that for the Baseline survey (AMD, 2016). For the southern sites, most (including the new control site C3) were not significantly different to each other, except for S4 vs S6 (R=0.49, P=2.9%).

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. 0.56 vs 0.65. Similar patterns of significance were found between the northern sites (including C2 vs S1 not being significantly different, R=0.19, P=17.1%), but S1 and S2 were now not significantly different from each other 32

MARL Update 1 survey Final Report April 2017

(R=0.52, P=5.7%) and the northern sites were not always different from the southern sites, i.e. C2 and S2 vs S5, S1 vs all southern sites. The R-statistic for S3 vs the northern sites was 0.76-1 (P=2.9%).

ANOSIM of the pen site (S3) vs the two compliance sites (S1 and S2) detected a significant difference at P=2.9% with both broad-scale (R=0.76 and 0.93, respectively) and family-level comparisons (R=0.52 and 0.92, respectively).

RELATE showed that the two matrices, i.e. broad-scale vs family level, had a relatively high and significant correlation with each other (Rho=0.61, P=0.1%). nMDS ordination of the broad-scale benthic macroinvertebrates showed that, similar to the baseline, the samples for C1 and C2 lay to the left of most other sites, those for the southern sites (S4-S6 and C3) formed a tight group on the lower right part of the plot and those for S1 were distributed near C1 and C2, while S2 lay closer to the southern sites (Figure 15a). Unlike baseline, however, the samples for S3 formed a discrete group in the upper right hand part of the plot, indicative of a difference in species composition (Figure 15a). SIMPER showed that the taxa that contributed to this difference comprised inter alia more ostracods and polychaetes at S3 than other northern sites and more ostracods and typically gastropods at S3 than in the southern sites. nMDS ordination of the family-level taxa of polychaete, molluscs and decapods showed a similar pattern to that of the broad-scale taxa, but with the samples for the southern sites being less tightly grouped and those for S3 now lying closer to the samples from the southern sites (Figure 15a,b).

This less marked difference between sites than for the broad-scale comparisons indicates that these taxa are not showing a marked variation in species composition (in contrast to that for all taxa). This may be related to the large numbers of families and the relative sparseness of the data, i.e. many zero values.

33

MARL Update 1 survey Final Report April 2017

Figure 15 - nMDS ordinations using data on the benthic macroinvertebrates 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 1 survey at Providence Bay.

Concluding Remarks Based on the above faunal patterns, and when comparing with the results from the Baseline survey, it is evident that S3 (the pen site), is now showing evidence of difference with respect to the northern control and compliance sites, reflecting increased numbers of benthic macroinvertebrates overall and of particular taxa such as ostracods and buccinid gastropods. These differences are likely to reflect the increased organic input (fish food and faeces) from farm operations. The analyses also demonstrate that these faunal differences are not present at either of the compliance sites (S1 and S2). In this case, the broad-scale analyses of the benthic macroinvertebrates were sufficient to detect such change, with these patterns being less pronounced at the family level for the polychaetes, molluscs and decapods, probably owing to the relative sparseness of the data, with zero individuals reported for many of the large number of families.

Although the number of broad scale and family level taxa were similar between Baseline and Update 1 surveys, the number of individuals in the Update 1 survey was more than two and a half times 34

MARL Update 1 survey Final Report April 2017

than that recorded for the Baseline survey. This is related to the addition of the further control site (C3 â&#x20AC;&#x201C; 1312 individuals from four replicate grabs) and likely to reflect seasonal increases in tanaids and other crustaceans, as these surveys occurred in different seasons, i.e. Spring (September 2016) for Baseline and Autumn (April 2017) for Update 1.

It is recommended that family-level identification occur in subsequent sampling events as the lease is still developing, with the two stocked pens, i.e. PB01 and PB04, currently located close to the pen site (S3). For example, buccinid gastropods were recorded at S3, which are known scavengers (Aguzzi et al., 2012), and their appearance in the present survey is likely to be of significance. It is also important to be able to quantify either any future changes in polychaete taxa, such as increases in capitellids and dorvilleids, which were collectively represented by 30 individuals on this sampling occasion at the pen sites, or any concomitant declines in the abundances of particular bivalve families (Macleod & Forbes, 2004; Edgar et al., 2010).

Remnants of the colonies of the barnacle M. coccopoma, which attach and grow on the fish nets and that are dislodged during wave action and routine net cleaning operations, were found only at S3, while the caprellid amphipods which occur on M. coccopoma (Platell, unpub. data) were essentially not recorded at this pen site. Thus, these barnacles do not apparently act as vectors for caprellids in this survey, however, they may be providing additional organic input, and possibly a food source for the observed buccinids.

The choice of control sites has been enhanced by the addition of the third control site (C3) in this survey, which is shown to be similar in taxonomic composition to the other southern sites, and it is recommended that sampling be continued at this site. Although C1 and C2 do not represent ideal â&#x20AC;&#x153;controlâ&#x20AC;? sites (also see Baseline), owing to their initial difference from the northern compliance sites (S1 and S2) and pen site (S3) and their typically finer grain size (this report and AMD, 2016), it is recommended that both C1 and C2 are retained in the future, as they represented only a small investment in processing time.

35

MARL Update 1 survey Final Report April 2017

8

References

Aguzzi, J., Jamieson, A.J., Fujii, T., Sbragaglia, V., Costa, C., Menesatti, P., Fujiwara, Y. (2012). Shifting feeding behaviour of deep-sea buccinid gastropods at natural and simulated food falls.

Marine Ecology Progress Series 458, 247-253.

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). MARI Lease â&#x20AC;&#x201C; Providence Bay Baseline Environmental Assessment. Report to Huon Aquaculture Company Pty Ltd. 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.

Crawford, C., MacLeod, C. & Mitchell, I. (2002). Evaluation of Techniques for Environmental Monitoring of Salmon Farms in Tasmania. May, 2002. Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, 134 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.

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 â&#x20AC;&#x201C; 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.

36

MARL Update 1 survey Final Report April 2017

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.

Wilson, R.S., Hutchings, P.A. & Glasby, C.J. (2017). Polychaetes: An interactive Identification Guide.

Retrieved from http://researchdata.museum.vic.gov.au/polychaetes

37

MARL Update 1 survey Final Report April 2017

Appendices Appendix 1 Survey coordinates for sediment 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 433772 433297 432791 432422 432586 432360 432519 432159 430273

6388151 6388520 6387476 6387434 6387272 6386763 6386592 6386558 6384854

38

MARL Update 1 survey Final Report April 2017 Appendix 2 Water Column results for all sites - Probe Data

Site C1 C1 C1 C1 C1 C2 C2 C2 C2 C2 C3 C3 C3 C3 C3 S1 S1 S1 S1 S1 S2 S2 S2 S2 S2 S3 S3 S3 S3 S3 S4 S4 S4 S4 S4 S5 S5 S5 S5 S5 S6 S6 S6 S6 S6

Depth 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20

% Saturation 101.5 100.7 99.6 98.9 98.3 101.6 100.6 99.7 98.9 97.4 101.7 101.2 100.2 98.8 98.5 101.8 100.9 100.1 98.7 98.2 101.8 101 100.1 99 98.1 101.7 100.7 99.7 99 97.1 101.8 100.5 99.8 98.4 94.6 101.7 100.5 97.8 93.4 93 101.7 100.9 99.6 97.8 95.5

Temp 22 22 21.9 21.9 21.9 21.9 21.9 21.9 21.9 21.8 22 21.9 21.9 22 22 21.9 21.9 21.9 21.9 21.9 21.9 21.9 21.9 21.9 21.9 22 22 21.9 21.9 21.9 21.9 21.9 22 21.9 21.8 21.9 22 21.9 21.9 21.9 21.9 21.9 22 21.9 21.9

39

MARL Update 1 survey Final Report April 2017 Appendix 3 Water Column results for all sites â&#x20AC;&#x201C; Nutrients

Sample date:

19/04/2017

SITE

Ammonia mg-N/L 0.005 0.006 0.006 0.005 0.006 0.013 0.005 0.005 0.006 0.006 0.01 0.01 0.01 0.009 0.008 0.009 0.008 0.009 0.007 0.008 0.002 0.006

NOx mg-N/L 0.002 0.002 0.002 0.002 <0.002 0.002 0.002 <0.002 0.002 0.004 0.047 0.059 0.02 0.008 0.004 0.027 0.033 0.032 0.009 0.016 0.018 0.002

Kjeldahl Nitrogen mg-N/L 0.1 0.23 0.29 0.22 0.25 0.26 0.25 0.28 0.21 0.26 0.24 0.28 0.28 0.21 0.3 0.23 0.23 0.28 0.25 0.253 0.028 0.250

Total Nitrogen mg-N/L 0.1 0.23 0.29 0.22 0.25 0.26 0.25 0.28 0.21 0.27 0.29 0.34 0.3 0.22 0.3 0.25 0.26 0.31 0.25 0.266 0.035 0.251

Total Phosphorus mg-P/L 0.01 0.02 0.04 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.03 0.02 0.03 0.03 0.02 0.023 0.007 0.021

Reactive Phosphorus mg-P/L 0.003 0.004 0.005 0.004 0.005 0.005 0.004 0.004 0.004 0.005 0.01 0.012 0.007 0.005 0.005 0.008 0.008 0.008 0.005 0.006 0.002 0.004

0.003 0.009

0.001 0.027

0.026 0.256

0.027 0.280

0.008 0.024

0.001 0.008

0.001

0.019

0.030

0.037

0.005

0.002

UNIT LOR C1 Surface C2 Surface S1 Surface S2 Surface S3 Surface S4 Surface S5 Surface S6 Surface C3 Surface C1 Seafloor C2 Seafloor S1 Seafloor S2 Seafloor S3 Seafloor S4 Seafloor S5 Seafloor S6 Seafloor C3 Seafloor Overall Mean Overall Standard Deviation Surface Mean Surface Standard Deviation Seafloor Mean Seafloor Standard Deviation

40

MARL Update 1 survey Final Report April 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 114 -52 134 204 252 252 162 191 220 235 236 247 261 240 245 235 213 241 202 192 210 225 223 238 231 198 225

41

MARL Update 1 survey Final Report April 2017 Appendix 5 Sulphide analysis, measured in sediments at 3 cm from sediment surface

Core or Grab

Sulphide 3cm

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

(uM) 2.86 0.09 1.70 0.00 0.00 0.00 0.00 0.00 5.24 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

42

MARL Update 1 survey Final Report April 2017 Appendix 6 Organic content â&#x20AC;&#x201C; raw data Date 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 20/04/2017 19/04/2017 19/04/2017 20/04/2017 19/04/2017 19/04/2017 19/04/2017

Site 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

Result 0.75 0.09 0.12 0.04 0.05 0.07 0.1 0.06 0.11 <0.02 0.02 <0.02 0.04 0.04 0.04 <0.02 <0.02 <0.02 0.07 0.04 0.04 0.06 0.04 0.03 0.02 <0.02 <0.02

43

MARL Update 1 survey Final Report April 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.

44

MARL Update 1 survey Final Report April 2017

Appendix 8 Raw data for benthic macroinvertebrates, obtained from four replicate grabs at each site during Update 1 survey at Providence Bay. Note: Red represents taxa present in only the Baseline survey, while grey represents those recorded in the Baseline survey but not Update 1 survey.

INVERTEBRATES

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

Cnidaria: Anthozoa: Actinaria 1

Bryozoa

1 1

Porifera

1

3

3

1

1

Phoronida Annelida: Oligochaeta

1

7 6

5

5

3

4

4

2

1

1

3

S4-1

S4-2

S4-3

S4-4

11

31

7

7

S5-1

S5-2

S5-3

S5-4

S6-1

S6-2

S6-3

S6-4

C3-1

C3-2

C3-3

5

3

3

1

1

5

13

18

9

2

1

8

2

9

1

3

1

1

2

2

25

34

36

45

2

1

8

C3-4

1 2

1

2

6 1

1

1

1

3

S3-4

4

2 3

1

Sipuncula

Polychaeta: Spionidae

S3-3

1

Nemertea

Polychaeta: Orbiniidae

S3-2

7

Cnidaria: Scleractinia

Annelida: Polychaeta TOTAL

S3-1

4

3

5

4

1 1

Polychaeta: Magelonidae

1

33

1

1

1

1

1

1

1

1

3

1

2

26

32

42

2

1

10

10

1

1

1

2 2

2 26

7

3

9

13

3 1

1

8

2

2

6

11

12

12

1

1

8

4

47

6

39

35

89

1

1

1

2

12

32

37

34

16

1 13

13

5

30

22

32

2 27

50

30

1

15

13

1

2

19

27

1 13

9

22 1 4

1

Polychaeta: Trochochaetidae

1

1

Polychaeta: Cirratulidae

1

Polychaeta: Arenicolidae

1

3

1

2

2

1

3

1

Polychaeta: Capitellidae

1

1

1

Polychaeta: Maldanidae

1

1

1

1

1

1

1

1

1

Polychaeta: Opheliidae

1

1

Polychaeta: Phyllodocidae

1

1

1

1

1 1

1

Polychaeta: Polynoidae Polychaeta: Sigalionidae

2

Polychaeta: Hesionidae

1

4

Polychaeta: Syllidae

1

1

3

1

1

1

17

2

1

2

1

2

2

3

43

2

5

3

3

1 1

1

1

1

3

3

Polychaeta: Pisionidae Polychaeta: Nereididae

2

2

Polychaeta: Glyceridae

32 1

29

19

1

Polychaeta: Nephtyidae 12

2

1

1

2

4

2

1

Polychaeta: Onuphidae

9

1 2

2

2

1

1 1

10

2

5

5

4

3

3

3

15

1 1

8

1

Polychaeta: Dorvilleidae

11

10

6

8

Polychaeta: Chaetopteridae

2 5

Polychaeta: Sabellidae

1

1

11

5

4

2

1

2

4 1

1 1

1

17

1

Polychaeta: Lumbrineridae Polychaeta: Eunicidae

14

1

1 15

3

2

2

3

1

1

1

1

2 1

1

2

1 2

2

2

45

1

MARL Update 1 survey Final Report April 2017

Polychaeta: Oweniidae

1

Polychaeta: Sabellongidae

1

Polychaeta: Sternapsidae

1

2

2

Polychaeta: Flabelligeridae

1

Polychaeta: Ampharetiidae

1

Polychaeta: Pectinariidae Polychaeta: Sabellariidae Polychaeta: Trichobranchidae Polychaeta: Terebellidae sensu lato Polychaeta: Terebellidae Polychaeta: Telothelepidinae

0

0

0

0

0

0

2

1

2

0

0

1

0

0

0

4

1

2

0

0

1

2

Polychaeta: Polycirridae Polychaeta: Unidentified ter. sl.

2

3

0

0

13

11

7

5

Mollusca: Bivalvia

10

7

4

5

1

4

2

2

3

65

2

1

3

1

2

0

4

5

0

3

4 1

3

1

7

1

4

4

5

6

13

3

14

1

2

2

5

6

3

3

Bivalvia: Mactridae

7

1

1

2

1

2

2

2

2

1

2

2

2

2

0

1

9

6

1

0

9

6

4

1

4

2

1

1

2

3

2

1

1 2

2

0

2

5

4

1

3

4

0

1

Bivalvia: Myochamidae

1

7

2

Bivalvia: Veneridae

1

1

1

17

12

5

7

10

1

2

1

4

1

3

5

2

1 2

2

1 1

1

1

1

Bivalvia: Tellinidae

1

21

3

Bivalvia: Mesodesmatidae

0

1

1

Mollusca TOTAL

3

1

1

1

2 1

1

Bivalvia: Galeommatidae

2

1

1

Bivalvia: Cardiidae Bivalvia: Corbulidae

0

1

1

Polychaeta: Unidentified

Bivalvia: Nuculanidae

0

1

1

1

1

1

2

1

1

1

Bivalvia: Psammobiidae Bivalvia: Mytilidae

2

Bivalvia: Hiatellidae

3

Bivalvia: Pectinidae

1

Bivalvia: Unidentified

1

Mollusca: Scaphopoda Scaphopoda: Gadiludae

1

1

1

1

1

1

1

Scaphopoda: Dentaliidae Mollusca: Gastropoda Gastropoda: Trochidae

2

1

1

1

2

1

1

1

1 3

3

3

0

3

64

1

60

1

2

2

2

2

1 1

6

3

2

3 1

0

7

0

9

7

2

1

1

1

3

1 0

0

4

2

0

0

4

1

4

0

2

1

1

1

3

1

11

5

10

1

1

Gastropoda: Turbinidae

3

1

46

MARL Update 1 survey Final Report April 2017

Gastropoda: Acteonidae

1

Gastropoda: Architectonidae

1

1

1

1

1

2

1 1

4 1

Gastropoda: Terebridae

1

1 1

1

1

4

4

1

5

2

6

1

Gastropoda: Turritellidae

1

1

Gastropoda: Scaphandridae

1

2

1

2

2

Gastropoda: Pyramidellidae Gastropoda: Rissoellidae Gastropoda: Naticidae Gastropoda: Muricidae Gastropoda: Buccinidae

5

Gastropoda: Turridae

1

8

6

1

Gastropoda: Fascolariidae

1

Gastropoda: Marginellidae Gastropoda: Olivellidae

3 1

1

2

1

2

2

2

1

3

1

1

1 2

Gastropoda: Olividae Gastropoda: Mitridae

1

1

15

22

1

2

12

14

12

14

1

Gastropoda: Aplysiidae Gastropoda: Unidentified Crustacea TOTAL Crustacea: Ostracoda Crustacea: Amphipoda TOTAL Crustacea: Amphipoda: Gammaridea Crustacea: Amphipoda: Caprellidea

1 25

52

106

47

1

1

2

20

40

79

30

42

33

16

25

9

16

38

25

52

20

40

77

30

42

33

16

25

9

16

38

25

1

1

2

2

1

1

1

1

38

16

26

62

51

234

159

139

234

366

103

42

10

16

2

49

45

87

84

167

100

95

42

38

119

52

49

44

87

84

167

100

95

42

38

4

14

10

18

2

2

1

10

17

4

2

2

2

4

6

3

6

2

3

89

97

1

518

158

135

651

238

228

252

2

2

52

67

73

23

119

52

67

73

6

7

9

9

3

5

4 2

4

120

162

228

494

307

187

1

1

4

3

2

2

88

69

72

101

132

61

30

23

88

69

72

101

132

61

30

1 1 1

Crustacea: Cumacea

5 1

Crustacea: Tanaidacea

1

Crustacea: Decapoda TOTAL

0

0

1

3

1

2

1

1

5

3

1

1

1

5

1

3

1

3

1

1

1

1

1

2

1

1

2

1

18

2

1 2

3

7

3

4

3

4

11

2

4

6

18

2

1

1

4

90

12

9

10

7

5

23

17

5

8

2

2

4

3

2

1

13

4

5

1

1

1

3 1

1

1

2

2

1

3

3

3

1

2

2

2

1

2

6

8

5

4

4

3

2

3

6

1

3

3

11

12

2

10

4

4

3

5

1

1

2

5

5

1

3

1

2

1

2

5

1

2

2

1

16

8

13

42

6

5

1

2

21

18

19

35

57

21

37

29

242

373

5

3

3

0

0

0

5

0

1

0

0

0

1

8

4

3

10

2

4

4

2

2

6

1

1

2

236

1

2

Crustacea: Leptostraca

Decapoda: Majiidae

24

1

Crustacea: Isopoda: Valvifera Crustacea: Isopoda: Flabellifera: Sphaeromatidae Crustacea: Isopoda: Flabellifera- Serolidae

Decapoda: Leucosiidae

80

2

Crustacea: Isopoda TOTAL Crustacea: Isopoda: Anthuridea Crustacea: Isopoda: Cymothoida: Gnathidae

Crustacea: Mysidacea

56

8

10

6

11

6

1

2

7

2

1

103

73

506

163

139

146

84

123

76

138

354

144

114

49

1

2

8

1

0

0

1

4

3

3

4

1

3

0

1 1

2 1

1

47

MARL Update 1 survey Final Report April 2017

Decapoda: Calappidae

1

Decapoda: Hexapodidae

1

Decapoda: Pasiphaeidae

3

3

2

1 1

1

1 4

1

1

1

5

1

1

2

1

Decapoda: Callianassidae Decapoda: Crangonidae

1

Decapoda: ?Pandalidae Decapoda: Palinuridae

1

Decapoda: Diogenidae

5

1

2

1

1

3

1

2

1

Decapoda: Unid. hermit crab Decapoda: Crab Megalopa

1

1

Decapoda: Scyllarid Nisto

1

2

1

1

2

Crustacea: Stomatopoda

1

Arthropoda: Pycnogonida Echinodermata TOTAL

2

Echinodermata: Asteroidea Echinodermata: Clypeasteroida Echinodermata: Ophiuroidea Fish remains (otoliths, bones) M. coccopoma remains

2

2

3

1

1

1

2

3

1

2

1

3

1

2

1 Y

Y

1

2

1

2

1

1 Y

1

1

3

1

2

3

1

3

1

1

2

6

3

1

1

1

3

1

2

3

1

3

1

1

2

6

3

1

Y Y

Y Y

Y

Y

48