Onslow Seawater Desalination Plant Desalination Brine Toxicity Assessment
CLIENT: Water Corporation STATUS: Revision 1 REPORT No.: R200092 ISSUE DATE: 16/01/2021
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20WAU-0008/ R200092 Water Corporation ii Onslow Seawater Desalination Plant: Desalination Brine Toxicity Assessment
WA Marine Pty Ltd t/as O2 Marine ACN 168 014 819 Originating Office – Townsville 48 Valencia St, Cranbrook, QLD 4814 T 1300 739 447 | [email protected]
Version Register
Version Status Author Reviewer Change from Authorised for Release Previous Version (signed and dated)
A Draft T. Hurley J. Abbott J.Abbott 29/05/2020
B Draft T. Hurley C. Lane C. Lane 11/06/2020
0 Final T. Hurley J. Abbott C. Lane 22/12/220
1 Final J. Abbott C. Lane C. Lane 14/01/2021
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Name Email Address
Russell Brown [email protected]
Julia Phillips [email protected]
20WAU-0008/ R200092 Water Corporation iii Onslow Seawater Desalination Plant: Desalination Brine Toxicity Assessment
Acronyms and Abbreviations
Acronyms/Abbreviation Description
ANZECC Australia and New Zealand Environment and Conservation Council ARMCANZ Agricultural and Resource Management Council of Australia and New Zealand ANZG Australia and New Zealand Guidelines for Fresh and Marine Water Quality BCH Benthic Communities and Habitats CSIRO Commonwealth Scientific and Industrial Research Organisation DAF Development Assessment Footprint DBT Dibutyltin DGV Default Guideline value from ANZECC/ARMCANZ (2000) or ANZG (2018) EC Effects Concentration EC/IC10 The concentration of a given contaminant that will cause an effect or the inhibition of growth or reproduction in 10% of a collection of organisms over a given period of time EC/IC50 The concentration of a given contaminant that will cause a sub-lethal effect or inhibition of growth or reproduction in 50% of a collection of organisms over a given period of time. Effects can be larval abnormalities, reproductive impairment, and growth inhibition or fertilisation success. EPA Western Australian Environmental Protection Authority ESA Ecotox Services Australasia FSW Filtered seawater IC Inhibition Concentration ISO/IEC International Organisation for Standards/International Electrotechnical Commission LAT Lowest Astronomical Tide LEP Level of Environmental Protection LOEC Lowest Observed Effect Concentration. The lowest concentration of a material used in a toxicity test that has a statistically significant adverse effect on the exposed population of test organisms as compared with the controls. OMEMMP Operational Marine Environmental Monitoring and Management Plan MBT Monobutyltin NATA National Association of Testing Authorities NOEC No Observed Effect Concentration. The highest concentration of a toxicant at which no statistically significant effect is observable, compared to the controls; the statistical significance is measured at the 95% confidence level. NSW New South Wales NTU Nephelometric Turbidity Units O&G TSE Oil and Grease Total Solvent Extractable ppt Parts Per Thousand PQL Practical Quantitative Limit QA/QC Quality Assurance/Quality Control QLD Queensland SA South Australia SPL Species Protection Limit
20WAU-0008/ R200092 Water Corporation iv Onslow Seawater Desalination Plant: Desalination Brine Toxicity Assessment
Acronyms/Abbreviation Description
SSD Species Sensitivity Distribution SSDP Southern Seawater Desalination Plant SWDP Seawater Desalination Plant SWRO Seawater Reverse Osmosis TBT Tributyltin TOC Total Organic Carbon Tp Wave period (seconds) TRH Total Recoverable Hydrocarbons WA Western Australia
20WAU-0008/ R200092 Water Corporation v Onslow Seawater Desalination Plant: Desalination Brine Toxicity Assessment
Table of Contents
1. Introduction 8 Proposal Description 8 Scope and Objectives 9 EPA Guidance 10 2. Environmental Setting 12 Climate 12 Marine Environment 12 3. Methods 16 Sample Information 16 Desalination Brine Toxicity Assessment 16 Data presentation and statistical analysis 18 4. Results 19 Physico-Chemistry Results 19 Desalination Brine Toxicity Assessment 19 Species Sensitivity Distribution 21 Species Protection Levels and Dilutions 22 QA/QC 23 5. Conclusions 24 6. Reference List 25
Figures
Figure 1-1 Project location – Onslow, Western Australia. 11 Figure 2-1 Levels of ecological protection in relation to diffuser outfall location. 15 Figure 4-1 Comparison of the brine concentration and sea urchin larval development test response data (range from the mean) on 2018, 2019 and 2020 brine samples 21 Figure 4-2 Burrlioz 2.0 SSD based on the chronic test results from the analysed bitterns effluent and the 95% Confidence Interval 22
Tables
Table 1-1 Short summary of the proposal 8 Table 1-2 Location and proposed extent of physical and operational elements. 9 Table 2-1 Monthly median baseline water quality data collected in Beadon Bay (June 2019 – August 2020). 13 Table 3-1 Analytical tests, temperature, acute/chronic tests and test protocols for SSDP brine samples submitted on 1 August 2018 (Intertek 2018, Intertek 2019a). 16 Table 3-2 Analytical tests, temperature, acute or chronic tests and test protocols for SSDP brine samples submitted on 1 April 2020 (Intertek 2020). 17 Table 4-1 Physico-chemical concentrations recorded during testing of the targeted brine samples 19
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Table 4-2 Ecotoxicity results for the proportion (%) of SSDP brine samples resulting in the NOEC, LOEC, 50% and 10% EC/IC 20 Table 4-3 Comparison of repeated SSDP brine sample ecotoxicity sea urchin larval development test results using Echinometra mathaei 21 Table 4-4 Recommended guideline values (±95%CI) and corresponding dilutions for concentration of the analysed bitterns effluent for each SPL and LEP 23
Appendices
Appendix A Intertek Ecotoxicology Laboratory Report 18/10/2018 27 Appendix B Intertek Ecotoxicology Laboratory Report 15/05/2020 28 Appendix C Burrlioz 2.0 Statistical Report 29
20WAU-0008/ R200092 Water Corporation vii Onslow Seawater Desalination Plant: Desalination Brine Toxicity Assessment
1. Introduction
Proposal Description
The Water Corporation propose to construct a seawater desalination plant (‘the project’) in the town of Onslow, Western Australia. This project will establish a reliable drinking water supply to the town, which is currently supplied from the Cane River Bore-field. The seawater desalination plant will supply fresh water to the town of Onslow and will replace the existing supply from the Cane River Bore-filed as the volume of fresh water has been decreasing. The project will involve the installation of permanent subtidal infrastructure in Beadon Bay (intake head, brine diffusers and transport pipes), a land-based processing plant, and piping to the existing town storage tanks. A short description of the proposal is included in Table 1-1.
1.1.1. Short Summary of the Proposal
Table 1-1 Short summary of the proposal
Project Title Onslow Seawater Desalination Plant.
Proponent Name Water Corporation.
The Proponent intends to construct and operate a SWRO desalination plant to supply potable Short Description water to the town of Onslow, Western Australia. Seawater intake and brine wastewater release will occur in Beadon Bay approximately 800 m seaward from the shoreline. The desalination processing facility is to be located at Lot 551-553 Beadon Creek Road and will feed the towns existing drinking water storage tanks at Lot 880 Onslow Road
The proposal includes the installation of infrastructure (intake/diffuser and pipeline) in the subtidal region of Beadon bay. The outfall is proposed to be located in approximately 5 m depth Lowest Astronomical Tide (LAT) approximately 800 m offshore of Onslow Beach in Beadon Bay (Figure 1-2). Pipelines will be trenched/tunnels below the shoreline and dune system to the processing plant between lots 551 and 553 Beadon Creek Road. A storage supply pipeline will run from the processing plant, south to the existing town storage tanks at Lot 880 Onslow Road. The physical and operational extent of these elements are summarised below in Table 1-2.
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Table 1-2 Location and proposed extent of physical and operational elements.
Element Location Proposed Extent
Physical Elements
Intake Head and Pipeline Figure 1-1 0.02 ha of intertidal and subtidal BCH, extending approximately 800m offshore into Beadon Bay.
Diffuser Pipeline Figure 1-1 Disturbance areas aligned with dilution plume of intertidal and subtidal BCH, extending approximately 800m offshore into Beadon Bay.
Desalination Plant Figure 1-1 Lots 551-553 Beadon Creek Rd, Onslow. Land clearing within Lots 551-553 of up to 3.5 ha.
Storage Supply Pipeline Figure 1-1 Within existing access track behind coastal dune out to storage tank facility.
Operational Elements
Seawater Intake Figure 1-1 Flow rate up to 4.44 ML/day
Brine Wastewater Figure 1-1 Flow rate up to 2.44 ML/day discharged of brine to Beadon Bay. Establishment of a Low Ecological Protection Area (LEPA) surrounding the brine outfall in Beadon Bay.
Scope and Objectives
The objective of this report is to address the anticipated requirement for environmental approval for the Onslow SWDP Project in relation to potential ecotoxicity impacts on the receiving environment, resulting from operational brine wastewater release. The scope involves conducting toxicity tests on brine effluent to determine and describe the toxic effects of the desalination brine discharge and predict the number of dilutions required to meet the different levels of ecological protection surrounding the outfall as shown in the Environmental Quality Plan.
Specifically, the document includes the following key elements:
undertake ecotoxicity testing of a prototype desalination brine discharge using a suite of targeted representative tropical/sub-tropical Australian species comprising several trophic levels and taxonomic groups use the results of ecotoxicity tests to develop species protection values using a species sensitivity distribution provide advice that informs hydrodynamic modelling and dilution factors for the proposed effluent.
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A prototype brine from the Southern Seawater Desalination Plant (SSDP) has been used as the brine sample for ecotoxicity testing in the absence of a Project specific brine sample.
EPA Guidance
The EPA (2016) provide a framework for protecting the quality of Western Australia’s marine environment via processes to spatially define, assess and manage potential impacts of proposals on marine environmental quality. One of the processes relevant to this report is the spatial designation of the area around the outfall into four Levels of Ecological Protection (LEPs): Maximum, High, Moderate and Low. The LEPs for the Onslow SWDP Project have been defined in the Marine Environmental Quality Monitoring and Management Plan (MEQMMP) (O2 Marine 2020a). The Low, Moderate and High LEP areas surrounding the outfall are presented in Figure 2-1. The ecotoxicity testing described in this report is used to determine the number of dilutions of the desalination brine required to protect 80%, 90% or 99% species to meet the different LEPs surrounding the outfall, respectively. In accordance with EPA (2016), the Low LEP extends 70 m around the outfall, the Moderate LEP extends 250 m from the Project infrastructure (including the berth pocket), and the area beyond the 250 m boundary is designated as a High LEP.
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Figure 1-1 Project location – Onslow, Western Australia.
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2. Environmental Setting
Climate
Onslow experiences an arid, semi-desert to subtropical climate with two distinct seasons. Summer heat begins from October to April (~24.4°C and 36.4°C), whilst milder winter temperatures (13.0°C – 25.4°C) are from May to September (Sudmeyer 2016). Rainfall in the region Is variable with a mean annual rainfall of 311.6 mm of which the majority (88%) of rainfall throughout the year is primarily due to tropical cyclones and thunderstorms that typically occur between Summer and Autumn (November-April) (Sudmeyer 2016). The dry season is typically categorised by light winds from the south, southeast and east. In the wet season or monsoon, the winds are typically stronger with a strong sea-breeze effect in operation and wind directions around the southwest, west and northwest directions (Sudmeyer 2016).
Marine Environment
Coastal Processes and Oceanography Oceanography and water circulation at the site are dominated by tidal flows, with a mean tidal range of 3.6 m in spring tides and 1 m during neap tides (Baird 2020). Current directions ebb and flow in a northeast and southwest direction during ebb and flood tides, respectively, with peak depth averaged speeds of between 0.3 m/s-1 and 0.4 m/s-1 in spring tides and 0.15m/s-1 and 0.25m/s-1 during neap tides (Baird 2020). The significant wave height is dominated by locally generated sea conditions within the range of 0.5 m to 1 m at short wave periods (Tp <5 s). In dry season months low amplitude swell from the Indian Ocean propagates to the site from the north and occurs in conjunction with locally generated sea waves of short period derived from east-southeasterly (morning) to northwesterly (afternoon) prevailing winds. In the wet season the wave climate is locally generated sea waves from strong west- northwest prevailing winds, with sea conditions protected by the chain of islands further offshore (Baird 2020).
Water Quality Baseline water quality data (physico-chemical parameters) has been recorded in Beadon Bay from September 2019 to current (proposed end of data collection June 2021). Monthly median results are summarised below in Table 2-1. These results indicate a relatively turbid marine environment, with notable increases in NTU during January and February. This is a typical seasonal trend for the majority of Pilbara waters with increasing winds due to prevailing winds and cyclone events (i.e. Tropical Cyclone Damian, 8 February 2020). Salinity, pH and dissolved oxygen measurements have been relatively consistent over the monitoring period, while seawater temperatures are highest between December and March. These baseline data are consistent with findings from previous studies in the Onslow region. MScience (2013) found that marine waters around Onslow are characterised by relatively turbid inshore/nearshore waters that are subject to strong tidal flows, while mid and outer waters are generally clear (Chevron 2010). The shallow inshore bathymetry combines with climatic and meteorological factors such as seasonal winds, rainfall and tropical cyclones to affect turbidity, salinity and temperature in the marine waters around Onslow.
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Table 2-1 Monthly median baseline water quality data collected in Beadon Bay (June 2019 – August 2020).
Parameter
Temperature Salinity Dissolved Turbidity pH Season (°C) (ppt) Oxygen (%) (NTU)
June 2019 21.74 37.48 79.66 8.20 4.45
July 2019 20.22 37.50 80.12 8.31 4.30
Dry August 2019 19.85 37.48 93.71 8.34 4.63
September 2019 23.40 37.59 90.09 8.35 2.25
October 2019 24.18 37.47 96.30 8.22 4.91
November 2019 26.47 37.54 93.15 8.21 5.87
December 2019 28.35 37.50 93.30 8.22 4.69
January 2020 28.76 37.59 88.79 8.08 6.90 Wet February 2020 27.39 37.61 83.68 8.25 11.56
March 2020 29.19 38.45 88.53 8.42 4.24
April 2020 28.39 38.09 94.05 8.92 7.67
May 2020 24.18 37.25 95.90 8.69 9.17
Dry June 2020 22.21 37.91 93.74 8.77 7.86
July 2020 21.42 37.59 93.93 8.42 5.77
August 2020 20.65 37.36 93.74 8.67 5.54
Natural background marine water quality in the Pilbara and the North-West shelf is generally deemed to be very good (99% species protection) with only localised elevations above default guideline values (DGVs) of some contaminants near industrial centres such as high tonnage ports like Dampier and Port Hedland (Wenziker et al. 2006). MScience (2013) undertook baseline marine water quality sampling in nearshore areas (0.5–1 km) in the area of Onslow. In general, baseline conditions met the 90% and 99% species protection level (SPL) DGVs (ANZECC & ARMCANZ 2000) for cadmium, chromium, manganese, molybdenum, nickel, vanadium and mercury, arsenic, copper, lead, aluminium and selenium. The 95th percentile zinc concentration exceeded the DGV for 99% SPL for the High LEP, but not the 90% SPL DGV for a Moderate LEP. Oil and Grease, Total Solvent Extractable (O&G TSE) was rarely detectable and median concentration was usually below 5 mg/L (MScience 2013).
Benthic Communities and Habitats Subtidal benthic communities and habitat (BCH) within the immediate vicinity of the outfall have been classified by O2 Marine (2020b) as generally comprising a flat benthos of fine sand/silt with large bare areas of substrate, with patchy zones of sparse to moderate mixed assemblages of macroalgae, filter
120WAU-0008/ R200092 Water Corporation 13 Onslow Seawater Desalination Plant: Desalination Brine Toxicity Assessment feeders and seagrass. Beadon Creek has previously been classified (O2 Marine 2017) as largely void of benthic habitat. BCH within the Project area is considered common throughout the Pilbara region, and therefore the small area of potential impact is not deemed regionally significant.
Sediment Quality O2 Marine undertook a sediment investigation within and adjacent to the proposed Development Assessment Footprint (DAF) in March 2020 (O2 Marine 2020c). This investigation involved the collection of surface cores (0–0.15 m) at 10 locations within the proposed DAF. Sediments were observed to be comprised of fine sand/silt with shell grit common throughout. Samples were analysed for heavy metals, Organotins (TBT, DTB and MBT), Total Recoverable Hydrocarbons (TRH), Total Organic Carbon (TOC), nutrients and particle size. Infauna samples were also collected, however these results are not available at the time of writing this document.
Metal results indicate that the majority of samples were below the ANZG (2018) DGVs. Aluminium, arsenic and iron were recorded in high concentrations but these analytes are known to be naturally elevated within the Pilbara region and concentrations were comparable to previous baseline investigation results undertaken in Onslow (DEC 2006). All sample results for Organotins and Hydrocarbons were below their respective Practical Quantitative Limit (PQL). TOC and nutrient levels were low and comparable to previous findings (DEC 2006). Particle size results show that surface sediments within the DAF are dominated by fine sands (62–250 µm) and to a lesser extent silts (4– 62 µm).
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Figure 2-1 Levels of ecological protection in relation to diffuser outfall location.
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3. Methods
Sample Information
Desalination brine effluent from Water Corporation’s Southern Seawater Desalination Plant (SSDP) was used as a surrogate sample to enable an impact assessment in the absence of a local (Onslow) SWRO brine product. Ecotoxicity testing was undertaken by Intertek Ecotoxicology Laboratory (Intertek).
This assessment combines ecotoxicity testing results obtained from two separate brine samples collected at the Southern Seawater Desalination Plant:
• Desalination brine received by Intertek Ecotoxicology on 1st August 2018 • Desalination brine received by Intertek Ecotoxicology on 1st April 2020
Desalination Brine Toxicity Assessment
3.2.1. Existing Relevant Toxicity On the 13 January 2020, Water Corporation requested O2 Marine to conduct a review of ecotoxicity testing reports conducted for the SSDP to determine the suitability of these results for impact assessment of a proposed Onslow SWRO brine product. The historical testing conducted on a brine sample from 1st August 2018 is presented in Table 3-1 and the complete report is provided in Appendix A (Intertek 2018, Intertek 2019a). The testing was conducted to assess the toxicity of the brine sample using marine test organisms representative of South Western Australian temperate waters. An additional sea urchin larval development test was performed on a brine sample submitted on 6 December 2019 (Intertek 2019b).
Table 3-1 Analytical tests, temperature, acute/chronic tests and test protocols for SSDP brine samples submitted on 1 August 2018 (Intertek 2018, Intertek 2019a).
Test Performed Temperature Acute/Chronic Test Protocols
Microtox® bacterial bioluminescence 15±1°C Acute Microbics (1992) bioassay: Vibrio fischeri. Microalgal growth inhibition bioassay: 21±1°C Chronic OECD Test Guideline 201 (1984) and T. Isochrysis galbana. the protocol of Stauber et al. (1994). Macroalgal germination and tube 21±1°C Chronic Burridge et al. (1999) growth bioassays: Ecklonia radiata. Mollusc larval development bioassay: 21±1°C Chronic ASTM E724-98 blue mussel Mytilis edulis Copepod Larval development 21±1°C Chronic ISO 16778 (2015) Bioassay: Gladioferens imparipes. Fish larvae development bioassays: 21±1°C Chronic USEPA Method 1001.0 (USEPA, Kingfish Seriola lalandi 2003) Sea Urchin Larval Development 25±1°C Chronic APHA and ASTM protocols Echinometra mathaei
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Review of the test species and discussion with Intertek indicated the microalgae (T. Isochrysis galbana) used was the tropical strain version, whilst Yellowtail Kingfish (Seriola lalandi) and the Sea Urchin (Echinometra mathaei) occur in both tropical and temperate waters in Australia. Intertek indicated there have been no observed differences in results for tests conducted between 21 ± 1°C or above 25 ± 1°C. Therefore, it was considered the results for these tests are representative for species found in tropical/sub-tropical waters of Australia.
3.2.2. Targeted Analysis ANZG (2018) describes the minimum data requirements for using a Species Sensitivity Distribution (SSD) is at least five species that belong to at least four taxonomic groups, but using toxicity data from at least 8 species is strongly encouraged and from more than 15 species is considered optimal.
O2 Marine provided recommendation to Water Corporation to conduct further testing on a suite of bioassays to assess the toxicity of the desalination brine sample. In addition, the existing test results representative of tropical/sub-tropical species were utilised to improve the statistical assessment for the derivation of SPLs. Species selected for testing were determined to be locally relevant and methodologies used for each species are outlined in Table 3-2.
Table 3-2 Analytical tests, temperature, acute or chronic tests and test protocols for SSDP brine samples submitted on 1 April 2020 (Intertek 2020).
Test Performed Temperature Acute/Chronic Test Protocols
72-hr marine algal growth test using 22°C Chronic ESA SOP 110 (ESA 2016), based Nitzschia closterium on Stauber et al. (1994). Sea Urchin Larval Development 25±1°C Chronic APHA and ASTM protocols Echinometra mathaei 48-hr larval development test using the 29±1°C Sub-chronic ESA SOP 106 (ESA 2016), based Milky oyster Saccostrea echinata1 on APHA (1998) and Krassoi (1995) 8-day Sea anemone pedal lacerate 25±1°C Chronic ESA SOP 128 (ESA 2014), based development test using Aiptasia on Howe et al (2014). pulchella 7-day fish imbalance and biomass 25±2°C Chronic ESA SOP 122 (ESA 2017), based toxicity test using barramundi Lates on USEPA (2002) calcarifer1 1 Test not covered by NATA accreditation
Intertek subcontracted Ecotox Services Australia (ESA) to perform some of the tests on tropical/sub- tropical species. Both laboratories undertook testing in compliance with ISO/IEC 17025 and Quality Assurance/Quality Control (QA/QC) test protocols, including use of diluted filtered seawater (FSW) control samples and reference toxicants. In addition, the laboratories are accredited by the National Association of Testing Authorities (NATA). The complete laboratory testing methodology and QA/QC undertaken by Intertek (& ESA) is provided in Appendix B.
All samples were serially diluted (5 x) with FSW to achieve the test concentrations of 3.1, 6.3, 12.5, 25, 50 and 100%.
All species selected are found in tropical/sub-tropical waters of Australia. The Milky oyster Saccostrea echinata, a farm-reared species from Mackay, QLD, was selected as representative of local milky oyster molluscs found in the Pilbara including Saccostrea cucullata and pearl oysters (e.g. Pinctada spp.). The sea anemone Aiptasia pulchella was sourced from Marine Ecology Research Centre at Southern Cross
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University, Lismore, NSW, and was tested to represent sessile benthic fauna species such as scleractinian corals, octo-corals and hydrozoans. The barramundi Lates calcarifer was hatchery reared in SA and represents a common tropical fish species. The Nitzschia closterium cultured at CSIRO in Tasmania is a common marine diatom found in Pilbara waters. A repeat test for the sea urchin (Echinometra mathaei) was undertaken on advice from the laboratory.
Data presentation and statistical analysis
The toxicity test data generated by Intertek are presented in several ways. The concentration at which no observed effects (no observed effect concentration, NOEC) is generally used as the most conservative measure of toxicity in that it is the lowest concentration at which no test organisms are affected. The lowest observed effects concentration (LOEC) is the concentration where the first statistically detectable toxicity is observed. The concentration that causes one or more specified effects in 50% of the test organisms in the prescribed test duration (EC50) or which inhibits growth or reproduction of 50% of the test organisms in the prescribed test duration (IC50) are statistically calculated. Similarly, IC/EC10 values are statistically calculated as the concentration that causes one or more specified effects/detectable inhibition in 10% of the test organisms. Three methods are used to calculate the EC/IC values dependent on the distribution of the data and the curve of best fit; 1) the integrated normal (probit) model, 2) the logistic (logit) model, and 3) the trimmed Spearman-Karber method (Hamilton et al. 1977).
The Burrlioz 2.0 program was used to analyse the toxicity results and to plot the SSD to derive the concentration of brine required to protect 80%, 90%, 95% and 99% SPLs. Burrlioz 2.0 is a statistical software package for use in environmental management of anthropogenic impacts regarding analysis of the effects of toxin levels in an environment. The Burrlioz 2.0 uses a Burr type III regression model for datasets that have eight or more datapoints to estimate the toxin concentrations at SPLs (Warne et al. 2014). Analysis by the Burrlioz 2.0 program was performed on EC/IC10 values derived from chronic toxicity tests to provide high reliability guideline values in accordance with Warne et al. (2014) and ANZG (2018).
The six test results conducted during the targeted analysis, combined with two test results recorded from a previous reference (microalgae T. Isochrysis galbana and Yellowtail Kingfish Seriola lalandi) on the brine samples collected from the same facility, resulted in eight tests of seven species that belong to five groups (i.e. algae, fish, Echinoderm, Mollusc, Cnidaria) used to develop the SSD.
The derived chronic species protection trigger values for the brine effluent are presented as the safe dilution factors required to achieve an adequate level of species protection in the mixing zone in the marine environment. The species protection levels were then used to determine the dilution factor(s) that will ensure the effluent remains below the required dilution target in the mixing zone.
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4. Results
Physico-Chemistry Results
Empirical testing of the brine sample for physico-chemical parameters in water was performed by Intertek for each submitted sample to determine the potential concentrations in the effluent. The results have been summarised in Table 4-1. Testing indicates the effluent concentration is hypersaline at >60 psu dropping rapidly with subsequent dilutions to near-seawater concentrations at 3.1% effluent concentrations (37.5 psu), whilst pH and dissolved oxygen remained relatively stable compared to seawater. Similar physico-chemical results were recorded during historical testing undertaken by Intertek and during the subcontractor ESA testing.
Table 4-1 Physico-chemical concentrations recorded during testing of the targeted brine samples
Parameter Control 3.1% 6.3% 12.5% 25% 50% 100%
pH 8.2 8.2 8.2 8.2 8.2 8.2 8.1 0 Salinity ( /00) 36.1 37.5 38.3 40.1 43.5 50.2 64.0 DO (%) 100 100 100 100 100 100 100
Desalination Brine Toxicity Assessment
A summary of the results of ecotoxicity testing of the brine effluent on seven species used for this assessment is presented in Table 4-2. The complete report for the two test results from the brine sample submitted on 1 August 2018 is provided in Appendix A. The test report for the brine sample submitted on 1 April 2020 is provided in Appendix B.
Toxicity was observed in all tests with NOEC’s ranging from <3.1% brine concentration for the sea urchin and up to 25% for microalgae T. Isochrysis galbana, the Kingfish and barramundi imbalance test results. The LOEC’s for each species varied between the different species and tests relative to the NOEC values for each species, with the sea urchin exhibiting effects at a lower concentration of brine than the other species (3.1%).
The EC50/IC50 and EC10/IC10 concentrations were lowest for the milky oyster (14.5% & 8.3%, respectively) and highest for microalgae T. Isochrysis galbana, growth inhibition results (<100% & 46.4%, respectively). Both species of microalgae tested were the only organisms tested which did not exhibit complete effects observed at the raw (100%) and 50% brine effluent concentration.
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Table 4-2 Ecotoxicity results for the proportion (%) of SSDP brine samples resulting in the NOEC, LOEC, 50% and 10% EC/IC
Test NOEC LOEC EC50/IC50 EC10/IC10
(%) (%) (%) (%)
Microalgal growth inhibition bioassay: T. Isochrysis galbana* 25 50 >100 46.4 Fish larvae development bioassays: Kingfish Seriola lalandi* 25 50 41.9 25.7 Microalgae Growth using Nitzschia closterium 3.1 6.3 46.6 21.3 Sea Urchin Development using Echinometra mathaei <3.1 3.1 17.2 13.0 48-hr larval development test using the Milky oyster 6.3 12.5 14.5 8.3 Saccostrea echinata. 8-day Sea anemone pedal lacerate development test using 12.5 25 17.5 13 Aiptasia pulchella 7-day fish imbalance toxicity test using barramundi Lates 25 50 35.4 32.6 calcarifer 7-day fish biomass toxicity test using barramundi Lates 12.5 25 35.8 22.6 calcarifer * Ecotoxicity test results for tropical/sub-tropical representative species undertaken on previous SSDP brine sample submitted 1 August 2018
The sea urchin test had been conducted previously on two separate brine samples:
• Sample received 01 August 2018 (Intertek 2019a) • Sample received 06 December 2019 (Intertek 2019b)
Figure 4-1 and Table 4-3 provide a comparison of the test results for 2020 against previous testing undertaken. Ecotoxicity results are relatively comparable between years with a significant negative change (69-82%) in sea urchin larval development observed between a brine concentration of 12.5% and 25%. However, in 2020 5% of sea urchin larvae still developed normally at 25% brine concentration, unlike previous years where all larval development was abnormal at this concentration. Conversely, normal larval development in 2020 remained consistently high between 87% to 91% from 12.5% to 3.1% brine concentration relative to the control sample, although during previous years normal development levels gradually increased from 12.5% to 3.1% brine concentration, recording results higher than the control sample at low concentrations. Therefore, the response curve generated in 2020 indicates the brine sample is both slightly more toxic at low concentrations (3.1% or 6.1%) and less toxic at higher concentrations (12.5% or 25%) than results from previous years. This is reflective in low
NOEC and LOEC values in 2020, yet the EC50/IC50 and EC10/IC10 concentrations are slightly higher than previous years.
The variation observed in test results may be indicative of repeat sample test error where it could be assumed the median value of the three results provides the most representative response level. The cause of the sampling error may be a complex mixture of naturally variable early life history development in invertebrates, intraspecies larval responses to changing environmental conditions, chemical properties of the sample, laboratory processes and accuracy. This variation provides a useful demonstration of why it is important to conduct toxicity testing on multiple species and why the NOEC value is not suitable for calculating the SSD using small sample numbers. If there is a similar level of variability within each test, the sample error effect is alleviated through a greater number of tests.
Ultimately, the difference in the EC10/IC10 values between these tests will not greatly affect the SSD curve, brine concentrations for SPLs and subsequent dilutions when considered in the context of the
120WAU-0008/ R200092 Water Corporation 20 Onslow Seawater Desalination Plant: Desalination Brine Toxicity Assessment broader results from other species (i.e. all results would be within the 95% confidence interval of the 2020 results).
Sea Urchin Larval Development Test Data
2020 2019 2018
120
100
80
60
40
20 Normal Development Response (%) Response Development Normal 0 0% 3.10% 6.30% 12.50% 25% 50% 100% Desalination Brine Concentration (%)
Figure 4-1 Comparison of the brine concentration and sea urchin larval development test response data (range from the mean) on 2018, 2019 and 2020 brine samples
Table 4-3 Comparison of repeated SSDP brine sample ecotoxicity sea urchin larval development test results using Echinometra mathaei
Sample Receipt NOEC (%) LOEC (%) EC50/IC50 (%) EC10/IC10 (%)
01 August 2018 6.3 12.5 13.8 10.9 06 December 2019 6.3 12.5 14.7 11.3 01 April 2020 <3.1 3.1 17.2 13.0
Species Sensitivity Distribution
ANZG (2018) guidelines state the minimum toxicity data requirements include at least five species that belong to at least four different taxonomic groups for the development of a SSD for input into the Burrlioz 2.0 software. This study involved eight separate chronic tests from seven species and five taxonomic groups.
All IC10 values used for the SSD are presented in Table 4-2. The percentage of species potentially affected by the discharge of desalination brine with variable concentrations is presented in Figure 4-2. The full Burrlioz 2.0 report is provided in Appendix C. Species protection level values range from 4.4% concentration of desalination brine diluted in seawater for 99% SPL to 13% brine for 80% SPL.
120WAU-0008/ R200092 Water Corporation 21 Onslow Seawater Desalination Plant: Desalination Brine Toxicity Assessment
Figure 4-2 Burrlioz 2.0 SSD based on the chronic test results from the analysed bitterns effluent and the 95% Confidence Interval
Species Protection Levels and Dilutions
Guideline concentrations for a range of SPLs (80%, 90%, 95% & 99%) are statistically derived and presented in Table 4-4. The table includes calculations corresponding to the number of dilutions required to achieve the SPLs in each LEP. The spatial layout of the LEP boundaries are presented in Figure 2-1.
Based on these results, in order to achieve 80% protection of species at the Project site, a brine concentration of 13% should be achieved on release from the outfall requiring a dilution of 1:8. In line with EPA (2016), an 80% SPL is acceptable in a Low Level of Environmental Protection (LEP) area, which has been allocated as a 70 m radius buffer around the diffuser in the MEQMMP for the Project (O2 Marine, 2020a). The area greater than 70 m from the outfall is afforded a High LEP and 99% SPL, indicating a bitterns concentration of 4.4% and a dilution of 1:23 must be achieved at the Low/High LEP boundary.
120WAU-0008/ R200092 Water Corporation 22 Onslow Seawater Desalination Plant: Desalination Brine Toxicity Assessment
Table 4-4 Recommended guideline values (±95%CI) and corresponding dilutions for concentration of the analysed bitterns effluent for each SPL and LEP
SPL LEP Estimated Guideline lower 95% upper 95% dilutions CI CI
99% High 23 4.4% 1.8% 12% 95% - 13 7.8% 5.3% 15% 90% Moderate 10 10% 7.7% 19% 80% Low 8 13% 9.0% 23%
QA/QC
QA/QC protocols and procedures were implemented for all testing undertaken for this study. Prototype brine desalination samples were sent with a Chain of Custody form and were received in apparent good condition. All water quality criteria were met throughout testing, all tests were valid according to test validity criteria and reference toxicity indicated that responses of all organisms were within the accepted parameters. Detailed QA/QC results are provided in Appendix B.
120WAU-0008/ R200092 Water Corporation 23 Onslow Seawater Desalination Plant: Desalination Brine Toxicity Assessment
5. Conclusions
Ecotoxicity testing of prototype desalination brine effluent for the Onslow SWDP Project was undertaken on a suite of representative species for the proposed Project site by Intertek, a NATA certified laboratory. The results of ecotoxicity testing were used to create an SSD in accordance with ANZG (2018) guidelines. This SSD was used to statistically derive guideline concentrations of brine in seawater for different SPLs. These guideline concentrations were used to predict the number of dilutions required to meet the LEPs surrounding the outfall.
• Ecotoxicity testing of SSDP sample brine was conducted by Intertek on the five marine organism groups to represent local marine indicator species (microalgae, echinoderm, cnidarian, mollusc and fish) using six discrete chronic tests • Historical testing of SSDP sample brine was also reviewed for this assessment to investigate species which are representative of tropical/sub-tropical waters of Australia • The SSDP brine sample was found to be highly saline and toxic to all tested organisms. Salinity levels >40 ppt is considered to be the primary causative agent of the toxic effects observed, with a notable reduction in effects at concentrations ≤40 ppt • The sea urchin development test was found to be the most sensitive species exhibiting effects at a lower concentration of brine than the other species, although comparison with previous testing indicates the NOEC and LOEC results were unusually low from the 2020 sample, whilst
the EC10/IC10 results were comparable to previous years
• The EC50/IC50 and EC10/IC10 concentrations were lowest for the milky oyster Saccostrea echinata. The microalgae T. Isochrysis galbana, the Kingfish and barramundi imbalance test results indicate the least sensitivity. Both species of microalgae tested were the only taxonomic group which did not exhibit complete effects observed on the raw (100%) brine sample • An SSD was derived using eight separate chronic tests from seven species and five taxonomic groups • The SSD derived 80%, 90%, 95% and 99% species protection guideline concentrations for the effluent of 4.4%, 7.8%, 10% and 13%, respectively • These concentrations for 80% and 99% SPLs were estimated to require 1:8, and 1:23 dilutions, respectively. These dilutions need to be achieved at the following locations within the Project area (see Figure 2-1): o 8 dilutions in the Low LEP area (<70 m from the outfall) o 23 dilutions on the Low/High LEP boundary (70 m from Project infrastructure).
The results imply that potential toxic effects on local marine indicator species could be minimised if the required dilutions can be achieved within LEP boundaries.
120WAU-0008/ R200092 Water Corporation 24 Onslow Seawater Desalination Plant: Desalination Brine Toxicity Assessment
6. Reference List
ANZECC/ARMCANZ (2000) Australian and New Zealand Guidelines for Fresh and Marine Water Quality. Volume 1: The Guidelines. Australian and New Zealand Environment and Conservation Council, Agriculture and Resource Management Council of Australia and New Zealand, Canberra, ACT, October 2000
ANZG 2018. Australian and New Zealand Guidelines for Fresh and Marine Water Quality. Australian and New Zealand Governments and Australian state and territory governments, Canberra ACT, Australia. Available at www.waterquality.gov.au/anz-guidelines
APHA (1998) Standard Methods for the Examination of Water and Wastewater. 20th Ed. American Public Health Association, American Water Works Association and the Water Environment Federation, Washington, DC.
Baird (2020). Onslow SWRO. Hydrodynamic Modelling Report. 15 June 2020. Report Number; 13166.301.R1.RevA.
Chevron Australia (2010) Draft Environmental Impact Statement/Environmental Review and Management Programme for the Proposed Wheatstone Project: Technical Appendices Q6, Q7, R1, S1, T1, U1, V1, and W1, July 2010
DEC (2006). Background quality of the marine sediments of the Pilbara coast. Department of Environment and Conservation, Marine Technical Report Series, No. MTR 1.
EPA (2016) Technical Guidance – Protecting the Quality of Western Australia’s Marine Environment. Environmental Protection Authority, December 2016.
ESA (2014) SOP 128 – Sea Anemone Pedal Lacerate Development Toxicity Test. Issue No 1. Ecotox Services Australasia, Sydney, NSW.
ESA (2016) SOP 106 – Bivalve Larval Development Test. Issue No. 15. Ecotox Services Australasia, Sydney, NSW.
ESA (2017) SOP 117 –Freshwater and Marine Fish Imbalance Test. Issue No 6. Ecotox Services Australasia, Sydney, NSW USEPA (2002) Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms. Fifth edition EPA-821-R-02-012. United States Environmental Protection Agency, Office of Research and Development, Washington FC, USA
Hamilton, M., Russo, R. & Thurston, R. (1977) Trimmed spearman-karber method for estimating the median lethal concentrations in toxicity bioassays. U.S. Environmental Protection Agency, Washington D.C., EPA/600/J-77/178 (NTIS PB81191918)
Howe, Pelli L., Reichelt-Brushett, Amanda J. and Clark, Malcolm W (2014) Development of a chronic, early life-stage sub-lethal toxicity test and recovery assessment for the tropical zooxanthellate sea anemone Aiptasia pulchella. Ecotoxicology and Environmental Safety 100: 138-147.
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Intertek Ecotoxicology Laboratory (2018). Comparative Toxicity Assessment of a Chemical Additive to Desalination Effluent. Prepared for Water Corporation Southern Seawater Joint Venture. Report No. ECX18-0801
Intertek Ecotoxicology Laboratory (2019a). Test Report 17/05/2019. Prepared for Water Corporation Southern Seawater Joint Venture. Report No. ECX18-0801-2
Intertek Ecotoxicology Laboratory (2019b). Test Report 16/12/2019. Prepared for Water Corporation Southern Seawater Joint Venture. Report No. 904538,904539
Krassoi, R (1995) Salinity adjustment of effluents for use with marine bioassays: effects on the larvae of the doughboy scallop Chlamys asperrimus and the Sydney rock oyster Saccostrea commercialis. Australasian Journal of Ecotoxicology, 1: 143-148.
MScience (2013) Wheatstone LNG Development: Water Quality Around the Proposed Nearshore Outfall. Unpublished Report to Chevron Australia by MScience Pty Ltd, MSA188R1, Perth, WA
O2 Marine (2017) Onslow Marine Support Base Stage 2 Capital Dredging: Ecological Site Investigation. Report No. 1702005
O2 Marine (2020a) Onslow Seawater Desalination Plant: Marine Environmental Quality Monitoring and Management Plan. Prepared for Water Corporation
O2 Marine (2020b) Benthic Communities and Habitat Assessment. Onslow Seawater Desalination Plant. Report No. R200065.
O2 Marine (2020c) Sediment and Infauna Sampling Report. Onslow Seawater Desalination Plant. Report No. R200085
Stauber, J.L., Tsai, J., Vaughan, G.T., Peterson, S.M. and Brockbank, C.I. (1994) Algae as indicators of toxicity of the effluent from bleached eucalypt kraft pulp mills. National Pulp Mills Research Program, Technical Report No. 3. CSIRO, Canberra, ACT
Sudmeyer, R 2016, ‘Climate in the Pilbara’, Bulletin 4873, Department of Agriculture and Food, Western Australia, Perth
USEPA (2002) Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms. Fifth Edition. United States Environmental Protection Agency, Office of Research and Development, Washington DC, EPA/600/4-90/027F.
Warne MStJ, Batley GE, van Dam RA, Chapman JC, Fox DR, Hickey CW and Stauber JL (2014). Revised Method for Deriving Australian and New Zealand Water Quality Guideline Values for Toxicants. Department of Science, Information Technology, Innovation and the Arts, Brisbane, Queensland. 36pp.
Wenziker K, McAlpine K, Apte S, and Masini R (2006) Background Quality for Coastal Marine Waters of the North West Shelf, Western Australia, North West Shelf Joint Environmental Management Study Technical Report 18
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Appendix A Intertek Ecotoxicology Laboratory Report 18/10/2018
120WAU-0008/ R200092 Water Corporation 27 Onslow Seawater Desalination Plant: Desalination Brine Toxicity Assessment
ECOTOXICOLOGY LABORATORY: COMPARATIVE TOXICITY ASSESSMENT OF A CHEMICAL ADDITIVE TO DESALINATION EFFLUENT
CLIENT NAME Attn: Grant Griffith Southern Seawater Joint Venture Lot 32 Taranto Road Binningup, WA 6233
REPORT NO. ECX18-0801
COMPILED BY Dr Tristan Stringer
PROJECT NAME Comparative toxicity assessment of a chemical additive to desalination effluent
DATE 18 October 2018
List of Revisions REV. DATE REVISION DETAILS AUTHOR ISSUE 0 18/10/18 Document Created Dr Tristan Stringer Dr Tristan Stringer
Issuing office: Intertek Welshpool 41-45 Furnace Road, Welshpool, 6106 Western Australia
Disclaimer This report has been prepared for the titled project or named part thereof and should not be relied upon or used for any other project without an independent check being carried out as to its suitability and prior written authority of Intertek being obtained. Intertek accepts no responsibility or liability for the consequences of this document being used for a purpose other than the purposes for which it was commissioned. Any person using or relying on the document for such other purposes agrees, and will by such use or reliance be taken to confirm his agreement to indemnify Intertek for all loss or damage resulting therefrom. Intertek accepts no responsibility or liability for this document to any party other than the person by whom it was commissioned.
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EXECUTIVE SUMMARY
The objective of this assessment was to determine if a proposed process chemical Memclean R increased the toxicity of the Southern Seawater Joint Venture desalination plant’s effluent. In addition to the assessment of Memclean R, a comparison of the desalination brine and the whole effluent (the brine effluent with additional waste streams) was also assessed.
Using the methodology outlined in ANZECC (2000) the toxicity of the four desalination brine effluents was assessed using a suite of marine organisms representative of South Western Australian temperate waters. Chronic endpoints for five species from different trophic levels were used the ecotoxicity assessment. The chronic toxicity tests included the, microalgae T. Isochrysis galbana, the macroalgae Ecklonia radiata, the mollusc Mytilis edulis, the copepod Gladioferens imparipes and the fish Seriola lalandi. To minimise variability between tests, all samples were tested in parallel. Comparisons of toxicity were based on the statistical endpoints for each bioassay and the species protection trigger values for each sample.
The desalination brine sample was used to establish a baseline toxicity level. The results of this study show that the addition of waste streams from the clean in place (CIP) and backwash sumps, creating the whole effluent sample, do not have a significant impact on toxicity. To assess if Memclean R clean increases the toxicity of the whole effluent, two pair wise comparisons were made between the whole effluent sample and the two whole effluent samples spiked with Memclean R. The results show that at 0.17 g/L, Memclean R does not increase the toxicity of the effluent. However, at 2.0g/L, Memclean R does significantly increase the toxicity of the effluent.
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CONTENTS
Introduction 6
Methodology 7 Sample Information 7 Ecotoxicity Testing 7 Diluent Seawater and Concentration Series 7 Microtox® bacterial bioluminescence bioassay 7 Microalgal growth inhibition bioassay 8 Macroalgal germination and tube growth bioassays 10 Mollusc larval development bioassay 12 Copepod Larval development Bioassay 14 Fish larvae development bioassays 15 Statistical Analysis 16 Calculation of Species Protection Trigger Values 17 Comparison of Toxicity 17
Results 18 Desalination Brine 18 Physicochemistry 18 Ecotoxicology Bioassay Results 18 Species Sensitivity Distribution 18 Whole Effluent 20 Physicochemistry 20 Ecotoxicology Bioassay Results 20 Species Sensitivity Distribution 20 Whole Effluent + 0.17 g/L Memclean R 22 Physicochemistry 22 Ecotoxicology Bioassay Results 22 Species Sensitivity Distribution 22 Whole Effluent + 2.0 g/L Memclean R 24 Physicochemistry 24 Ecotoxicology Bioassay Results 24 Species Sensitivity Distribution 24 Comparison of Toxicity 26 Microtox 27 Microalgae Growth 27 Macroalgae Zoospore Germination 27 Mollusc Larvae Development 28 Page 4 of 32
Copepod Larval Development 28 Fish Larvae Development 28 Trigger values 28
Conclusions 29
References 31
Appendix 32
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INTRODUCTION
The Southern Seawater Joint Venture Binningup desalination plant is considering using a new production chemical, Memclean R. To ensure that this new chemical does not change the toxicity of the desalination effluent discharged in to the Indian Ocean a comparative ecotoxicity study was undertaken.
This comparative ecotoxicity assessment was performed with four different desalination effluent samples described below:
1. Desalination brine – This sample is the raw brine created during the desalination process.
2. Whole effluent – This sample consists of the desalination brine with the addition of waste streams from the Clean In Place (CIP) and Backwash Sumps.
3. Whole effluent + 0.17 g/L Memclean R – The whole effluent spiked with the proposed cleaning chemical (Memclean R) at a loading rate of 0.17 g/L
4. Whole effluent + 2.0 g/L Memclean R – The whole effluent spiked with Memclean R at a loading rate of 2.0 g/L
The four-desalination brine effluent samples were supplied (including the spiked samples) to Intertek by Southern Seawater Joint Venture for toxicity testing.
The toxicity of the four desalination brine effluents was assessed using local marine test organisms representative of South Western Australian temperate waters. Chronic endpoints for five species from different trophic levels were used to assess toxicity. The chronic toxicity tests included the, microalgae T. Isochrysis galbana, the macroalgae Ecklonia radiata, the mollusc Mytilis edulis, the copepod Gladioferens imparipes and the fish Seriola lalandi. In addition, a range finding test, Microtox (the bacteria Vibrio fischeri) was used to screen toxicity prior to testing with the chronic bioassays. To minimise variability between tests, all four samples were tested in parallel using the same cohort of individuals for each bioassay.
In accordance with ANZECC/ARMCANZ (2000), results from the toxicity testing for each of the four samples were used to derive species protection trigger values for 99, 95, 90 and 80% levels of species protection for each sample. The derived chronic species protection trigger values for the brine effluent are presented as the safe dilution factors required to achieve an adequate level of species protection in the mixing zone in the marine environment.
To determine any differences in toxicity between the four samples, a pair wise comparison was used on a per bioassay basis. In addition to the pair wise comparison the resulting 99, 95, 90 and 80% trigger values for each sample is also assessed to look any potential impact on the resulting dilution factors.
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METHODOLOGY
Sample Information The desalination brine was received by Intertek Ecotoxicology on 1st August 2018 and was stored at 4°C in acid-washed polyethylene containers prior to toxicity testing.
Ecotoxicity Testing Diluent Seawater and Concentration Series
All toxicity tests were performed using filtered seawater as the diluent. Sea water was sourced from a seawater bore at the Australian Centre for Applied Aquaculture Research, Challenger Institute of Technology, Fremantle, Western Australia. Prior to use the seawater was filtered through a 0.45 µm filter, aerated to allow full saturation of dissolved oxygen and allowed to equilibrate to room temperature (~ 21°C).
For all toxicity tests, five concentrations of the simulated brine effluent were prepared using a 1:2 dilution series (v/v) with filtered seawater. The concentration series included a seawater control and brine concentrations of 6.3, 12.5, 25, 50 and 100%. The physio-chemistry of the simulated brine effluent was not adjusted prior to testing to ensure the toxicity would be representative of in situ release and exposure. Appropriate volumes of the concentration series were made prior to initiation of each bioassay which are described below.
Microtox® bacterial bioluminescence bioassay
The effect of the four samples on the Microtox bioluminescent bacteria was assessed using the standard Microtox WET test methodology. This test measures the decrease of light output from the bacterium over 15-minutes in response to contaminant exposure and is based on the methods outlined by Microbics (1992). The test protocol is summarised in Table 1.
Microtox test organism
The standard marine bacterium Vibrio fischeri was used in the Microtox bioassay. The commercially available Microtox Reagent (containing approximately 108 lyophilised V. fischeri cells) was rehydrated with 1 mL Microtox Reconstitution Solution to activate the bacteria. The bacterial suspension was stored in a cuvette at 4°C on the day of testing.
Microtox bioassay
Bioassays were performed using the Microtox standard test kit (Modern Water Inc.). The Microtox Analyser (Model 500) was set to the initial light reading of the reconstituted bacterial suspension. A 1 mL aliquot of each control, concentration and reference toxicant solution was dispensed into each cuvette in triplicate. 0.45µm filtered seawater was used to prepare the concentration series. Each replicate then received a 10 µL aliquot of the bacterial suspension at test initiation. Cuvettes were incubated at 15°C in the Microtox Analyser and the relative light output determined after the 15-minute incubation period. Inhibition of bioluminescence was reported as a percentage of the control after the 15-minute exposure interval.
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Quality assurance
A phenol reference bioassay was performed concurrently to ensure V. fischeri was responding as expected to a known toxicant. The spiked phenol concentration series consisted of a Control (0), 2.5, 5.1, 10.25, 20.5, 41 ppm treatments. Test acceptability was achieved when (i) bioluminescence in the control treatments was >90% and (ii) inhibition of cellular activity from the reference toxicant was within the expected response range.
TABLE 1: SUMMARY OF THE TEST PROTOCOL FOR THE MICROTOX BACTERIAL BIOLUMINESCENCE BIOASSAY Test Parameter Specification Organism Vibrio fischeri Test Type Static Test Duration 15-minutes Temperature 15 ± 1°C Instrument Microtox Analyser (Model 500) Test Chamber Size 3 mL Test Solution Volume 1 mL Renewal of Test Solutions None No. of Replicates 3 Age of Test Organism Lyophilised and Reconstituted (<8-hours) Shaking Rate Once initially by hand Dilution Water 0.45µm filtered seawater Osmotic Adjustment Modern Water Inc. MOAS (22% NaCl) Concentration Series Control (0), 6.3, 12.5, 25, 50, 100% Dilution Factor 1:2 Endpoint Inhibition of light output
Control bioluminescence at t15 >90% and Test Acceptability Phenol reference EC50 15.9 – 20.4 mg/L
Microalgal growth inhibition bioassay
The effect of the four samples on microalgal growth was tested using the marine dinoflagellate T. Isochrysis galbana. This test determines the inhibition of cell division over 72-hours and is based on the OECD Test Guideline 201 (1984) and the protocol of Stauber et al. (1994). The test protocol is summarized in Table 2.
Microalgal stock culture
The marine microalgae I. galbana was cultured in f2 nutrient medium (Guillard and Ryther, 1962). The in- house culture was maintained axenically on a 12-h light:12-h dark cycle at 21°C. Cells in log phase growth were used in the test. Prior to test start algal cells were centrifuged and washed three times to remove culturing medium before use in the microalgal bioassay.
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Microalgal bioassay
A 20 mL aliquot of each control, sample concentration, reference toxicant and matrix matched blank were dispensed in triplicate into 120 mL acid washed polycarbonate vials. To each vial, 0.2 mL of both 25 mM sodium nitrate and 1.6 mM potassium dihydrogen phosphate were added as nutrients. Each vial was inoculated to 3 × 105 cells/mL of the prewashed I. galbana suspension. Vials were then incubated at 21°C on a 12-h light:12-h dark photoperiod at 150 µmol photons s-1 m-2 for 72 hours. The final algal cell density was determined using an automated microplate reader. Microalgal growth inhibition was reported as a percentage of the control.
Quality assurance
A spiked copper reference bioassay was performed concurrently to ensure I. galbana was responding as expected to a known toxicant. The spiked copper concentration series consisted of a Control (0), 5, 10, 20 and 40 µg/L treatments. Test acceptability was achieved when (i) the cell division in the control treatments was greater than 3x replications (ii) microalgal growth inhibition from the reference toxicant was within the expected response range.
TABLE 2: SUMMARY OF THE TEST PROTOCOL FOR THE ISOCHRYSIS GALBANA GROWTH INHIBITION BIOASSAY Test Parameter Specification Organism Isochrysis galbana Test Type Static Test Duration 72-h Temperature 21 ± 1°C Light Quality Daylight Fluorescent Lighting Light Intensity 150 µmol photons s-1 m-2 Photoperiod 12 hour light : 12 hour dark Test Chamber Size 120 mL Test Solution Volume 20 mL Renewal of Test Solutions None Age of Test Organisms 4-6 days Initial Cell Density 3 × 104 cells/mL No. of Replicates 3 Shaking Rate Twice daily by hand Dilution Water 0.45 µm filtered seawater Concentration Series Control (0), 6.3, 12.5, 25, 50 and 100% Dilution Factor 1:2 Endpoint Algal Growth Inhibition Control >3x Cell Divisions and Test Acceptability Copper Reference EC50 within 21 – 55 µg/L
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Macroalgal germination and tube growth bioassays
The effect of the four samples on macroalgal zoospore germination and germination tube growth was tested using the marine kelp Ecklonia radiata. This test determines the inhibition of zoospore germination after 72-hours of exposure and is based on the methods described by Burridge et al. (1999) and is summarised in Table 3.
Macroalgal stock culture
Fertile blades of E. radiata were collected near Rockingham, WA. On delivery to the laboratory, the blades were rinsed with deionised water and air dried under ambient laboratory conditions. Once dried, the sori were dissected and placed into filtered seawater, cooled to 15ºc, to initiate the release of zoospores. After zoospores were released into the water column, the seawater was decanted off, the sori and the zoospore density was determined prior to use in the bioassays.
Macroalgal bioassay
A 25 mL aliquot of each control, concentration and reference toxicant were dispensed into 30 mL acid washed borosilicate glass beakers in triplicate. A microscope cover slip was placed into each beaker to allow zoospores to settle and germinate. Each beaker was then inoculated with 0.25 mL of the zoospore solution and incubated at 21°C under ambient light conditions. After 72-hours, the cover slips were removed, mounted on a slide and the proportion of germinated zoospore (indicated by the presence or absence of a germination tube) were determined on visual inspection using a microscope.
Quality assurance
A spiked copper reference bioassay was performed concurrently to ensure E. radiata was responding as expected to a known toxicant. The spiked copper concentration series consisted of a Control (0), 78, 156, 312, 625 and 1250 µg/L treatments. Test acceptability was achieved when (i) germination in the control treatments was >80% and (ii) germination inhibition from the reference toxicant was within the expected response range.
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TABLE 3: SUMMARY OF THE TEST PROTOCOL FOR THE ACUTE AND CHRONIC ECKLONIA RADIATA BIOASSAYS Test Parameter Acute Specification Organism Ecklonia radiata Test Type Static Test Duration 72-h Temperature 21 ± 1°C Light Quality Ambient illumination Test Chamber Size 30 mL Test Solution Volume 25 mL Renewal of Test None Solutions Age of Test Organisms < 1 hour No. of Replicates 3 Dilution Water 0.45 µm filtered seawater Concentration Series Control (0), 6.3, 12.5, 25, 50 and 100% Dilution Factor 1:2 Endpoint Inhibition of germination >70% germination in controls and Test Acceptability Copper Reference EC50 within 59 -225 µg/L
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Mollusc larval development bioassay
The effect of the four samples on mollusc larval development was assessed using the blue mussel Mytilis edulis. This test determines the inhibition of development over 48-hours. The methods are based on ASTM E724-98 and is summarised in Table 4.
Mussel stock culture
Adult Mussels were sourced from Cockburn Sound, Western Australia. Mussels were induced to spawn by being thermal cycled between 15°C and 30°C. Once individuals started to spawn they were isolated into individual beakers and identified as male or female by visual inspection using a microscope. After collection, eggs and sperm quality was assessed and then were mixed to allow for fertilization. After fertilization eggs were washed with filtered seawater on a 20 µm sieve to remove excess sperm. Fertilized eggs were used within 3 hours of fertilization.
Mollusc bioassay
After eggs and sperm were collected, and the eggs fertilized, a 3 mL aliquot of each control, sample concentration and reference toxicant solution was dispensed into 5 mL acid washed borosilicate glass test tubes in triplicate. Each vial was inoculated with 0.1 mL of the fertilised egg suspension to a final concentration of100eggs/mL. Vials were then incubated at 21°C under ambient light conditions. After the 48-h exposure duration, the proportion of normally developed larvae (D-veliger) were assessed and reported as a percentage of the control.
Quality assurance
A spiked copper reference bioassay was performed concurrently to ensure mussel larvae were responding as expected to a known toxicant. The spiked copper concentration series consisted of a Control (0), 10, 20, 40 and 80 µg/L treatments. Test acceptability was achieved when (i) normal development in the control treatments is >80% and (ii) inhibition of larval development from the reference toxicant was within the expected response range.
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TABLE 4: SUMMARY OF THE TEST PROTOCOL FOR THE ACUTE AND CHRONIC MYTILIS EDULIS BIOASSAYS Test Parameter Chronic Specification Organism Mytilis edulis Test Type Static Test Duration 48-h Temperature 21 ± 1°C Light Quality Ambient illumination Photoperiod 12 hour light : 12 hour dark Test Chamber Size 5 mL Test Solution Volume 3 mL Renewal of Test Solutions None Age of Test Organisms < 1 hour Density 30 eggs/mL No. of Replicates 3 Dilution Water 0.45 µm filtered seawater Concentration Series Control (0), 6.3, 12.5, 25, 50 and 100%
Dilution Factor 1:2 Endpoint Inhibition of larval development >80% development in controls and Test Acceptability Copper Reference EC50 within 4.0 – 12.9 µg/L
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Copepod Larval development Bioassay
The effect of the four samples on copepod larval development was tested using the estuarine calanoid Gladioferens imparipes. The 5-7 day chronic larval development bioassay is based on the ISO 16778 (2015) methodology and is summarised in Table 5.
Copepod stock culture
The copepod G. imparipes was cultured in-house under ambient light conditions at 21°C. Culturing water was renewed every second day with 0.45 µm filtered seawater followed by feeding with marine microalgae (I. galbana). Prior to testing (<24 hours) newly hatched nauplii were isolated from the stock cultures by sieving.
Copepod bioassays
A 100 mL aliquot of each control, concentration and reference toxicant solution was dispensed into acid washed 250 ml borosilicate glass beakers. A microalgae food source was added to final concentration of 2.5 × 104 cells/mL. To each treatment approximately 50-80 newly hatched nauplii were added and incubated at 21°C under ambient light conditions. The control seawater treatments were monitored daily until 60-80% of the copepods had reached the copepodite life stage (5-7 days after test initiation). Once the control development had reached this stage the bioassay was terminated with the addition of Lugol’s solution. The number of copepodites and nauplii was then assessed to determine the proportion development and reported as a percentage of the control.
Quality assurance
A spiked copper reference bioassay was performed concurrently to ensure G. imparipes was responding as expected to a known toxicant. The spiked copper concentration series consisted of a Control (0), 2.5, 5, 10, 20, and 40 µg/L treatments. Test acceptability was achieved when (i) copepod development in the control treatments was 60-80% and (ii) copepod developmental effects from the reference toxicant were within the expected response range.
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TABLE 5: SUMMARY OF THE TEST PROTOCOL FOR THE GLADIOFERENS IMPARIPES LARVAL DEVELOPMENT TEST Test Parameter Chronic Specification Organism Gladioferens imparipes Test Type Static Test Duration 5-7 day Temperature 21 ± 1°C Light Quality Ambient illumination Photoperiod 12 hour light : 12 hour dark Test Chamber Size 250 mL Test Solution Volume 100 ml Renewal of Test Solutions Nil Age of Test Organisms Nauplii < 24-h Organisms per Replicate Min 50 Nauplii No. of Replicates 3 replicates Feeding At initiation and day 3 Dilution Water 0.45 µm filtered seawater
Concentration Series Control (0), 6.3, 12.5, 25, 50 and 100%
Dilution Factor 1:2 Endpoint Larval development >80% survival in controls and Test Acceptability Copper Reference EC50 within 8.7 – 23.7 µg/L
Fish larvae development bioassays
The effect of the four samples on the development of fish larvae was tested using fertilised eggs of the Kingfish (Seriola lalandi). The 7-day chronic growth endpoints were based on USEPA Method 1001.0 (USEPA, 2003). Test protocols are summarised in Table 6.
Fish stock culture
The fertilised Seriola lalandi eggs were obtained from broodstock maintained in a low-density flow through system by the Australian Centre for Applied Aquaculture Research in Fremantle, WA. Broodstock were fed a varied diet and only high viability batches of fertilized eggs (>80%) were accepted for use in the bioassays.
Fish bioassays
A 400 mL aliquot of each control, sample concentration and reference toxicant solution were dispensed into acid washed 500 mL borosilicate glass beakers., 20 fertilised eggs (<24-hours old) were added to each of the three replicates per treatment. Hatched larvae were fed juvenile copepods every other day after Day 3. The test was incubated at 21°C on a 12-h light: 12-h dark photoperiod. Survival and development of the fish larvae was determined daily over 7 days. Dead fish were removed to prevent deterioration of water quality. Fish development was reported as a percentage of the control.
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Quality assurance
A spiked copper reference bioassay was performed concurrently to ensure P. auratus was responding as expected to a known toxicant. The spiked copper concentration series consisted of a Control (0), 10, 25, 50, 100 and 150 µg/L treatments. Test acceptability was achieved when (i) survival in the control treatments was >80% and (ii) fish larvae survival / growth effects from the reference toxicant were within the expected response range.
TABLE 6: SUMMARY OF THE TEST PROTOCOL FOR THE SERIOLA LALANDI LARVAL DEVELOPMENT Test Parameter Chronic Specification Organism Seriola lalandi Test Type Static Test Duration 7-day Temperature 21 ± 1°C Light Quality Ambient illumination Photoperiod 12 hour light : 12 hour dark Test Chamber Size 500 mL Test Solution Volume 400 mL Renewal of Test None Solutions Age of Test Organisms < 24-h fertilised eggs Organisms per 20 Replicate No. of Replicates 3 Feeding Every second day after day 3 Dilution Water 0.45 µm filtered seawater Concentration Series Control (0), 6.3, 12.5, 25, 50 and 100% Dilution Factor 1:2 Endpoint Survival / Development >80% survival in controls and Test Acceptability Copper Reference EC50 within 14 – 61 µg/L
Statistical Analysis
The EC10 and EC50 values (i.e. the inhibitory or effective concentration of simulated brine which gave a 10% or 50% reduction in cell division, reproduction, growth, development or survival compared to the controls) were calculated using ToxCalc Version 5.0.23 (Tidepool Software). Dunnett’s Multiple Comparison Test was used to determine which concentrations were significantly different to the controls in order to estimate the No Observable Effect Concentrations (NOEC) and Lowest Observable Effect Concentrations (LOEC).
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It should be noted that NOEC and LOEC values have been reported for historical comparisons only. The NOEC and LOEC values are not statistically robust and should not be used independently. For this reason, the reported IC10/EC10 values should be used in the place of NOEC/LOEC values.
For all bioassays, percentage data were Arcsin transformed and tested for normality and equality of variance. Logit-maximum Likelihood or Linear Interpolation analyses were used to determine the EC50 and EC10 concentrations.
Calculation of Species Protection Trigger Values
Following the protocol outlined in ANZECC/ARMCANZ (2000), species protection trigger values were calculated using the BurrliOZ statistics program (BurrliOZ v2.0) to determine the dilution factor(s) that will ensure the effluent remains below the required dilution target in the mixing zone. Species protection trigger values at 99, 95, 90 and 80% species protection levels were derived using the EC10 data from the 5 chronic bioassays (4 different trophic levels).
Previous studies for Southern Seawater Joint Venture have used the previous version of BurrliOZ (v1). The new version of BurrliOZ (v2.0) uses an updated in methodology for deriving trigger values (now referred to as guideline values) for small data sets with less than 8 data points (Warne et al. 2014). In the old BurrliOZ (v1) all species sensitivity distributions (SSDs) were calculated using a Burr type III regression, in the updated v2.0 of BurrliOZ a Burr type III regression is only used on data sets that have 8 or more data points. For data sets with 5-7 data points a Log logistic regression is used.
Due to the differences in statistical methodology used in the two versions of BurrliOZ (for small data sets) the calculated trigger values will differ between the two versions of BurrliOZ. Therefore, the species protection trigger values derived in this study are not directly comparable to those calculated in previous toxicity assessments Intertek has conducted for SSJV as they utilise different methodologies used by the two versions of BurrliOZ.
Please note that while a range of trigger values and safe dilution factors are presented, the appropriate level of species protection and statistical method must be implemented as stipulated by the governing regulatory authority.
Comparison of Toxicity To determine if there were any differences in toxicity between the four samples, a pair wise comparison on a per bioassay basis was used. Statistical difference in toxicity was determined by using the resulting EC10 and EC50 values and their corresponding 95% confidence intervals for each bioassay. Statistically significant toxicity was determined when the samples did not have overlapping 95% confidence intervals when comparing either the EC10 and EC50 values.
In addition to the bioassay-based pair wise comparison, the resulting 99, 95, 90 and 80% trigger values for each sample were also compared to assess any impacts on the resulting dilution factors.
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RESULTS
Desalination Brine All raw data and statistical calculations for all bioassay are detailed in the appendix. A synopsis of the results is detailed below.
Physicochemistry
The physicochemistry of the desalination brine dilution series is detailed in Table 7.
TABLE 7: DESALINATION BRINE PHYSICOCHEMISTRY PARAMETER CONTROL 6.3% 12.5% 25% 50% 100% pH 7.95 7.98 7.95 8.05 7.96 8.26 Salinity (‰) 34.4 36.9 38.4 41.9 53^ 68^ DO (%) 100 100 100 100 100 100 ^ Refractometer reading - to be used for indicative purposes only
Ecotoxicology Bioassay Results
A summary of the statistical effects data for each bioassay is detailed in Table 8.
TABLE 8: SUMMARY OF CHRONIC STATISTICAL EFFECTS DATA OF THE DESALINATION BRINE
BIOASSAY EC10 (%) EC50 (%) NOEC (%) LOEC (%) Microtox* 29.8 93.3 50 100 Microalgae Growth* 46.4 >100 25 50 Macroalgae Zoospore Germination * 59.1 86.5 25 50 Mollusc Larvae Development* 11.5 15.1 <6.3 6.3 Copepod Larval Development 16.3 32.0 12.5 25 Fish Larvae Development* 25.7 41.9 25 50 * NATA Accredited method – Accreditation Number 5646
Species Sensitivity Distribution
Species protection trigger values have been calculated to determine the dilution factor which will ensure the desalination brine remains below the required level of species protection in the mixing zone. For example, dilution factors calculated for the 99% species protection trigger value based on the EC10 biological effects data will theoretically result in 1% of the exposed species showing a 10% reduction in chronic effects if the dilution target is exceeded.
The dilution factors calculated to achieve 99, 95, 90 and 80% species protection was determined to be 21.7, 11.6, 9.1, and 6.3 respectively (Figure 1, Table 9).
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FIGURE 1: SPECIES SENSITIVITY DISTRIBUTION FOR THE DESALINATION BRINE
TABLE 9: CALCULATED TRIGGER VALUES AND DILUTION FACTORS FOR 80, 90, 95, AND 99% SPECIES PROTECTION FOR THE DESALINATION BRINE LEVEL OF SPECIES PROTECTION TRIGGER VALUE (% SAMPLE) DILUTION FACTOR REQUIRED 99% 4.6 (2.8 – 23) 21.7 95% 8.6 (5.5 – 29) 11.6 90% 11 (7.4 – 33) 9.1 80% 16 (10 – 38) 6.3
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Whole Effluent All raw data and statistical calculations for all bioassay are detailed in the appendix. A synopsis of the results is detailed below.
Physicochemistry
The physicochemistry of the whole effluent dilution series is detailed in Table 10.
TABLE 10: WHOLE EFFLUENT PHYSICOCHEMISTRY PARAMETER CONTROL 6.3% 12.5% 25% 50% 100% pH 7.95 7.86 7.99 8.07 8.00 8.21 Salinity (‰) 34.4 36.5 37.7 40.7 48^ 62^ 86 DO (%) 100 100 100 100 100 100 ^ Refractometer reading - to be used for indicative purposes only
Ecotoxicology Bioassay Results
A summary of the statistical effects data for each bioassay is detailed in Table 11.
TABLE 11: SUMMARY OF CHRONIC STATISTICAL EFFECTS DATA OF THE WHOLE EFFLUENT BIOASSAY EC10 (%) EC50 (%) NOEC (%) LOEC (%) Microtox* 16.1 >100 12.5 25 Microalgae Growth* 73.9 >100 50 100 Macroalgae Zoospore Germination * 60.8 84.3 50 100 Mollusc Larvae Development* 11.6 16.3 <6.3 6.3 Copepod Larval Development 13.1 25.4 12.5 25 Fish Larvae Development* 43.9 55.4 25 50 * NATA Accredited method – Accreditation Number 5646
Species Sensitivity Distribution
Species protection trigger values have been calculated to determine the dilution factor which will ensure the whole effluent remains below the required level of species protection in the mixing zone. For example, dilution factors calculated for the 99% species protection trigger value based on the EC10 biological effects data will theoretically result in 1% of the exposed species showing a 10% reduction in chronic effects if the dilution target is exceeded.
The dilution factors calculated to achieve 99, 95, 90 and 80% species protection was determined to be 29.4, 13.0, 9.1, and 5.9 respectively (Figure 2, Table 12 )
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FIGURE 2: SPECIES SENSITIVITY DISTRIBUTION FOR THE WHOLE EFFLUENT
TABLE 12: CALCULATED TRIGGER VALUES AND DILUTION FACTORS FOR 80, 90, 95, AND 99% SPECIES PROTECTION FOR THE WHOLE EFFLUENT LEVEL OF SPECIES PROTECTION TRIGGER VALUE (% SAMPLE) DILUTION FACTOR REQUIRED 99% 3.4 (1.9 – 39) 29.4 95% 7.7 (4.6 – 48) 13.0 90% 11 (6.7 – 51) 9.1 80% 17 (9 – 54) 5.9
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Whole Effluent + 0.17 g/L Memclean R All raw data and statistical calculations for all bioassay are detailed in the appendix. A synopsis of the results is detailed below.
Physicochemistry
The physicochemistry of the whole effluent + 0.17 g/L Memclean R concentration series is detailed in Table 13.
TABLE 13: WHOLE EFFLUENT + 0.17 G/L MEMCLEAN R PHYSICOCHEMISTRY PARAMETER CONTROL 6.3% 12.5% 25% 50% 100% pH 7.95 8.07 7.92 7.88 7.86 8.21 Salinity (‰) 34.4 36.4 37.7 40.4 49^ 63^ 86 DO (%) 100 100 100 100 100 100 ^ Refractometer reading - to be used for indicative purposes only
Ecotoxicology Bioassay Results
A summary of the statistical effects data for each bioassay is detailed in Table 14.
TABLE 14: SUMMARY OF CHRONIC STATISTICAL EFFECTS DATA OF THE WHOLE EFFLUENT + 0.17 G/L MEMCLEAN R BIOASSAY EC10 (%) EC50 (%) NOEC (%) LOEC (%) Microtox* 51.6 >100 50 100 Microalgae Growth* 85.7 >100 50 100 Macroalgae Zoospore Germination * 53.5 75.4 25 50 Mollusc Larvae Development* 11.6 15.8 <6.3 6.3 Copepod Larval Development 18.1 27.0 12.5 25 Fish Larvae Development* 30.7 44 25 50 * NATA Accredited method – Accreditation Number 5646
Species Sensitivity Distribution
Species protection trigger values have been calculated to determine the dilution factor which will ensure the whole effluent + 0.17 g/L Memclean R remains below the required level of species protection in the mixing zone. For example, dilution factors calculated for the 99% species protection trigger value based on the EC10 biological effects data will theoretically result in 1% of the exposed species showing a 10% reduction in chronic effects if the dilution target is exceeded.
The dilution factors calculated to achieve 99, 95, 90 and 80% species protection was determined to be 24.4, 11.9, 8.3, and 5.9 respectively (Figure 3, Table 15).
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FIGURE 3: SPECIES SENSITIVITY DISTRIBUTION FOR THE WHOLE EFFLUENT + 0.17 G/L Memclean R
TABLE 15: CALCULATED TRIGGER VALUES AND DILUTION FACTORS FOR 80, 90, 95, AND 99% SPECIES PROTECTION FOR THE WHOLE EFFLUENT + 0.17 G/L MEMCLEAN R LEVEL OF SPECIES PROTECTION TRIGGER VALUE (% SAMPLE) DILUTION FACTOR REQUIRED 99% 4.1 (2.3 – 23) 24.4 95% 8.4 (4.7 – 34) 11.9 90% 12 (6.7 – 40) 8.3 80% 17 (9.8 – 48) 5.9
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Whole Effluent + 2.0 g/L Memclean R All raw data and statistical calculations for all bioassay are detailed in the appendix. A synopsis of the results is detailed below.
Physicochemistry
The physicochemistry of the whole effluent + 2.0 g/L Memclean R concentration series is detailed in Table 16.
TABLE 16: WHOLE EFFLUENT + 2.0 G/L MEMCLEAN R PHYSICOCHEMISTRY PARAMETER CONTROL 6.3% 12.5% 25% 50% 100% pH 7.95 8.07 7.92 7.88 7.86 8.21 Salinity (‰) 34.4 36.4 37.7 40.4 49^ 63^ 86 DO (%) 100 100 100 100 100 100 ^ Refractometer reading - to be used for indicative purposes only
Ecotoxicology Bioassay Results
A summary of the statistical effects data for each bioassay is detailed in Table 17: SUMMARY of chronic statistical effects data of the Whole EFFLUENT + 2.0 g/L Memclean R
TABLE 17: SUMMARY OF CHRONIC STATISTICAL EFFECTS DATA OF THE WHOLE EFFLUENT + 2.0 G/L MEMCLEAN R BIOASSAY EC10 (%) EC50 (%) NOEC (%) LOEC (%) Microtox* 13.0 15.7 12.5 25 Microalgae Growth* 7.0 8.2 6.3 12.5 Macroalgae Zoospore Germination * 10.1 13.2 6.3 12.5 Mollusc Larvae Development* 1.7 2.4 0.8 1.6 Copepod Larval Development 14.6 16.9 12.5 25 Fish Larvae Development* 11.0 14.3 6.3 12.5 * NATA Accredited method – Accreditation Number 5646
Species Sensitivity Distribution
Species protection trigger values have been calculated to determine the dilution factor which will ensure the whole effluent + 2.0 g/L Memclean R remains below the required level of species protection in the mixing zone. For example, dilution factors calculated for the 99% species protection trigger value based on the EC10 biological effects data will theoretically result in 1% of the exposed species showing a 10% reduction in chronic effects if the dilution target is exceeded.
The dilution factors calculated to achieve 99, 95, 90 and 80% species protection was determined to be 76.9, 40, 29.4, and 21.3 respectively (Figure 4, Table 18).
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FIGURE 4: SPECIES SENSITIVITY DISTRIBUTION FOR THE WHOLE EFFLUENT + 2.0 G/L Memclean R
TABLE 18: CALCULATED TRIGGER VALUES AND DILUTION FACTORS FOR 80, 90, 95, AND 99% SPECIES PROTECTION FOR THE WHOLE EFFLUENT + 2.0 G/L MEMCLEAN R LEVEL OF SPECIES PROTECTION TRIGGER VALUE (% SAMPLE) DILUTION FACTOR REQUIRED 99% 1.3 (0.24 – 8.1) 76.9 95% 2.5 (0.61 – 9.2) 40 90% 3.4 (0.92 – 9.7) 29.4 80% 4.7 (1.5 – 10) 21.3
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Comparison of Toxicity The comparison of the toxicity of the four samples was conducted by a pair wise comparison on a per bioassay basis using the EC10 and EC50 values and their corresponding 95% confidence intervals. The EC50 will be the primary basis of the comparison as the EC50 (the median value for toxicity) generally has the narrowest 95% confidence intervals. A comparison of the EC10 has been included as the EC10 are the values that are used in the species sensitivity distribution and thus have an impact on the calculated trigger values.
The results of all bioassays EC10 and EC50 values and corresponding 95% confidence intervals are presented in Table 19 and
Table 20. Table 21 details the comparison of the trigger values for the four samples. Individual comparisons for each bioassay and trigger values are detailed in the sections below.
TABLE 19: COMPARISON OF THE EC10 VALUES AND 95% CONFIDENCE INTERVALS (IN PARENTHESES) FOR THE FOUR SAMPLES TESTED WHOLE EFFLUENT WHOLE EFFLUENT DESALINATION BIOASSAY WHOLE EFFLUENT + 0.17 G/L + 2.0 G/L BRINE MEMCLEAN R MEMCLEAN R Microtox 29.8 (NC) 16.1 (NC) 51.6 (NC) 13.0 (10.0 - 11.2) Microalgae Growth 46.4 (NC) 73.9 (NC) 85.7 (NC) 7.0 (NC) Macroalgae Zoospore Germination 59.1 (48.7 - 66.2) 60.8 (57.6 - 63.6) 53.5 (46.3 - 59.2) 10.1 (9.7 - 10.4) Mollusc Larvae Development 11.5 (9.9 - 12.7) 11.6 (9.9 - 12.9) 11.6 (10.2 -12.6) 1.7 (1.6 - 1.7) Copepod Larval Development 16.3 (10.2 - 21.0) 13.1 (12.3 - 13.6) 18.1 (17.4 - 18.9) 14.6 (NC) Fish Larvae Development 25.7 (16.7 - 31.8) 43.9 (34.5 - 47.2) 30.7 (27.6 - 33.1) 11.0 (9.5 - 10.7) NC - Not able to be calculated
TABLE 20: COMPARISON OF THE EC50 VALUES AND 95% CONFIDENCE INTERVALS (IN PARENTHESES) FOR THE FOUR SAMPLES TESTED WHOLE EFFLUENT WHOLE EFFLUENT DESALINATION BIOASSAY WHOLE EFFLUENT + 0.17 G/L + 2.0 G/L BRINE MEMCLEAN R MEMCLEAN R Microtox 93.3 (NC) >100 (NC) >100 (NC) 15.7 (15.1 - 16.3) Microalgae Growth >100 (NC) >100 (NC) >100 (NC) 8.2 (NC) Macroalgae Zoospore Germination 86.5 (80.6 - 91.6) 84.3 (82.3 - 86.2) 75.4 (69.7 - 81.0) 13.2 (13.0 - 13.4) Mollusc Larvae Development 15.1 (13.7 - 17.0) 16.3 (14.5 - 17.8) 15.8 (14.6 -17.1) 2.4 (2.4 - 2.5) Copepod Larval Development 32.0 (26.2 - 37.9) 25.4 (24.6 - 26.2) 27.0 (26.4 -27.6) 16.9 (NC) Fish Larvae Development 41.9 (34.6 - 48.8) 55.4 (52.1 - 65.2) 44.0 (42.0 - 45.9) 14.3 (13.8 - 15.0) NC - Not able to be calculated
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TABLE 21: COMPARISON OF THE SPECIES PROTECTION TRIGGER VALUES AND 95% CONFIDENCE INTERVALS (IN PARENTHESES) FOR THE FOUR SAMPLES TESTED WHOLE EFFLUENT + WHOLE EFFLUENT + 2.0 LEVEL OF SPECIES DESALINATION BRINE WHOLE EFFLUENT (%) 0.17 G/L G/L PROTECTION (%) MEMCLEAN R (%) MEMCLEAN R (%) 99% 4.6 (2.8 – 23) 3.4 (1.9 – 39) 4.1 (2.3 – 23) 1.3 (0.24 – 8.1) 95% 8.6 (5.5 – 29) 7.7 (4.6 – 48) 8.4 (4.7 – 34) 2.5 (0.61 – 9.2) 90% 11 (7.4 – 33) 11 (6.7 – 51) 12 (6.7 – 40) 3.4 (0.92 – 9.7) 80% 16 (10 – 38) 17 (9 – 54) 17 (9.8 – 48) 4.7 (1.5 – 10)
Microtox
Microtox is predominantly used as a screening test to set the concentration series for the other bioassays and is not used to calculate trigger values. However, comparing the results is still useful. There was very minimal toxicity observed to Microtox and no statically significant toxicity between the desalination brine, whole effluent and whole effluent + 0.17 g/L Memclean R when comparing the EC50s. The whole effluent + 2.0 g/L Memclean R sample showed increased toxicity that was statically significant compared to the other three samples. When comparing the EC10 values for Microtox there is more variation in the results and this is likely due to the variation of the Microtox test and highlight the instability of Microtox for use in species sensitivity distribution.
Microalgae Growth
There was very minimal toxicity observed to the microalgal growth bioassay for the desalination brine, whole effluent and whole effluent + 0.17 g/L Memclean R samples. EC50 Values for these three bioassays was >100 and no statically significant toxicity between them. Comparing these three samples EC10 values show some slight increased toxicity in the desalination brine (note 95% CI were not able to be calculated due to incomplete dose response curves). This is likely due to the slightly higher salinity of the desalination brine compared to the whole effluent and whole effluent + 0.17 g/L Memclean R samples.
The whole effluent + 2.0 g/L Memclean R sample showed increased toxicity compared to the other three samples at both the EC10 and EC50.
Macroalgae Zoospore Germination
The macroalgae zoospore germination test showed a similar trend with the desalination brine, whole effluent and whole effluent + 0.17 g/L Memclean R samples sowing similar levels of toxicity. Comparing the EC50 values and 95% confidence intervals show some slight significance in toxicity between the whole effluent and whole effluent + 0.17 g/L Memclean R samples. There was no significance in toxicity between these two samples and the desalination brine sample. The EC10 values for these three samples show no significance difference in toxicity. Despite the slight differences in toxicity at the EC50 it is very minimal.
As with the previous bioassays, the whole effluent + 2.0 g/L Memclean R sample showed increased toxicity compared to the other three samples at both the EC10 and EC50.
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Mollusc Larvae Development
The mollusc larval development test was the most sensitive bioassay to the four samples tested. There was no statistically significant toxicity between the desalination brine, whole effluent, and whole effluent + 0.17 g/L Memclean R samples at both the EC10 and EC50.
As with the previous bioassays the whole effluent + 2.0 g/L Memclean R sample showed increased toxicity compared to the other three samples at both the EC10 and EC50.
Copepod Larval Development
The copepod larval development test was the second most sensitive test to the four samples tested. There was no statistically significant toxicity between the desalination brine, whole effluent, and whole effluent + 0.17 g/L Memclean R samples at both the EC10 and EC50.
As with the previous bioassays the whole effluent + 2.0 g/L Memclean R sample showed increased toxicity compared to the other three samples at both the EC10 and EC50.
Fish Larvae Development
Comparing the desalination brine, whole effluent, and whole effluent + 0.17 g/L Memclean R samples for the fish larval development bioassay showed some differences in toxicity at the EC50 and EC10. The desalination brine and whole effluent + 0.17 g/L Memclean R samples showed no significant differences in toxicity. However, when compared to the whole effluent sample both samples were slightly more toxic. Though only slightly more toxic, it is not known if this is due to intra-test variability or due to a slight difference in toxicity.
As with the previous bioassays the whole effluent + 2.0 g/L Memclean R sample showed increased toxicity compared to the other three samples at both the EC10 and EC50.
Trigger values
As the species sensitivity distribution are an integration of all the EC10s, any differences in sensitivity between the four samples will be highlighted in the resulting trigger values. Therefore, comparing the trigger values, especially the 99% species protection trigger value is useful. However, it is noted that given the small number data points (only 5), the regression analysis is not considered to be “high reliability” (Waren et al. 2015) and is prone to a poor the fit of the SSD to the toxicity data and large 95% confidence intervals.
Comparison of the trigger values for the four samples show an expected trend based on the comparison of the individual bioassay results. The desalination brine, whole effluent, and whole effluent + 0.17 g/L Memclean R samples show similar 99% species protection trigger values of 4.6, 3.4, 4.1% respectively and the whole effluent + 2.0 g/L Memclean R sample was significantly more toxic with a 99% species protection trigger value of 1.3%.
The whole effluent sample shows a slightly lower 99% species protection trigger value of 3.4% compared to the desalination brine and whole effluent + 0.17 g/L Memclean R samples trigger values of 4.6% and 4.1% respectively. Comparing the 95% confidence intervals show a much wider range for the whole effluent sample compared to the other two samples. Based on a visual assessment of the fit of the SSDs to the toxicity data, the fit of the SSD for the whole effluent sample is considered poor fit whereas the fit of the SSDs for the desalination brine and whole effluent + 0.17 g/L Memclean R samples are considered to be a good fit. This poor fit explains the larger 95% confidence intervals and the difference in the trigger
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values. This is likely due to the slight variation in the bioassay response compared to the other two samples (mainly the copepod and fish bioassays) which is enough to changing the fit the regression. This ultimately impacted the trigger values for this sample and with small data sets such as these, it is not uncommon.
CONCLUSIONS
Four-desalination samples were supplied to Intertek by Southern Seawater Joint Venture for comparative toxicity testing. The four different desalination effluent samples tested are described below:
1. Desalination brine – This sample is the raw brine created during the desalination process.
2. Whole effluent – This sample consists of the desalination brine with the addition of waste streams from the Clean In Place (CIP) and Backwash Sumps.
3. Whole effluent + 0.17 g/L Memclean R – The whole effluent spiked with the proposed cleaning chemical (Memclean R) at a loading rate of 0.17 g/L
4. Whole effluent + 2.0 g/L Memclean R – The whole effluent spiked with Memclean R at a loading rate of 2.0 g/L
The toxicity of the four desalination brine effluents was assessed using local marine test organisms representative of South Western Australian temperate waters. Chronic endpoints for five species from different trophic levels were used to assess toxicity. The chronic toxicity tests included the, microalgae T. Isochrysis galbana, the macroalgae Ecklonia radiata, the mollusc Mytilis edulis, the copepod Gladioferens imparipes and the fish Seriola lalandi. To minimise variability between tests, all samples were tested in parallel.
All the bioassays tested showed a toxic response to the four samples. The least sensitive tests were Microtox and the microalgal growth bioassay. The most sensitive test was the mollusc larval development test. All of the four samples show the same pattern of the most and least sensitive bioassays.
Comparing the four samples on a per bioassay basis and based on the trigger values show that the whole effluent + 2.0 g/L Memclean R was the most toxic sample, showing a statistically significant higher toxicity than the other three samples for all bioassays. As expected all of the species protection trigger values were lower than the others three samples. The desalination brine, whole effluent, and whole effluent + 0.17 g/L Memclean R samples showed very little difference in toxicity. However, there were some differences seen in the fish larval development bioassay and the macroalgal zoospore germination bioassay. These differences did not show a consistent pattern with one sample always slightly more toxic and the two most sensitive bioassays, the mollusc and copepod bioassays, did not show any difference in toxicity. Therefore, the slight differences in toxicity between these three samples is likely due to intra-test variability and not true indications of increased toxicity. When considering the comparisons based on the trigger values, these three samples were similar and the differences in trigger values (for the whole effluent sample) is more a product of the poor SSD fit than a true difference in toxicity as discussed previously.
The objective of this assessment was to determine if two different loading ratios of the process chemical Memclean R increased the toxicity of the Southern Seawater Joint Venture desalination plant’s effluent. In addition to the assessment of Memclean R, a comparison of the desalination brine and the final whole effluent was assessed. This was to investigate whether the other waste streams that are added to the desalination brine prior to discharge have an impact on toxicity.
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Using the desalination brine sample to establish a baseline toxicity level the other samples were then compared to see if there was any difference in toxicity. The results of this study show that the addition of waste streams from the clean in place (CIP) and backwash sumps (creating the whole effluent sample) do not have a significant impact on toxicity.
To assess if Memclean R clean increases the toxicity of the whole effluent, two pair wise comparisons were made between the whole effluent sample and the two samples spiked with Memclean R. The results of this show that at 0.17 g/L, Memclean R does not increase the toxicity of the effluent. However, at 2.0g/L, Memclean R does significantly increase the toxicity of the effluent.
In conclusion, the use of Memclean R at 0.17 g/L will not change the toxicity of the whole effluent discharged. However, with the increased toxicity seen at 2.0 g/L it is unknown at what loading ratio Memclean R will start to increase the toxicity of the whole effluent. It is therefore recommended that if concentrations of Memclean R are expected to be greater than 0.17 g/L further testing should be conducted to ensure no increase in toxicity.
Page 30 of 32
REFERENCES
ANZECC/ARMCANZ (2000). Australian and New Zealand guidelines for fresh and marine water quality. Australian and New Zealand Environment and Conservation Council/ Agriculture and Resource Management Council of Australia and New Zealand, Canberra, ACT.
ASTM E724-98 Standard (2004). ‘Standard Guide for Conducting Static Acute Toxicity Tests Starting with Embryos of Four Species of Saltwater Bivalve Molluscs’. ASTM International, West Conshohocken, PA, 2004, DOI: 10.1520/E0724-98R04.
Burridge, T.R., M. Karistianos and J.R. Bidwell (1999). The use of aquatic macrophyte ecotoxicological assays in monitoring coastal effluent discharges in Southern Australia. Marine Pollution Bulletin, 39:89-96.
Campbell, E., Palmer, M.J., Shao, Q., Warne, M. StJ. and Wilson, D. (2000). BurrliOZ: A computer program for calculating toxicant trigger values for the ANZECC and ARMCANZ water quality guidelines. In: ANZECC and ARMCANZ (2000) Australian and New Zealand Guidelines for Fresh and Marine Water Quality, Australian and New Zealand Environment and Conservation Council and Agriculture and Resource Management Council of Australia and New Zealand, Canberra.
Guillard, R.R.L. and Ryther, J.H. (1962). Studies of marine planktonic diatoms. I. Cyclotella nana and Detonula confervaceae Hustedt (Cleve) Gran. Canadian Journal of Microbiology, 8, 229-339.
OECD (2005) OECD Draft guidelines for testing of chemicals. Proposal for a new guideline. Calanoid Copepod development and reproduction test with Acartia tonsa. Organisation for Economic Corperation and Development, Paris, France
OECD (1984). Guidelines for testing of chemicals. Algal growth inhibition test. Test Guideline No. 201. Organisation for Economic Cooperation and Development.
Simon, J. and Laginestra, E. (1997) Bioassay for testing sublethal toxicity in effluents using gametes of the sea urchin Heliocidaris tuberculata. National Pulp Mills Research Program Technical Report Series No. 20, Canberra: CSIRO, pp. 13-18.
Southern Seawater Alliance (2010) Whole effluent toxicity monitoring Program. Doc no: 00-EN-PRO-0004, Revision: C, 15 December 2010
Stauber, J.L., Tsai, J., Vaughan, G., Peterson, S.M. and Brockbank, C.I. (1994). Algae as indicators of toxicity of BEKM effluents. National Pulp Mills Research Program Technical Report Series No. 3, Canberra: CSIRO, pp. 1-82.
Warne, M., (2001) Derivation of the ANZECC and ARMCANZ Water Quality Guidelines for Toxicants. Australasian Journal of Ecotoxicology, 7, 123−136
Warne, M., Batley, G., van Dam, R., Chapman, J., Fox, DR., Hickey, C., & Stauber, J. (2015). Revised Method for Deriving Australian and New Zealand Water Quality Guideline Values for Toxicants.
USEPA (2003). Short-term methods for assessing the chronic toxicity of effluents and receiving water to marine and estuarine organisms. US Environmental Protection Authority, Washington DC, USA
Page 31 of 32
APPENDIX
Comparative toxicity assessment of a chemical additive to desalination effluent Report No: ECX18-0801 32 of 32
ECOTOXICOLOGY LABORATORY TEST REPORT
CLIENT ATTN: GRANT GRIFFITH SOUTHERN SEAWATER JOINT VENTURE LOT 32 TARANTO ROAD BINNINGUP, WA 6233
JOB INFORMATION JOB REFERENCE : ECX18-0801-2 NO. SAMPLES : 1 CLIENT ORDER NO. : PO #94350 PROJECT : Comparative toxicity assessment SAMPLED BY : S. McNeil DATE RECEIVED : 01/08/18 DATE REPORTED : 18/10/2018
REPORT NOTES Comparative toxicity assessment of a four desalination effluent samples
TESTED BY COMPANY APPROVED SIGNATORY
Intertek Ecotoxicology Laboratory Intertek Geotech 41-45 Furnace Road Welshpool, Western Australia 6106
Tel: +61 8 9263 0263 DR TRISTAN STRINGER [email protected] PRINCIPAL ECOTOXICOLOGIST
This report relates specifically to the sample(s) tested that were drawn and/or provided by the client or their nominated third party to Intertek. The reported result(s) provide no warranty or verification on the sample(s) representing any specific goods and/or shipment. This report was prepared solely for the use of the client named in this report. Intertek accepts no responsibility for any loss, damage or liability suffered by a third party as a result of any reliance upon or use of this report.
Except where explicitly agreed in writing, all work and services performed by Intertek is subject to our standard Terms and Conditions which can be obtained at our website: intertek.com/terms
Page 1 of 10 Report Ref: ECX18-0801-2
Sample Information SAMPLE REFERENCE INTERTEK REFERENCE Desalination Brine ECX18-0801-2
Bioassay Details BIOASSAY PROTOCOL REFERENCE TEST SPECIES TEMPERATURE Microtox* WIECX-17 Microbics 1992 V. fischeri 15°C Microalgae Growth* WIECX-06 Stauber et al. 1994 I. galbana 22°C Macroalgae Zoospore Germination * WIECX-08 Burridge et al. 1999 E. radiata 22°C Mollusc Larvae Development* WIECX-23 ASTM E724 M. edulis 22°C Copepod Larval Development WIECX-26 ISO 16778 G. imparipes 22°C Fish Larvae Development* WIECX-16 USEPA 1004.0 S. lalandi 22°C * NATA Accredited method – Accreditation Number 5646
Physicochemistry
PARAMETER CONTROL 6.3% 12.5% 25% 50% 100% pH 7.95 7.98 7.95 8.05 7.96 8.26 Salinity (‰) 34.4 36.9 38.4 41.9 53 68 DO (%) 100 100 100 100 100 100 ^ Refractometer reading - to be used for indicative purposes only
Concentration-Response Data
BIOASSAY (% CONTROL) CONTROL 6.3% 12.5% 25% 50% 100% Microtox* 100 ± 17 98 ± 20 94 ± 4 100 ± 4 92 ± 2 48 ± 5 Microalgae Growth* 100 ± 2 100 ± 1 96 ± 6 99 ± 5 88 ± 5 59 ± 10 Macroalgae Zoospore Germination * 100 ± 1 98 ± 6 98 ± 1 95 ± 6 93 ± 3 29 ± 6 Mollusc Larvae Development* 100 ± 2 86 ± 3 76 ± 1 1 ± 0 0 ± 0 0 ± 0 Copepod Larval Development 100 ± 14 114 ± 12 104 ± 4 69 ± 12 21 ± 8 0 ± 0 Fish Larvae Development* 100 ± 6 88 ± 16 96 ± 3 82 ± 6 35 ± 8 0 ± 0 * NATA Accredited method – Accreditation Number 5646
Page 2 of 10 Report Ref: ECX18-0801-2
Statistical Effects Data
BIOASSAY EC10 (%) EC50 (%) NOEC (%) LOEC (%) Microtox* 29.8 93.3 50 100 Microalgae Growth* 46.4 >100 25 50 Macroalgae Zoospore Germination * 59.1 56.5 25 50 Mollusc Larvae Development* 11.5 15.1 <6.3 6.3 Copepod Larval Development 16.3 32.0 12.5 25 Fish Larvae Development* 25.7 41.9 25 50 * NATA Accredited method – Accreditation Number 5646
Quality Assurance Limits BIOASSAY REFERENCE OBSERVED CUSUM CHART COEFFICIENT OF CONTROL TEST TOXICANT EC50 LIMITS VARIANCE RESPONSE ACCEPTABILITY Microtox* Phenol 17.0 mg/L 15.9 – 20.4 mg/L 6.32% < 90% PASS Microalgae Growth* > 3 x cell Copper 46.2 µg/L 21.9 – 55 µg/L 24.2% PASS divisions Macroalgae Zoospore >80% Copper 143 µg/L 59 – 225 µg/L 33.2% PASS Germination * Germinated Mollusc Larvae >80% Copper 5.9 µg/L 4.0 – 12.9 µg/L 30.6% PASS Development* Development 50-80% Copepod Larval Copper 23.2 µg/L 8.6 – 23.7 µg/L 26.2% copepodite PASS Development life stage Fish Larvae >80% Copper 45.2 µg/L 21.6 – 82.9 µg/L 33.1% PASS Development* Development * NATA Accredited method – Accreditation Number 5646
General Comments Quality assurance and quality control criteria were within acceptable limits for all bioassays. A detailed interpretation of the results and species sensitivity distribution is included in the interpretative report (Report No. ECX18-0801)
Page 3 of 10 Report Ref: ECX18-0801-2
STATISTICAL DATA
Page 4 of 10 Report Ref: ECX18-0801-2
MICROTOX Abalone Larval Development Test-Proportion Normal Start Date: Test ID: ECX18-0801 Sample ID: ECX0801-2 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Brine Sample Date: Protocol: WIECX-17 Test Species: Microtox Comments: Conc-% 1 2 3 B-Control 1.0000 1.0000 0.8287 6.3 1.0000 0.7300 0.8000 12.5 0.8100 0.8700 25 0.8600 0.8800 0.9300 50 0.8300 0.8000 0.8300 100 0.4200 0.4700 0.3900
Transform: Arcsin Square Root 1-Tailed Isotonic Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Mean N-Mean B-Control 0.9429 1.0000 1.3952 1.1440 1.5208 15.590 3 0.9433 1.0000 6.3 0.8433 0.8944 1.2174 1.0244 1.5208 21.844 3 1.441 2.718 0.3352 0.8578 0.9093 12.5 0.8400 0.8909 1.1609 1.1198 1.2019 5.005 2 1.699 2.718 0.3748 0.8578 0.9093 25 0.8900 0.9439 1.2358 1.1873 1.3030 4.863 3 1.292 2.718 0.3352 0.8578 0.9093 50 0.8200 0.8697 1.1329 1.1071 1.1458 1.970 3 2.127 2.718 0.3352 0.8200 0.8693 *100 0.4267 0.4525 0.7116 0.6745 0.7554 5.740 3 5.542 2.718 0.3352 0.4267 0.4523
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.94964 0.851 0.27744 1.71539 Bartlett's Test indicates equal variances (p = 0.03) 12.0241 15.0863 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Bonferroni t Test 50 100 70.7107 2 0.20851 0.21507 0.15847 0.02282 0.0037 5, 11
Log-Logit Interpolation (200 Resamples) Point % SD 95% CL(Exp) Skew IC05* 2.619 11.275 0.799 81.264 2.3166 IC10 29.771 18.718 0.000 81.235 0.6009 IC15 52.332 17.974 0.000 67.943 -1.0194 1.0 IC20 58.141 4.250 43.552 72.708 0.4049 0.9 IC25 63.753 3.939 49.654 77.578 0.4296 IC40 80.740 3.635 67.819 95.508 0.4115 0.8 IC50 93.300 * indicates IC estimate less than the lowest concentration 0.7 0.6
0.5
0.4 Response 0.3
0.2
0.1
0.0 1 10 100 Dose % Dose-Response Plot
1
0.9
0.8 1-tail, 0.05 level 0.7 of significance
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ProportionNormal 0.3
0.2
0.1
0
25 50
6.3
12.5 *100
B-Control
Page 5 of 10 Report Ref: ECX18-0801-2
MICROALGAE GROWTH -Proportion Normal Start Date: Test ID: ECX18-0801 Sample ID: ECX0801-2 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Brine Sample Date: Protocol: WIECX-06 Test Species: TISO-Isochrysis sp. Comments: Conc-% 1 2 3 B-Control 1.0000 1.0000 0.9850 6.3 0.9901 1.0000 1.0000 12.5 0.8974 0.9934 1.0000 25 1.0000 0.9503 1.0000 50 0.8510 0.8477 0.9404 100 0.5132 0.5497 0.7020
Transform: Arcsin Square Root 1-Tailed Isotonic Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Mean N-Mean B-Control 0.9950 1.0000 1.4966 1.4481 1.5208 2.803 3 0.9967 1.0000 6.3 0.9967 1.0017 1.5042 1.4710 1.5208 1.912 3 -0.098 2.500 0.1937 0.9967 1.0000 12.5 0.9636 0.9684 1.4183 1.2447 1.5208 10.658 3 1.011 2.500 0.1937 0.9733 0.9766 25 0.9834 0.9884 1.4625 1.3460 1.5208 6.898 3 0.439 2.500 0.1937 0.9733 0.9766 *50 0.8797 0.8841 1.2228 1.1699 1.3242 7.179 3 3.533 2.500 0.1937 0.8800 0.8829 *100 0.5883 0.5913 0.8757 0.7986 0.9933 11.817 3 8.013 2.500 0.1937 0.5867 0.5886
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.96062 0.858 -0.4194 -0.2784 Bartlett's Test indicates equal variances (p = 0.42) 4.93778 15.0863 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 25 50 35.3553 4 0.06459 0.06495 0.18078 0.009 1.9E-05 5, 12
Log-Logit Interpolation (200 Resamples) Point % SD 95% CL(Exp) Skew IC05 34.140 7.196 0.000 60.070 -1.1051 IC10 46.449 5.026 31.561 74.098 0.7116 IC15 56.299 5.096 42.912 80.030 0.4486 1.0 IC20 65.084 4.859 51.247 91.598 0.4511 0.9 IC25 73.385 5.082 58.169 99.509 0.5107 IC40 98.047 0.8 IC50 >100 0.7
0.6
0.5
0.4 Response 0.3
0.2
0.1
0.0 1 10 100 Dose % Dose-Response Plot
1 1-tail, 0.05 level 0.9 of significance
0.8
0.7
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ProportionNormal 0.3
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B-Control
Page 6 of 10 Report Ref: ECX18-0801-2
MACROALGAE ZOOSPORE GERMINATION -Proportion Normal Start Date: Test ID: ECX18-0801 Sample ID: ECX0801-2 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Brine Sample Date: Protocol: WIECX-08 Test Species: E. radiata Comments: Conc-% 1 2 3 B-Control 1.0000 1.0000 0.9885 6.3 1.0000 0.9385 0.9503 12.5 0.9754 0.9839 0.9723 25 1.0000 0.9124 0.9215 50 0.9260 0.9101 0.9638 100 0.2892 0.3498 0.2361
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9962 1.0000 1.5016 1.4633 1.5208 2.209 3 1 300 6.3 0.9629 0.9666 1.3957 1.3202 1.5208 7.818 3 1.580 2.500 0.1676 11 300 12.5 0.9772 0.9810 1.4202 1.4037 1.4436 1.469 3 1.215 2.500 0.1676 7 300 25 0.9446 0.9483 1.3593 1.2702 1.5208 10.306 3 2.123 2.500 0.1676 17 300 *50 0.9333 0.9369 1.3137 1.2663 1.3795 4.473 3 2.803 2.500 0.1676 20 300 *100 0.2917 0.2928 0.5694 0.5074 0.6329 11.019 3 13.905 2.500 0.1676 212 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.92596 0.858 0.94468 0.50029 Bartlett's Test indicates equal variances (p = 0.23) 6.905 15.0863 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 25 50 35.3553 4 0.05025 0.05049 0.35531 0.00674 9.5E-08 5, 12
Maximum Likelihood-Logit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 13.2518 1.1647 9.54522 16.9584 0.00333 8.17852 7.81473 0.04 17 Intercept -25.67 2.29645 -32.979 -18.362 TSCR 0.0294 0.00816 0.00344 0.05536 1.0 Point Logits % 95% Fiducial Limits EC01 -4.595 38.9374 27.4567 47.5295 0.9 EC05 -2.944 51.8716 40.7677 59.6438 0.8 EC10 -2.197 59.063 48.7033 66.1708 EC15 -1.735 64.0067 54.3346 70.6146 0.7 EC20 -1.386 68 58.9656 74.1996 0.6 EC25 -1.099 71.4855 63.0508 77.3415 EC40 -0.405 80.6351 73.8154 85.792 0.5 EC50 0.000 86.5209 80.576 91.5747 0.4 EC60 0.405 92.8363 87.4342 98.3304 Response EC75 1.099 104.719 98.8361 112.965 0.3 EC80 1.386 110.086 103.462 120.276 0.2 EC85 1.735 116.954 109.088 130.081 EC90 2.197 126.744 116.737 144.721 0.1 EC95 2.944 144.315 129.815 172.487 0.0 EC99 4.595 192.254 163.218 255.614 1 10 100 1000 Dose % Dose-Response Plot
1 1-tail, 0.05 level 0.9 of significance
0.8
0.7
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ProportionNormal 0.3
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Page 7 of 10 Report Ref: ECX18-0801-2
MOLLUSC LARVAL DEVELOPMENT -Proportion Normal Start Date: Test ID: ECX18-0801 Sample ID: ECX0801-2 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Brine Sample Date: Protocol: WIECX-23 Test Species: M. edulis Comments: Conc-% 1 2 3 B-Control 1.0000 1.0000 0.9784 6.3 0.8335 0.8851 0.8485 12.5 0.7704 0.7562 0.7636 25 0.0410 0.0000 0.0000 50 0.0000 0.0000 0.0000 100 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9928 1.0000 1.4883 1.4233 1.5208 3.782 3 2 300 *6.3 0.8557 0.8619 1.1821 1.1505 1.2249 3.253 3 6.679 2.420 0.1109 43 300 *12.5 0.7634 0.7689 1.0628 1.0543 1.0711 0.790 3 9.281 2.420 0.1109 71 300 *25 0.0137 0.0138 0.1013 0.0500 0.2039 87.686 3 30.255 2.420 0.1109 296 300 50 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300 100 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.95178 0.805 0.64484 0.43434 Bartlett's Test indicates equal variances (p = 0.11) 6.01693 11.3449 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test <6.3 6.3 0.03017 0.03037 1.07632 0.00315 8.8E-09 3, 8
Maximum Likelihood-Logit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 18.975 2.27379 11.7388 26.2112 0.00667 20.4143 7.81473 1.4E-04 18 Intercept -22.346 2.58761 -30.581 -14.111 TSCR 0.07407 0.02807 -0.0153 0.16341 1.0 Point Logits % 95% Fiducial Limits 0.9 EC01 -4.595 8.62 6.33022 10.0201 EC05 -2.944 10.5317 8.63343 11.741 0.8 EC10 -2.197 11.5313 9.87613 12.69 EC15 -1.735 12.1972 10.6929 13.3664 0.7 EC20 -1.386 12.7238 11.3201 13.9385 0.6 EC25 -1.099 13.1758 11.8389 14.4624 EC40 -0.405 14.332 13.0603 15.9621 0.5 EC50 0.000 15.0548 13.7454 17.0174 0.4 EC60 0.405 15.814 14.4113 18.2121 Response EC75 1.099 17.2017 15.5265 20.5817 0.3 EC80 1.386 17.8129 15.9883 21.6887 0.2 EC85 1.735 18.5819 16.5514 23.1293 EC90 2.197 19.6549 17.3107 25.2198 0.1 EC95 2.944 21.5204 18.5773 29.0559 0.0 EC99 4.595 26.2931 21.6182 39.9018 1 10 100 Significant heterogeneity detected (p = 1.39E-04) Dose % Dose-Response Plot
1 1-tail, 0.05 level of significance 0.9
0.8
0.7
0.6
0.5
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ProportionNormal 0.3
0.2
0.1
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50
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*6.3 *12.5
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Page 8 of 10 Report Ref: ECX18-0801-2
COPEPOD LARVAL DEVELOPMENT -Proportion Normal Start Date: Test ID: ECX18-0801 Sample ID: ECX18-0801-2 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Brine Sample Date: Protocol: WIECX-26 Test Species: GI-Gladioferens Imparipes Comments: Conc-% 1 2 3 B-Control 0.9615 0.8846 1.0000 6.3 1.0000 1.0000 1.0000 12.5 1.0000 0.7692 0.8077 25 0.6923 0.8077 0.5769 50 0.2692 0.2308 0.1154 100 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9487 1.0000 1.3728 1.2242 1.5208 10.801 3 16 300 6.3 1.0000 1.0541 1.5208 1.5208 1.5208 0.000 3 -1.219 2.470 0.2999 0 300 12.5 0.8590 0.9054 1.2358 1.0697 1.5208 20.064 3 1.129 2.470 0.2999 42 300 *25 0.6923 0.7297 0.9874 0.8626 1.1168 12.879 3 3.174 2.470 0.2999 92 300 *50 0.2051 0.2162 0.4644 0.3466 0.5455 22.487 3 7.482 2.470 0.2999 238 300 100 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.92702 0.835 0.66535 0.32301 Equality of variance cannot be confirmed Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 12.5 25 17.6777 8 0.18936 0.19698 0.51393 0.02211 4.8E-05 4, 10
Maximum Likelihood-Logit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 7.5029 0.82845 4.8664 10.1394 0.05333 36.6515 7.81473 5.5E-08 10 Intercept -11.297 1.28 -15.371 -7.2237 TSCR 0.03883 0.02797 -0.0502 0.12784 1.0 Point Logits % 95% Fiducial Limits 0.9 EC01 -4.595 7.82123 3.34308 11.9188 EC05 -2.944 12.9802 7.21415 17.5466 0.8 EC10 -2.197 16.3257 10.175 20.9933 EC15 -1.735 18.8161 12.5555 23.5221 0.7 EC20 -1.386 20.9389 14.6768 25.6805 0.6 EC25 -1.099 22.8716 16.6632 27.6671 EC40 -0.405 28.293 22.3587 33.5022 0.5 EC50 0.000 32.0421 26.2361 37.9253 0.4 EC60 0.405 36.2879 30.4018 43.4748 Response EC75 1.099 44.8896 37.926 56.6235 0.3 EC80 1.386 49.033 41.185 63.7798 0.2 EC85 1.735 54.5647 45.2786 74.0381 EC90 2.197 62.8884 51.0468 90.7968 0.1 EC95 2.944 79.0972 61.4237 127.334 0.0 EC99 4.595 131.27 90.8918 273.371 1 10 100 1000 Significant heterogeneity detected (p = 5.45E-08) Dose % Dose-Response Plot
1
0.9
0.8 1-tail, 0.05 level of significance 0.7
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ProportionNormal 0.3
0.2
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Page 9 of 10 Report Ref: ECX18-0801-2
FISH LARVAL DEVELOPMENT -Proportion Normal Start Date: Test ID: ECX18-0805 Sample ID: ECX18-0805-2 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Brine Sample Date: Protocol: WIECX-16 Test Species: S. lalandi Comments: Conc-% 1 2 3 B-Control 1.0000 0.9412 1.0000 6.3 1.0000 0.9412 0.7059 12.5 0.9412 0.9412 1.0000 25 0.8235 0.8824 0.7647 50 0.2941 0.4118 100 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9804 1.0000 1.4558 1.3258 1.5208 7.732 3 6 300 6.3 0.8824 0.9000 1.2814 0.9976 1.5208 20.634 3 1.397 2.685 0.3352 35 300 12.5 0.9608 0.9800 1.3908 1.3258 1.5208 8.093 3 0.521 2.685 0.3352 12 300 25 0.8235 0.8400 1.1408 1.0644 1.2207 6.858 3 2.524 2.685 0.3352 54 300 *50 0.3529 0.3600 0.6350 0.5732 0.6967 13.753 2 5.881 2.685 0.3747 130 200 100 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.95877 0.825 -0.3919 1.0761 Bartlett's Test indicates equal variances (p = 0.51) 3.27123 13.2767 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Bonferroni t Test 25 50 35.3553 4 0.2081 0.21088 0.24225 0.02337 0.00201 4, 9
Maximum Likelihood-Logit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 10.3727 1.29265 6.25886 14.4864 0.02 30.2368 7.81473 1.2E-06 15 Intercept -16.827 2.14897 -23.666 -9.9884 TSCR 0.06213 0.02626 -0.0214 0.14571 1.0 Point Logits % 95% Fiducial Limits 0.9 EC01 -4.595 15.1105 7.03838 21.3226 EC05 -2.944 21.798 12.7745 28.0319 0.8 EC10 -2.197 25.7308 16.6674 31.8488 0.7 EC15 -1.735 28.5137 19.6057 34.5485 EC20 -1.386 30.8058 22.115 36.7975 0.6 EC25 -1.099 32.8373 24.3881 38.8283 EC40 -0.405 38.2994 30.5859 44.6067 0.5 EC50 0.000 41.9065 34.5987 48.8241 0.4 EC60 0.405 45.8534 38.7683 53.9498 Response EC75 1.099 53.4806 45.9563 65.5715 0.3 EC80 1.386 57.0073 48.9252 71.6722 0.2 EC85 1.735 61.59 52.5318 80.1964 EC90 2.197 68.2512 57.4055 93.6425 0.1 EC95 2.944 80.5652 65.6806 121.326 0.0 EC99 4.595 116.221 86.9074 218.785 1 10 100 1000 Significant heterogeneity detected (p = 1.23E-06) Dose % Dose-Response Plot
1
0.9
0.8 1-tail, 0.05 level of significance 0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
25
6.3
*50
100 12.5
B-Control
Page 10 of 10 Report Ref: ECX18-0801-2
ECOTOXICOLOGY LABORATORY TEST REPORT
CLIENT ATTN: GRANT GRIFFITH SOUTHERN SEAWATER JOINT VENTURE LOT 32 TARANTO ROAD BINNINGUP, WA 6233
JOB INFORMATION JOB REFERENCE : ECX18-0801-1 NO. SAMPLES : 1 CLIENT ORDER NO. : PO #94350 PROJECT : Comparative toxicity assessment SAMPLED BY : S. McNeil DATE RECEIVED : 01/08/18 DATE REPORTED : 18/10/2018
REPORT NOTES Comparative toxicity assessment of a four desalination effluent samples
TESTED BY COMPANY APPROVED SIGNATORY
Intertek Ecotoxicology Laboratory Intertek Geotech 41-45 Furnace Road Welshpool, Western Australia 6106
Tel: +61 8 9263 0263 DR TRISTAN STRINGER [email protected] PRINCIPAL ECOTOXICOLOGIST
This report relates specifically to the sample(s) tested that were drawn and/or provided by the client or their nominated third party to Intertek. The reported result(s) provide no warranty or verification on the sample(s) representing any specific goods and/or shipment. This report was prepared solely for the use of the client named in this report. Intertek accepts no responsibility for any loss, damage or liability suffered by a third party as a result of any reliance upon or use of this report.
Except where explicitly agreed in writing, all work and services performed by Intertek is subject to our standard Terms and Conditions which can be obtained at our website: intertek.com/terms
Page 1 of 10 Report Ref: ECX18-0801-1
Sample Information SAMPLE REFERENCE INTERTEK REFERENCE Whole Effluent ECX18-0801-1
Bioassay Details BIOASSAY PROTOCOL REFERENCE TEST SPECIES TEMPERATURE Microtox* WIECX-17 Microbics 1992 V. fischeri 15°C Microalgae Growth* WIECX-06 Stauber et al. 1994 T I. galbana 22°C Macroalgae Zoospore Germination * WIECX-08 Burridge et al. 1999 E. radiata 22°C Mollusc Larvae Development* WIECX-23 ASTM E724 M. edulis 22°C Copepod Larval Development WIECX-26 ISO 16778 G. imparipes 22°C Fish Larvae Development* WIECX-16 USEPA 1001.0 S. lalandi 22°C * NATA Accredited method – Accreditation Number 5646
Physicochemistry PARAMETER CONTROL 6.3% 12.5% 25% 50% 100% pH 7.95 7.86 7.99 8.07 8.00 8.21 Salinity (‰) 34.4 36.5 37.7 40.7 48^ 62^ 86 DO (%) 100 100 100 100 100 100 ^ Refractometer reading - to be used for indicative purposes only
Concentration-Response Data
BIOASSAY (% CONTROL) CONTROL 6.3% 12.5% 25% 50% 100% Microtox* 100 ± 16 84 ± 2 95 ± 12 78 ± 3 78 ± 4 72 ± 8 Microalgae Growth* 100 ± 2 79 ± 10 112 ± 10 106 ± 7 117 ± 17 85 ± 17 Macroalgae Zoospore Germination * 100 ± 1 99 ± 3 99 ± 4 100 ± 8 96 ± 2 24 ± 3 Mollusc Larvae Development* 100 ± 2 90 ± 8 81 ± 1 6 ± 3 0 ± 0 0 ± 0 Copepod Larval Development 100 ± 14 99 ± 12 89 ± 8 46 ± 2 9 ± 2 1 ± 2 Fish Larvae Development* 100 ± 6 108 ± 9 94 ± 10 98 ± 9 71 ± 10 0 ± 0 * NATA Accredited method – Accreditation Number 5646
Page 2 of 10 Report Ref: ECX18-0801-1
Statistical Effects Data
BIOASSAY EC10 (%) EC50 (%) NOEC (%) LOEC (%) Microtox* 16.1 >100 12.5 25 Microalgae Growth* 73.9 >100 50 100 Macroalgae Zoospore Germination * 60.8 84.3 50 100 Mollusc Larvae Development* 11.6 16.3 <6.3 6.3 Copepod Larval Development 13.1 25.4 12.5 25 Fish Larvae Development* 43.9 55.4 25 50 * NATA Accredited method – Accreditation Number 5646
Quality Assurance Limits BIOASSAY REFERENCE OBSERVED CUSUM CHART COEFFICIENT OF CONTROL TEST TOXICANT EC50 LIMITS VARIANCE RESPONSE ACCEPTABILITY Microtox* Phenol 17.0 mg/L 15.9 – 20.4 mg/L 6.32% < 90% PASS Microalgae Growth* > 3 x cell Copper 46.2 µg/L 21.9 – 55 µg/L 24.2% PASS divisions Macroalgae Zoospore >80% Copper 143 µg/L 59 – 225 µg/L 33.2% PASS Germination * Germinated Mollusc Larvae >80% Copper 5.9 µg/L 4.0 – 12.9 µg/L 30.6% PASS Development* Development 50-80% Copepod Larval Copper 23.2 µg/L 8.6 – 23.7 µg/L 26.2% copepodite PASS Development life stage Fish Larvae >80% Copper 45.2 µg/L 21.6 – 82.9 µg/L 33.1% PASS Development* Development * NATA Accredited method – Accreditation Number 5646
General Comments Quality assurance and quality control criteria were within acceptable limits for all bioassays. A detailed interpretation of the results and species sensitivity distribution is included in the interpretative report (Report No. ECX18-0801)
Page 3 of 10 Report Ref: ECX18-0801-1
STATISTICAL DATA
Page 4 of 10 Report Ref: ECX18-0801-1
MICROTOX
Start Date: Test ID: ECX18-0801 Sample ID: ECX0801-1 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent Sample Date: Protocol: WIECX-17 Test Species: Microtox Comments: Conc-% 1 2 3 B-Control 1.0000 1.0000 0.8287 6.3 0.8392 0.8392 0.8182 12.5 0.7867 1.0000 0.9441 25 0.8077 0.8497 0.7448 50 0.8077 0.8182 0.7343 100 0.7867 0.7343 0.6294
Transform: Arcsin Square Root 1-Tailed Isotonic Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Mean N-Mean B-Control 0.9429 1.0000 1.3952 1.1440 1.5208 15.590 3 0.9433 1.0000 6.3 0.8322 0.8826 1.1489 1.1303 1.1581 1.400 3 2.236 2.500 0.2754 0.8717 0.9240 12.5 0.9103 0.9654 1.3145 1.0907 1.5208 16.398 3 0.732 2.500 0.2754 0.8717 0.9240 *25 0.8007 0.8492 1.1102 1.0412 1.1726 5.942 3 2.587 2.500 0.2754 0.8000 0.8481 *50 0.7867 0.8344 1.0921 1.0292 1.1303 5.026 3 2.751 2.500 0.2754 0.7867 0.8339 *100 0.7168 0.7602 1.0121 0.9163 1.0907 8.744 3 3.478 2.500 0.2754 0.7167 0.7597
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.93659 0.858 -0.6797 0.94231 Bartlett's Test indicates equal variances (p = 0.06) 10.776 15.0863 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 12.5 25 17.6777 8 0.15947 0.16449 0.0637 0.0182 0.03503 5, 12
Log-Logit Interpolation (200 Resamples) Point % SD 95% CL(Exp) Skew IC05* 3.292 7.580 0.604 46.647 1.2750 IC10 16.058 15.623 0.000 106.502 1.3865 IC15 24.633 1.0 IC20 69.841 IC25 >100 0.9 IC40 >100 0.8 IC50 >100 * indicates IC estimate less than the lowest concentration 0.7 0.6
0.5
0.4 Response 0.3
0.2
0.1
0.0 1 10 100 Dose % Dose-Response Plot
1
0.9
0.8 1-tail, 0.05 level of significance 0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
6.3
*25 *50
12.5 *100
B-Control
Page 5 of 10 Report Ref: ECX18-0801-1
MICROALGAE GROWTH -Proportion Normal Start Date: Test ID: ECX18-0801 Sample ID: ECX0801-1 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent Sample Date: Protocol: WIECX-06 Test Species: TISO-Isochrysis sp. Comments: Conc-% 1 2 3 B-Control 1.0000 1.0000 0.9850 6.3 0.6922 0.8985 0.7687 12.5 1.0000 1.0000 1.0000 25 0.9817 1.0000 1.0000 50 1.0000 1.0000 1.0000 100 0.7454 1.0000 0.7587
Transform: Arcsin Square Root 1-Tailed Isotonic Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Mean N-Mean B-Control 0.9950 1.0000 1.4966 1.4481 1.5208 2.803 3 0.9967 1.0000 *6.3 0.7865 0.7904 1.0994 0.9827 1.2466 12.237 3 3.838 2.500 0.2587 0.9450 0.9482 12.5 1.0000 1.0050 1.5208 1.5208 1.5208 0.000 3 -0.234 2.500 0.2587 0.9450 0.9482 25 0.9939 0.9989 1.4922 1.4351 1.5208 3.315 3 0.042 2.500 0.2587 0.9450 0.9482 50 1.0000 1.0050 1.5208 1.5208 1.5208 0.000 3 -0.234 2.500 0.2587 0.9450 0.9482 *100 0.8347 0.8389 1.2067 1.0419 1.5208 22.551 3 2.801 2.500 0.2587 0.8367 0.8395
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates non-normal distribution (p <= 0.01) 0.84248 0.858 1.34451 3.98004 Equality of variance cannot be confirmed Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test <6.3 6.3 0.1013 0.10186 0.10442 0.01606 0.0038 5, 12
Log-Logit Interpolation (200 Resamples) Point % SD 95% CL(Exp) Skew IC05* 6.120 IC10 73.850 IC15 95.599 1.0 IC20 >100 0.9 IC25 >100 IC40 >100 0.8 IC50 >100 0.7 * indicates IC estimate less than the lowest concentration 0.6 0.5 0.4
Response 0.3 0.2 0.1 0.0 -0.1 1 10 100 Dose % Dose-Response Plot
1
0.9 1-tail, 0.05 level of significance 0.8
0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
25 50
*6.3
12.5
*100 B-Control
Page 6 of 10 Report Ref: ECX18-0801-1
MACROALGAE ZOOSPORE GERMINATION -Proportion Normal Start Date: Test ID: ECX18-0801 Sample ID: ECX0801-1 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent Sample Date: Protocol: WIECX-08 Test Species: E. radiata Comments: Conc-% 1 2 3 B-Control 1.0000 1.0000 0.9885 6.3 0.9638 1.0000 0.9802 12.5 1.0000 1.0000 0.9397 25 0.9240 0.9839 1.0000 50 0.9638 0.9331 0.9708 100 0.2567 0.2010 0.2486
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9962 1.0000 1.5016 1.4633 1.5208 2.209 3 1 300 6.3 0.9813 0.9851 1.4433 1.3795 1.5208 4.964 3 0.917 2.500 0.1591 6 300 12.5 0.9799 0.9837 1.4548 1.3228 1.5208 7.858 3 0.736 2.500 0.1591 6 300 25 0.9693 0.9730 1.4187 1.2915 1.5208 8.222 3 1.304 2.500 0.1591 10 300 50 0.9559 0.9596 1.3626 1.3092 1.3991 3.466 3 2.185 2.500 0.1591 14 300 *100 0.2354 0.2363 0.5060 0.4649 0.5313 7.105 3 15.649 2.500 0.1591 229 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.93961 0.858 -0.6334 -0.0524 Bartlett's Test indicates equal variances (p = 0.44) 4.77292 15.0863 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 50 100 70.7107 2 0.04641 0.04663 0.43884 0.00607 1.5E-08 5, 12
Maximum Likelihood-Logit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 15.4682 0.80582 13.8888 17.0476 0.00333 3.18605 7.81473 0.36 15 Intercept -29.792 1.592 -32.912 -26.672 TSCR 0.01898 0.00397 0.01121 0.02675 1.0 Point Logits % 95% Fiducial Limits 0.9 EC01 -4.595 42.5538 38.7603 45.9361 EC05 -2.944 54.4068 50.9131 57.4632 0.8 EC10 -2.197 60.8078 57.5852 63.6121 EC15 -1.735 65.1429 62.1355 67.7573 0.7 EC20 -1.386 68.6096 65.7874 71.0662 0.6 EC25 -1.099 71.6116 68.9554 73.9309 EC40 -0.405 79.3952 77.1638 81.3854 0.5 EC50 0.000 84.3348 82.3324 86.1723 0.4 EC60 0.405 89.5818 87.7393 91.3528 Response EC75 1.099 99.3186 97.4115 101.361 0.3 EC80 1.386 103.664 101.568 106.002 0.2 EC85 1.735 109.181 106.734 112.015 EC90 2.197 116.965 113.875 120.67 0.1 EC95 2.944 130.726 126.238 136.291 0.0 EC99 4.595 167.138 158.101 178.814 1 10 100 1000 Dose % Dose-Response Plot
1 1-tail, 0.05 level 0.9 of significance
0.8
0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
25 50
6.3
12.5 *100
B-Control
Page 7 of 10 Report Ref: ECX18-0801-1
MOLLUSC LARVAL DEVELOPMENT -Proportion Normal Start Date: Test ID: ECX18-0801 Sample ID: ECX0801-1 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent Sample Date: Protocol: WIECX-23 Test Species: M. edulis Comments: Conc-% 1 2 3 B-Control 1.0000 1.0000 0.9784 6.3 0.8023 0.9646 0.9209 12.5 0.8177 0.8068 0.7911 25 0.0295 0.0718 0.0740 50 0.0000 0.0000 0.0000 100 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9928 1.0000 1.4883 1.4233 1.5208 3.782 3 2 300 *6.3 0.8959 0.9024 1.2591 1.1100 1.3816 10.940 3 3.494 2.420 0.1588 32 300 *12.5 0.8052 0.8111 1.1139 1.0961 1.1297 1.513 3 5.707 2.420 0.1588 58 300 *25 0.0584 0.0588 0.2397 0.1725 0.2754 24.304 3 19.032 2.420 0.1588 283 300 50 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300 100 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.91056 0.805 -0.6411 1.42378 Bartlett's Test indicates equal variances (p = 0.13) 5.62709 11.3449 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test <6.3 6.3 0.0503 0.05064 0.89396 0.00646 3.1E-07 3, 8
Maximum Likelihood-Logit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 14.8336 1.14072 11.2033 18.4639 0.00667 14.6259 7.81473 2.2E-03 16 Intercept -17.994 1.38987 -22.417 -13.57 TSCR 0.0542 0.02088 -0.0123 0.12067 1.0 Point Logits % 95% Fiducial Limits 0.9 EC01 -4.595 8.00285 6.20076 9.41534 EC05 -2.944 10.3401 8.62769 11.6715 0.8 EC10 -2.197 11.6118 9.99012 12.9007 EC15 -1.735 12.4763 10.9216 13.7482 0.7 EC20 -1.386 13.1694 11.6663 14.4396 0.6 EC25 -1.099 13.7708 12.3077 15.0511 EC40 -0.405 15.3352 13.9376 16.7097 0.5 EC50 0.000 16.3314 14.9344 17.8287 0.4 EC60 0.405 17.3923 15.9546 19.0798 Response EC75 1.099 19.368 17.7455 21.5666 0.3 EC80 1.386 20.2525 18.5081 22.7387 0.2 EC85 1.735 21.3776 19.4505 24.2746 EC90 2.197 22.9693 20.7409 26.5219 0.1 EC95 2.944 25.7941 22.9403 30.69 0.0 EC99 4.595 33.3273 28.4571 42.6722 1 10 100 Significant heterogeneity detected (p = 2.17E-03) Dose % Dose-Response Plot
1 1-tail, 0.05 level 0.9 of significance
0.8
0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
50
*25
100
*6.3 *12.5
B-Control
Page 8 of 10 Report Ref: ECX18-0801-1
COPEPOD LARVAL DEVELOPMENT -Proportion Normal Start Date: Test ID: ECX18-0805 Sample ID: ECX18-0805-1 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent Sample Date: Protocol: WIECX-26 Test Species: GI-Gladioferens Imparipes Comments: Conc-% 1 2 3 B-Control 0.8514 1.0000 1.0000 6.3 1.0000 1.0000 0.8514 12.5 0.8108 0.9730 0.8919 25 0.4459 0.4865 0.4459 50 0.0811 0.0811 0.1216 100 0.0405 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9505 1.0000 1.4055 1.1750 1.5208 14.204 3 15 300 6.3 0.9505 1.0000 1.4055 1.1750 1.5208 14.204 3 0.000 2.500 0.2765 15 300 12.5 0.8919 0.9384 1.2541 1.1208 1.4056 11.427 3 1.369 2.500 0.2765 33 300 *25 0.4595 0.4834 0.7448 0.7312 0.7719 3.151 3 5.974 2.500 0.2765 161 300 *50 0.0946 0.0995 0.3112 0.2887 0.3562 12.519 3 9.894 2.500 0.2765 272 300 *100 0.0135 0.0142 0.1009 0.0500 0.2027 87.360 3 11.796 2.500 0.2765 296 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.90124 0.858 -0.7359 0.04702 Bartlett's Test indicates equal variances (p = 0.12) 8.69506 15.0863 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 12.5 25 17.6777 8 0.15573 0.16006 0.98426 0.01835 8.6E-08 5, 12
Maximum Likelihood-Logit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 7.67017 0.24677 7.18649 8.15384 0.05 2.02693 7.81473 0.57 3 Intercept -10.771 0.35822 -11.473 -10.069 TSCR 0.04338 0.009 0.02574 0.06101 1.0 Point Logits % 95% Fiducial Limits 0.9 EC01 -4.595 6.3859 5.74241 7.01623 EC05 -2.944 10.4817 9.72589 11.2048 0.8 EC10 -2.197 13.1174 12.3373 13.8589 0.7 EC15 -1.735 15.0717 14.2885 15.815 EC20 -1.386 16.733 15.9535 17.4737 0.6 EC25 -1.099 18.2423 17.4688 18.9798 EC40 -0.405 22.4621 21.7002 23.2034 0.5 EC50 0.000 25.3695 24.5937 26.142 0.4 EC60 0.405 28.6533 27.8234 29.5052 Response EC75 1.099 35.2813 34.2017 36.452 0.3 EC80 1.386 38.4637 37.2063 39.8534 0.2 EC85 1.735 42.7033 41.165 44.4363 EC90 2.197 49.0655 47.0339 51.4003 0.1 EC95 2.944 61.4037 58.243 65.1249 0.0 EC99 4.595 100.786 93.1129 110.183 1 10 100 1000 Dose % Dose-Response Plot
1
0.9 1-tail, 0.05 level 0.8 of significance 0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
6.3
*25 *50
12.5
*100 B-Control
Page 9 of 10 Report Ref: ECX18-0801-1
FISH LARVAL DEVELOPMENT -Proportion Normal Start Date: Test ID: ECX18-0805 Sample ID: ECX18-0805-1 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent Sample Date: Protocol: WIECX-16 Test Species: S. lalandi Comments: Conc-% 1 2 3 B-Control 1.0000 0.9412 1.0000 6.3 1.0000 1.0000 1.0000 12.5 1.0000 0.8235 1.0000 25 1.0000 0.8824 1.0000 50 0.5882 0.7647 0.7647 100 0.0100 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9804 1.0000 1.4558 1.3258 1.5208 7.732 3 6 300 6.3 1.0000 1.0200 1.5208 1.5208 1.5208 0.000 3 -0.603 2.500 0.2695 0 300 12.5 0.9412 0.9600 1.3929 1.1373 1.5208 15.896 3 0.583 2.500 0.2695 18 300 25 0.9608 0.9800 1.4207 1.2207 1.5208 12.195 3 0.325 2.500 0.2695 12 300 *50 0.7059 0.7200 1.0009 0.8741 1.0644 10.974 3 4.219 2.500 0.2695 89 300 *100 0.0033 0.0034 0.0667 0.0500 0.1002 43.383 3 12.884 2.500 0.2695 299 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.88574 0.858 -1.0512 0.38433 Equality of variance cannot be confirmed Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 25 50 35.3553 4 0.12756 0.12926 0.93521 0.01744 8.6E-08 5, 12
Maximum Likelihood-Logit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 21.8752 4.17904 8.57568 35.1748 0.02 19.6189 7.81473 2.0E-04 17 Intercept -38.133 7.12623 -60.811 -15.454 TSCR 0.02982 0.0126 -0.0103 0.06992 1.0 Point Logits % 95% Fiducial Limits 0.9 EC01 -4.595 34.1291 18.349 39.915 EC05 -2.944 40.6054 28.4324 44.7039 0.8 EC10 -2.197 43.928 34.5391 47.2305 0.7 EC15 -1.735 46.1201 38.8113 49.054 EC20 -1.386 47.8423 42.1694 50.7169 0.6 EC25 -1.099 49.3132 44.8739 52.4664 EC40 -0.405 53.0457 50.0102 59.3402 0.5 EC50 0.000 55.3586 52.0996 65.22 0.4 EC60 0.405 57.7724 53.9053 72.1758 Response EC75 1.099 62.1451 56.7787 86.3715 0.3 EC80 1.386 64.0557 57.9525 93.1555 0.2 EC85 1.735 66.4478 59.3791 102.133 EC90 2.197 69.7635 61.2968 115.467 0.1 EC95 2.944 75.4721 64.4775 140.885 0.0 EC99 4.595 89.7936 71.9857 218.997 1 10 100 1000 Significant heterogeneity detected (p = 2.04E-04) Dose % Dose-Response Plot
1
0.9 1-tail, 0.05 level 0.8 of significance
0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
25
6.3
*50
100
12.5 B-Control
Page 10 of 10 Report Ref: ECX18-0801-1
ECOTOXICOLOGY LABORATORY TEST REPORT
CLIENT ATTN: GRANT GRIFFITH SOUTHERN SEAWATER JOINT VENTURE LOT 32 TARANTO ROAD BINNINGUP, WA 6233
JOB INFORMATION JOB REFERENCE : ECX18-0801-3 NO. SAMPLES : 1 CLIENT ORDER NO. : PO #94350 PROJECT : Comparative toxicity assessment SAMPLED BY : S. McNeil DATE RECEIVED : 01/08/18 DATE REPORTED : 18/10/2018
REPORT NOTES Comparative toxicity assessment of a four desalination effluent samples
TESTED BY COMPANY APPROVED SIGNATORY
Intertek Ecotoxicology Laboratory Intertek Geotech 41-45 Furnace Road Welshpool, Western Australia 6106
Tel: +61 8 9263 0263 DR TRISTAN STRINGER [email protected] PRINCIPAL ECOTOXICOLOGIST
This report relates specifically to the sample(s) tested that were drawn and/or provided by the client or their nominated third party to Intertek. The reported result(s) provide no warranty or verification on the sample(s) representing any specific goods and/or shipment. This report was prepared solely for the use of the client named in this report. Intertek accepts no responsibility for any loss, damage or liability suffered by a third party as a result of any reliance upon or use of this report.
Except where explicitly agreed in writing, all work and services performed by Intertek is subject to our standard Terms and Conditions which can be obtained at our website: intertek.com/terms
Page 1 of 10 Report Ref: ECX18-0801-3
Sample Information SAMPLE REFERENCE INTERTEK REFERENCE Whole Effluent + 0.17 g/L Memclean R ECX18-0801-3
Bioassay Details BIOASSAY PROTOCOL REFERENCE TEST SPECIES TEMPERATURE Microtox* WIECX-17 Microbics 1992 V. fischeri 15°C Microalgae Growth* WIECX-06 Stauber et al. 1994 I. galbana 22°C Macroalgae Zoospore Germination * WIECX-08 Burridge et al. 1999 E. radiata 22°C Mollusc Larvae Development* WIECX-23 ASTM E724 M. edulis 22°C Copepod Larval Development WIECX-26 ISO 16778 G. imparipes 22°C Fish Larvae Development* WIECX-16 USEPA 1004.0 S. lalandi 22°C * NATA Accredited method – Accreditation Number 5646
Physicochemistry PARAMETER CONTROL 6.3% 12.5% 25% 50% 100% pH 7.95 8.07 7.92 7.88 7.86 8.21 Salinity (‰) 34.4 36.4 37.7 40.4 49^ 63^ 86 DO (%) 100 100 100 100 100 100 ^ Refractometer reading - to be used for indicative purposes only
Concentration-Response Data
BIOASSAY (% CONTROL) CONTROL 6.3% 12.5% 25% 50% 100% Microtox* 100 ± 3 93 ± 12 97 ± 2 91 ± 11 90 ± 5 69 ± 3 Microalgae Growth* 100 ± 2 123 ± 9 137 ± 12 117 ± 20 126 ± 8 73 ± 8 Macroalgae Zoospore Germination * 100 ± 1 99 ± 1 98 ± 5 100 ± 0 92 ± 5 14 ± 8 Mollusc Larvae Development* 100 ± 2 90 ± 3 80 ± 3 4 ± 3 0 ± 0 0 ± 0 Copepod Larval Development 100 ± 14 114 ± 23 98 ± 19 58 ± 8 3 ± 2 0 ± 0 Fish Larvae Development* 100 ± 6 96 ± 21 102 ± 7 96 ± 3 31 ± 15 0 ± 0 * NATA Accredited method – Accreditation Number 5646
Page 2 of 10 Report Ref: ECX18-0801-3
Statistical Effects Data
BIOASSAY EC10 (%) EC50 (%) NOEC (%) LOEC (%) Microtox* 51.6 >100 50 100 Microalgae Growth* 85.7 >100 50 100 Macroalgae Zoospore Germination * 53.5 75.4 25 50 Mollusc Larvae Development* 11.6 15.8 <6.3 6.3 Copepod Larval Development 18.1 27.0 12.5 25 Fish Larvae Development* 30.7 44 25 50 * NATA Accredited method – Accreditation Number 5646
Quality Assurance Limits
BIOASSAY REFERENCE OBSERVED CUSUM CHART COEFFICIENT OF CONTROL TEST TOXICANT EC50 LIMITS VARIANCE RESPONSE ACCEPTABILITY Microtox* Phenol 17.0 mg/L 15.9 – 20.4 mg/L 6.32% < 90% PASS Microalgae Growth* > 3 x cell Copper 46.2 µg/L 21.9 – 55 µg/L 24.2% PASS divisions Macroalgae Zoospore >80% Copper 143 µg/L 59 – 225 µg/L 33.2% PASS Germination * Germinated Mollusc Larvae >80% Copper 5.9 µg/L 4.0 – 12.9 µg/L 30.6% PASS Development* Development 50-80% Copepod Larval Copper 23.2 µg/L 8.6 – 23.7 µg/L 26.2% copepodite PASS Development life stage Fish Larvae >80% Copper 45.2 µg/L 21.6 – 82.9 µg/L 33.1% PASS Development* Development * NATA Accredited method – Accreditation Number 5646
General Comments Quality assurance and quality control criteria were within acceptable limits for all bioassays. A detailed interpretation of the results and species sensitivity distribution is included in the interpretative report (Report No. ECX18-0801)
Page 3 of 10 Report Ref: ECX18-0801-3
STATISTICAL DATA
Page 4 of 10 Report Ref: ECX18-0801-3
MICROTOX Abalone Larval Development Test-Proportion Normal Start Date: Test ID: ECX18-0801 Sample ID: ECX0801-3 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent + 0.17g/L Sample Date: Protocol: WIECX-17 Test Species: Microtox Comments: Conc-% 1 2 3 B-Control 0.9681 1.0000 1.0000 6.3 1.0000 0.9468 0.7979 12.5 0.9681 0.9894 0.9468 25 1.0000 0.7979 0.9255 50 0.8936 0.8511 0.9468 100 0.6809 0.6702 0.7234
Transform: Arcsin Square Root 1-Tailed Isotonic Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Mean N-Mean B-Control 0.9894 1.0000 1.4776 1.3912 1.5208 5.064 3 0.9900 1.0000 6.3 0.9149 0.9247 1.3211 1.1045 1.5208 15.794 3 1.452 2.500 0.2694 0.9433 0.9529 12.5 0.9681 0.9785 1.3989 1.3381 1.4675 4.650 3 0.730 2.500 0.2694 0.9433 0.9529 25 0.9078 0.9176 1.3066 1.1045 1.5208 15.951 3 1.587 2.500 0.2694 0.9100 0.9192 50 0.8972 0.9068 1.2504 1.1746 1.3381 6.588 3 2.108 2.500 0.2694 0.8967 0.9057 *100 0.6915 0.6989 0.9822 0.9591 1.0170 3.124 3 4.597 2.500 0.2694 0.6900 0.6970
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.94621 0.858 -0.0361 0.68676 Bartlett's Test indicates equal variances (p = 0.18) 7.67163 15.0863 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 50 100 70.7107 2 0.11718 0.1182 0.08677 0.01742 0.01063 5, 12
Log-Logit Interpolation (200 Resamples) Point % SD 95% CL(Exp) Skew IC05 13.451 10.809 0.000 62.411 0.8394 IC10 51.553 14.987 0.000 71.941 -0.3177 IC15 64.271 7.482 35.007 81.287 -2.3785 1.0 IC20 76.130 4.556 52.097 89.387 -0.2348 0.9 IC25 87.687 3.690 70.680 99.714 -0.1108 IC40 >100 0.8 IC50 >100 0.7
0.6
0.5
0.4 Response 0.3
0.2
0.1
0.0 1 10 100 Dose % Dose-Response Plot
1
0.9 1-tail, 0.05 level of significance 0.8
0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
25 50
6.3
12.5
*100 B-Control
Page 5 of 10 Report Ref: ECX18-0801-3
MICROALGAE GROWTH -Proportion Normal Start Date: Test ID: ECX18-0801 Sample ID: ECX0801-3 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent + 0.17g/L Sample Date: Protocol: WIECX-06 Test Species: TISO-Isochrysis sp. Comments: Conc-% 1 2 3 B-Control 1.0000 1.0000 0.9850 6.3 1.0000 1.0000 1.0000 12.5 1.0000 1.0000 1.0000 25 1.0000 0.9868 1.0000 50 1.0000 1.0000 1.0000 100 0.6358 0.7881 0.7616
Transform: Arcsin Square Root 1-Tailed Isotonic Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Mean N-Mean B-Control 0.9950 1.0000 1.4966 1.4481 1.5208 2.803 3 0.9989 1.0000 6.3 1.0000 1.0050 1.5208 1.5208 1.5208 0.000 3 -0.683 2.500 0.0886 0.9989 1.0000 12.5 1.0000 1.0050 1.5208 1.5208 1.5208 0.000 3 -0.683 2.500 0.0886 0.9989 1.0000 25 0.9956 1.0006 1.4990 1.4555 1.5208 2.516 3 -0.069 2.500 0.0886 0.9983 0.9994 50 1.0000 1.0050 1.5208 1.5208 1.5208 0.000 3 -0.683 2.500 0.0886 0.9983 0.9994 *100 0.7285 0.7321 1.0253 0.9229 1.0924 8.790 3 13.296 2.500 0.0886 0.7300 0.7308
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates non-normal distribution (p <= 0.01) 0.84674 0.858 -1.1955 3.12588 Equality of variance cannot be confirmed Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 50 100 70.7107 2 0.02078 0.0209 0.1186 0.00188 3.4E-08 5, 12
Log-Logit Interpolation (200 Resamples) Point % SD 95% CL(Exp) Skew IC05 78.139 3.293 66.187 92.764 0.8911 IC10 85.866 2.657 75.356 95.918 0.3382 IC15 91.059 2.422 81.204 99.373 -0.1198 1.0 IC20 95.184 2.435 84.734 103.318 -0.2058 0.9 IC25 98.736 IC40 >100 0.8 IC50 >100 0.7 0.6 0.5 0.4
Response 0.3 0.2 0.1 0.0 -0.1 1 10 100 Dose % Dose-Response Plot
1 1-tail, 0.05 level of significance 0.9
0.8
0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
25 50
6.3
12.5 *100
B-Control
Page 6 of 10 Report Ref: ECX18-0801-3
MACROALGAE ZOOSPORE GERMINATION -Proportion Normal Start Date: Test ID: ECX18-0801 Sample ID: ECX0801-3 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent + 0.17g/L Sample Date: Protocol: WIECX-08 Test Species: E. radiata Comments: Conc-% 1 2 3 B-Control 1.0000 1.0000 0.9885 6.3 0.9839 0.9763 0.9962 12.5 0.9231 1.0000 0.9943 25 1.0000 0.9991 1.0000 50 0.8996 0.9802 0.8802 100 0.2147 0.1647 0.0537
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9962 1.0000 1.5016 1.4633 1.5208 2.209 3 1 300 6.3 0.9855 0.9893 1.4564 1.4164 1.5092 3.277 3 0.611 2.500 0.1849 4 300 12.5 0.9725 0.9762 1.4352 1.2898 1.5208 8.820 3 0.898 2.500 0.1849 9 300 25 0.9997 1.0035 1.5272 1.5208 1.5400 0.727 3 -0.346 2.500 0.1849 0 300 *50 0.9200 0.9235 1.2984 1.2173 1.4296 8.832 3 2.747 2.500 0.1849 24 300 *100 0.1444 0.1449 0.3778 0.2338 0.4817 34.078 3 15.191 2.500 0.1849 258 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.96943 0.858 -0.3595 -0.0628 Bartlett's Test indicates equal variances (p = 0.09) 9.45391 15.0863 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 25 50 35.3553 4 0.05842 0.0587 0.58711 0.00821 1.6E-08 5, 12
Maximum Likelihood-Logit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 14.7446 1.03403 11.4539 18.0354 0.00333 12.5175 7.81473 5.8E-03 13 Intercept -27.681 1.97699 -33.973 -21.39 TSCR 0.01173 0.00645 -0.0088 0.03224 1.0 Point Logits % 95% Fiducial Limits EC01 -4.595 36.7904 28.8943 43.1073 0.9 EC05 -2.944 47.6086 40.0298 53.5327 0.8 EC10 -2.197 53.5011 46.3102 59.1553 EC15 -1.735 57.5093 50.6304 62.9941 0.7 EC20 -1.386 60.7241 54.1059 66.098 0.6 EC25 -1.099 63.5144 57.1188 68.8211 EC40 -0.405 70.7754 64.8683 76.1057 0.5 EC50 0.000 75.4017 69.6737 80.9584 0.4 EC60 0.405 80.3305 74.6286 86.3589 Response EC75 1.099 89.514 83.3525 97.1053 0.3 EC80 1.386 93.6271 87.0603 102.19 0.2 EC85 1.735 98.8609 91.6311 108.87 EC90 2.197 106.267 97.8744 118.665 0.1 EC95 2.944 119.42 108.497 136.849 0.0 EC99 4.595 154.536 135.17 188.981 1 10 100 1000 Significant heterogeneity detected (p = 5.81E-03) Dose % Dose-Response Plot
1 1-tail, 0.05 level 0.9 of significance
0.8
0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
25
6.3
*50
12.5 *100
B-Control
Page 7 of 10 Report Ref: ECX18-0801-3
MOLLUSC LARVAL DEVELOPMENT -Proportion Normal Start Date: Test ID: ECX18-0801 Sample ID: ECX0801-3 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent + 0.17 Sample Date: Protocol: WIECX-23 Test Species: M. edulis Comments: Conc-% 1 2 3 B-Control 1.0000 1.0000 0.9784 6.3 0.9358 0.8733 0.9041 12.5 0.8221 0.8134 0.7718 25 0.0709 0.0140 0.0206 50 0.0000 0.0000 0.0000 100 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9928 1.0000 1.4883 1.4233 1.5208 3.782 3 2 300 *6.3 0.9044 0.9109 1.2591 1.2068 1.3145 4.283 3 4.793 2.420 0.1157 29 300 *12.5 0.8024 0.8082 1.1107 1.0727 1.1353 3.008 3 7.896 2.420 0.1157 60 300 *25 0.0352 0.0354 0.1774 0.1186 0.2695 45.528 3 27.416 2.420 0.1157 290 300 50 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300 100 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.94389 0.805 0.28433 -0.8765 Bartlett's Test indicates equal variances (p = 0.75) 1.1967 11.3449 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test <6.3 6.3 0.03199 0.03221 0.99416 0.00343 1.7E-08 3, 8
Maximum Likelihood-Logit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 16.411 1.24745 12.4411 20.3809 0.00667 12.6967 7.81473 5.3E-03 15 Intercept -19.652 1.47085 -24.333 -14.971 TSCR 0.05004 0.0186 -0.0092 0.10924 1.0 Point Logits % 95% Fiducial Limits 0.9 EC01 -4.595 8.26966 6.70408 9.46806 EC05 -2.944 10.4249 9.01477 11.5164 0.8 EC10 -2.197 11.5772 10.2738 12.6259 EC15 -1.735 12.3536 11.1187 13.3913 0.7 EC20 -1.386 12.9723 11.7844 14.017 0.6 EC25 -1.099 13.5066 12.3508 14.5717 EC40 -0.405 14.8862 13.7612 16.0794 0.5 EC50 0.000 15.7576 14.6067 17.0946 0.4 EC60 0.405 16.68 15.4633 18.2219 Response EC75 1.099 18.3837 16.9579 20.4291 0.3 EC80 1.386 19.1409 17.5935 21.454 0.2 EC85 1.735 20.0996 18.3787 22.7841 EC90 2.197 21.4475 19.4533 24.7079 0.1 EC95 2.944 23.8182 21.2812 28.2199 0.0 EC99 4.595 30.0256 25.8258 38.0338 1 10 100 Significant heterogeneity detected (p = 5.34E-03) Dose % Dose-Response Plot
1 1-tail, 0.05 level of significance 0.9
0.8
0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
50
*25
100
*6.3 *12.5
B-Control
Page 8 of 10 Report Ref: ECX18-0801-3
COPEPOD LARVAL DEVELOPMENT -Proportion Normal Start Date: Test ID: ECX18-0805 Sample ID: ECX18-0805-3 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent + 0.17g/L Sample Date: Protocol: WIECX-26 Test Species: GI-Gladioferens Imparipes Comments: Conc-% 1 2 3 B-Control 0.8514 1.0000 1.0000 6.3 1.0000 0.8919 1.0000 12.5 0.9324 1.0000 0.8108 25 0.6081 0.6486 0.4865 50 0.0000 0.0405 0.0405 100 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9505 1.0000 1.4055 1.1750 1.5208 14.204 3 15 300 6.3 0.9640 1.0142 1.4258 1.2358 1.5208 11.541 3 -0.159 2.470 0.3151 11 300 12.5 0.9144 0.9621 1.3165 1.1208 1.5208 15.202 3 0.698 2.470 0.3151 26 300 *25 0.5811 0.6114 0.8675 0.7719 0.9363 9.849 3 4.217 2.470 0.3151 125 300 *50 0.0270 0.0284 0.1518 0.0500 0.2027 58.071 3 9.827 2.470 0.3151 292 300 100 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.91845 0.835 -0.5233 -0.8493 Bartlett's Test indicates equal variances (p = 0.72) 2.10407 13.2767 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 12.5 25 17.6777 8 0.18648 0.19167 0.88293 0.02441 6.4E-06 4, 10
Maximum Likelihood-Logit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 12.7973 0.55593 11.7076 13.8869 0.05 4.25673 7.81473 0.24 11 Intercept -18.318 0.79845 -19.883 -16.753 TSCR 0.05162 0.00792 0.0361 0.06714 1.0 Point Logits % 95% Fiducial Limits 0.9 EC01 -4.595 11.8112 10.8893 12.6549 EC05 -2.944 15.8957 15.0424 16.6647 0.8 EC10 -2.197 18.1831 17.4007 18.8877 0.7 EC15 -1.735 19.7615 19.0348 20.4186 EC20 -1.386 21.0395 20.3585 21.66 0.6 EC25 -1.099 22.1573 21.514 22.7495 EC40 -0.405 25.1004 24.5262 25.6555 0.5 EC50 0.000 27 26.4288 27.5781 0.4 EC60 0.405 29.0434 28.4278 29.6981 Response EC75 1.099 32.9011 32.0844 33.8296 0.3 EC80 1.386 34.649 33.7049 35.7425 0.2 EC85 1.735 36.89 35.7602 38.2217 EC90 2.197 40.0921 38.6645 41.8048 0.1 EC95 2.944 45.8614 43.828 48.3523 0.0 EC99 4.595 61.7211 57.7222 66.7858 1 10 100 Dose % Dose-Response Plot
1
0.9
0.8 1-tail, 0.05 level of significance 0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
6.3
*25 *50
100
12.5 B-Control
Page 9 of 10 Report Ref: ECX18-0801-3
FISH LARVAL DEVELOPMENT -Proportion Normal Start Date: Test ID: ECX18-0805 Sample ID: ECX18-0805-3 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent + 0.17 g/L Sample Date: Protocol: WIECX-16 Test Species: S. lalandi Comments: Conc-% 1 2 3 B-Control 1.0000 0.9412 1.0000 6.3 1.0000 0.9412 12.5 0.9412 1.0000 1.0000 25 1.0000 0.9412 0.9412 50 0.1765 0.2941 0.4706 100 0.0100 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9804 1.0000 1.4558 1.3258 1.5208 7.732 3 6 300 6.3 0.9706 0.9900 1.4233 1.3258 1.5208 9.686 2 0.306 2.718 0.2888 6 200 12.5 0.9804 1.0000 1.4558 1.3258 1.5208 7.732 3 0.000 2.718 0.2583 6 300 25 0.9608 0.9800 1.3908 1.3258 1.5208 8.093 3 0.684 2.718 0.2583 12 300 *50 0.3137 0.3200 0.5876 0.4335 0.7560 27.519 3 9.137 2.718 0.2583 206 300 *100 0.0033 0.0034 0.0667 0.0500 0.1002 43.383 3 14.618 2.718 0.2583 299 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.95537 0.851 -0.0395 -1.0185 Bartlett's Test indicates equal variances (p = 0.62) 3.51303 15.0863 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Bonferroni t Test 25 50 35.3553 4 0.11982 0.12142 1.03121 0.01354 3.7E-08 5, 11
Maximum Likelihood-Probit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 8.14932 0.71297 6.75191 9.54674 0.02 1.21983 7.81473 0.75 1.64379 0.12271 6 Intercept -8.3958 1.19811 -10.744 -6.0475 TSCR 0.02183 0.00509 0.01186 0.03181 1.0 Point Probits % 95% Fiducial Limits 0.9 EC01 2.674 22.8205 19.4251 25.6202 EC05 3.355 27.6663 24.459 30.2571 0.8 EC10 3.718 30.6571 27.6395 33.0825 0.7 EC15 3.964 32.8556 30.0036 35.1507 EC20 4.158 34.7148 32.0146 36.8993 0.6 EC25 4.326 36.3935 33.8352 38.482 EC40 4.747 40.9923 38.806 42.8761 0.5 EC50 5.000 44.0342 42.0305 45.8785 0.4 EC60 5.253 47.3019 45.3806 49.2451 Response EC75 5.674 53.279 51.1227 55.8608 0.3 EC80 5.842 55.8554 53.4621 58.8756 0.2 EC85 6.036 59.0161 56.2521 62.676 EC90 6.282 63.2483 59.8875 67.902 0.1 EC95 6.645 70.0857 65.5966 76.595 0.0 EC99 7.326 84.968 77.5929 96.2893 1 10 100 Dose % Dose-Response Plot
1
0.9 1-tail, 0.05 level 0.8 of significance
0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
25
6.3
*50
100 12.5
B-Control
Page 10 of 10 Report Ref: ECX18-0801-3
ECOTOXICOLOGY LABORATORY TEST REPORT
CLIENT ATTN: GRANT GRIFFITH SOUTHERN SEAWATER JOINT VENTURE LOT 32 TARANTO ROAD BINNINGUP, WA 6233
JOB INFORMATION JOB REFERENCE : ECX18-0801-4 NO. SAMPLES : 1 CLIENT ORDER NO. : PO #94350 PROJECT : Comparative toxicity assessment SAMPLED BY : S. McNeil DATE RECEIVED : 01/08/18 DATE REPORTED : 18/10/2018
REPORT NOTES Comparative toxicity assessment of a four desalination effluent samples
TESTED BY COMPANY APPROVED SIGNATORY
Intertek Ecotoxicology Laboratory Intertek Geotech 41-45 Furnace Road Welshpool, Western Australia 6106
Tel: +61 8 9263 0263 DR TRISTAN STRINGER [email protected] PRINCIPAL ECOTOXICOLOGIST
This report relates specifically to the sample(s) tested that were drawn and/or provided by the client or their nominated third party to Intertek. The reported result(s) provide no warranty or verification on the sample(s) representing any specific goods and/or shipment. This report was prepared solely for the use of the client named in this report. Intertek accepts no responsibility for any loss, damage or liability suffered by a third party as a result of any reliance upon or use of this report.
Except where explicitly agreed in writing, all work and services performed by Intertek is subject to our standard Terms and Conditions which can be obtained at our website: intertek.com/terms
Page 1 of 11 Report Ref: ECX18-0801-4
Sample Information SAMPLE REFERENCE INTERTEK REFERENCE Whole Effluent + 2.0 g/L Memclean R ECX18-0801-4
Bioassay Details BIOASSAY PROTOCOL REFERENCE TEST SPECIES TEMPERATURE Microtox* WIECX-17 Microbics 1992 V. fischeri 15°C Microalgae Growth* WIECX-06 Stauber et al. 1994 I. galbana 22°C Macroalgae Zoospore Germination * WIECX-08 Burridge et al. 1999 E. radiata 22°C Mollusc Larvae Development* WIECX-23 ASTM E724 M. edulis 22°C Copepod Larval Development WIECX-26 ISO 16778 G. imparipes 22°C Fish Larvae Development* WIECX-16 USEPA 1001.0 S. lalandi 22°C * NATA Accredited method – Accreditation Number 5646
Physicochemistry PARAMETER CONTROL 6.3% 12.5% 25% 50% 100% pH 7.95 8.07 7.92 7.88 7.86 8.21 Salinity (‰) 34.4 36.4 37.7 40.4 49^ 63^ 86 DO (%) 100 100 100 100 100 100 ^ Refractometer reading - to be used for indicative purposes only
Page 2 of 11 Report Ref: ECX18-0801-4
Concentration-Response Data
BIOASSAY (% CONTROL) CONTROL 0.8% 1.6% 3.1% 6.3% 12.5% 25% 50% 100% Microtox* 100 ± 10 - - - 97± 15 92 ± 9 0 ± 0 0 ± 0 0 ± 0 Microalgae Growth* 100 ± 2 - 95 ± 2 102 ± 16 99 ± 12 124 ± 7 0 ± 0 - - Macroalgae Zoospore Germination * 100 ± 1 - - - 98 ± 5 60 ± 7 0 ± 0 0 ± 0 0 ± 0 Mollusc Larvae Development* 100 ± 2 101 ± 1 91 ± 3 22 ± 1 0 ± 0 0 ± 0 0 ± 0 0 ± 0 - Copepod Larval Development 100 ± 14 - 123 ± 14 110 ± 19 117 ± 8 129 ± 3 0 ± 0 - - Fish Larvae Development* 100 ± 6 - 94 ± 6 98 ± 9 92 ± 9 71 ± 12 0 ± 0 - - * NATA Accredited method – Accreditation Number 5646
Page 3 of 11 Report Ref: ECX18-0801-4
Statistical Effects Data
BIOASSAY EC10 (%) EC50 (%) NOEC (%) LOEC (%) Microtox* 13.0 15.7 12.5 25 Microalgae Growth* 7.0 8.2 6.3 12.5 Macroalgae Zoospore Germination * 10.1 13.2 6.3 12.5 Mollusc Larvae Development* 1.7 2.4 0.8 1.6 Copepod Larval Development 14.6 16.9 12.5 25 Fish Larvae Development* 11.0 14.3 6.3 12.5 * NATA Accredited method – Accreditation Number 5646
Quality Assurance Limits
BIOASSAY REFERENCE OBSERVED CUSUM CHART COEFFICIENT OF CONTROL TEST TOXICANT EC50 LIMITS VARIANCE RESPONSE ACCEPTABILITY Microtox* Phenol 17.0 mg/L 15.9 – 20.4 mg/L 6.32% < 90% PASS Microalgae Growth* > 3 x cell Copper 46.2 µg/L 21.9 – 55 µg/L 24.2% PASS divisions Macroalgae Zoospore >80% Copper 143 µg/L 59 – 225 µg/L 33.2% PASS Germination * Germinated Mollusc Larvae >80% Copper 5.9 µg/L 4.0 – 12.9 µg/L 30.6% PASS Development* Development 50-80% Copepod Larval Copper 23.2 µg/L 8.6 – 23.7 µg/L 26.2% copepodite PASS Development life stage Fish Larvae >80% Copper 45.2 µg/L 21.6 – 82.9 µg/L 33.1% PASS Development* Development * NATA Accredited method – Accreditation Number 5646
General Comments Quality assurance and quality control criteria were within acceptable limits for all bioassays. A detailed interpretation of the results and species sensitivity distribution is included in the interpretative report (Report No. ECX18-0801)
Page 4 of 11 Report Ref: ECX18-0801-4
STATISTICAL DATA
Page 5 of 11 Report Ref: ECX18-0801-4
MICROTOX
Start Date: Test ID: ECX18-0801 Sample ID: ECX0801-4 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent + 2g/L Sample Date: Protocol: WIECX-17 Test Species: Microtox Comments: Conc-% 1 2 3 B-Control 0.9700 0.8900 0.7900 6.3 0.8600 0.9900 0.7300 12.5 0.9100 0.7900 0.7500 25 0.0000 0.0000 0.0100 50 0.0000 0.0000 0.0000 100 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.8833 1.0000 1.2414 1.0948 1.3967 12.177 3 35 300 6.3 0.8600 0.9736 1.2274 1.0244 1.4706 18.397 3 0.115 2.420 0.2930 42 300 12.5 0.8167 0.9245 1.1360 1.0472 1.2661 10.135 3 0.870 2.420 0.2930 55 300 *25 0.0033 0.0038 0.0667 0.0500 0.1002 43.383 3 9.703 2.420 0.2930 299 300 50 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300 100 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.95973 0.805 0.43154 0.03338 Bartlett's Test indicates equal variances (p = 0.19) 4.78772 11.3449 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 12.5 25 17.6777 8 0.2352 0.26269 0.97253 0.02198 2.5E-05 3, 8
Maximum Likelihood-Logit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 27.4329 1.85183 23.8033 31.0625 0.11667 0.36497 7.81473 0.95 13 Intercept -32.787 2.13082 -36.964 -28.611 TSCR 0.12832 0.01366 0.10155 0.15509 1.0 Point Logits % 95% Fiducial Limits 0.9 EC01 -4.595 10.6582 10.0403 11.1956 EC05 -2.944 12.2421 11.697 12.741 0.8 EC10 -2.197 13.0345 12.5116 13.5336 EC15 -1.735 13.5506 13.0339 14.0599 0.7 EC20 -1.386 13.9526 13.4353 14.4762 0.6 EC25 -1.099 14.2936 13.7719 14.8338 EC40 -0.405 15.1499 14.601 15.7507 0.5 EC50 0.000 15.6743 15.098 16.3249 0.4 EC60 0.405 16.2169 15.6043 16.9283 Response EC75 1.099 17.1884 16.4936 18.0292 0.3 EC80 1.386 17.6085 16.8724 18.5124 0.2 EC85 1.735 18.1309 17.3393 19.1185 EC90 2.197 18.8488 17.9744 19.9599 0.1 EC95 2.944 20.0688 19.0396 21.4093 0.0 EC99 4.595 23.0512 21.5883 25.0339 1 10 100 Dose % Dose-Response Plot
1
0.9
0.8
0.7 1-tail, 0.05 level 0.6 of significance
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
50
6.3
*25
100 12.5
B-Control
Page 6 of 11 Report Ref: ECX18-0801-4
MICROALGAE GROWTH -Proportion Normal Start Date: Test ID: ECX18-0801 Sample ID: ECX0801-4 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent + 2g/L Sample Date: Protocol: WIECX-06 Test Species: TISO-Isochrysis sp. Comments: Conc-% 1 2 3 B-Control 0.9801 0.9371 0.9470 1.6 0.8311 1.0000 1.0000 3.1 0.9503 0.8907 1.0000 6.3 1.0000 1.0000 1.0000 12.5 0.0100 0.0000 0.0000 25 0.0000 0.0000 0.0000 50 0.0000 0.0000 0.0000 100 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Isotonic Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Mean N-Mean B-Control 0.9547 1.0000 1.3617 1.3173 1.4294 4.373 3 0.9617 1.0000 1.6 0.9437 0.9884 1.3963 1.1473 1.5208 15.442 3 -0.353 2.470 0.2416 0.9617 1.0000 3.1 0.9470 0.9919 1.3669 1.2339 1.5208 10.577 3 -0.053 2.470 0.2416 0.9617 1.0000 6.3 1.0000 1.0474 1.5208 1.5208 1.5208 0.000 3 -1.626 2.470 0.2416 0.9617 1.0000 *12.5 0.0033 0.0035 0.0667 0.0500 0.1002 43.383 3 13.237 2.470 0.2416 0.0033 0.0035 25 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 0.0000 0.0000 50 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 0.0000 0.0000 100 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 0.0000 0.0000
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.90956 0.835 -0.7983 1.69 Equality of variance cannot be confirmed Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 6.3 12.5 8.87412 15.873 0.14667 0.15327 1.09739 0.01436 1.9E-07 4, 10
Log-Logit Interpolation (200 Resamples) Point % SD 95% CL(Exp) Skew IC05 6.7479 0.3978 6.0329 7.2669 -11.2515 IC10 7.0175 0.1851 6.1768 7.6763 0.3148 IC15 7.2204 0.2132 6.2467 7.9499 0.2292 1.0 IC20 7.3889 0.2375 6.3176 8.1642 0.1458 0.9 IC25 7.5369 0.2596 6.3774 8.3458 0.0776 IC40 7.9219 0.3200 6.5243 8.7975 -0.0603 0.8 IC50 8.1632 0.3598 6.6114 9.0745 -0.1198 0.7 0.6 0.5 0.4
Response 0.3 0.2 0.1 0.0 -0.1 1 10 100 Dose % Dose-Response Plot
1
0.9
0.8 1-tail, 0.05 level of significance 0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
25 50
1.6 3.1 6.3
100 12.5
B-Control
Page 7 of 11 Report Ref: ECX18-0801-4
MACROALGAE ZOOSPORE GERMINATION -Proportion Normal Start Date: Test ID: ECX18-0801 Sample ID: ECX0801-4 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent + 2g/L Sample Date: Protocol: WIECX-08 Test Species: E. radiata Comments: Conc-% 1 2 3 B-Control 1.0000 1.0000 0.9885 6.3 1.0000 0.9295 1.0000 12.5 0.5293 0.6280 0.6536 25 0.0100 0.0000 0.0000 50 0.0000 0.0000 0.0000 100 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9962 1.0000 1.5016 1.4633 1.5208 2.209 3 1 300 6.3 0.9765 0.9803 1.4479 1.3020 1.5208 8.723 3 0.881 2.420 0.1477 7 300 *12.5 0.6036 0.6060 0.8904 0.8147 0.9415 7.511 3 10.013 2.420 0.1477 119 300 *25 0.0033 0.0033 0.0667 0.0500 0.1002 43.383 3 23.504 2.420 0.1477 299 300 50 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300 100 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.91168 0.805 -1.1046 1.23504 Bartlett's Test indicates equal variances (p = 0.22) 4.46469 11.3449 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 6.3 12.5 8.87412 15.873 0.04154 0.04174 1.33302 0.00559 3.7E-08 3, 8
Maximum Likelihood-Logit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 19.0698 1.35421 16.4156 21.7241 0.00333 2.2825 7.81473 0.52 18 Intercept -21.353 1.49447 -24.282 -18.424 TSCR 0.01142 0.00462 0.00237 0.02047 1.0 Point Logits % 95% Fiducial Limits 0.9 EC01 -4.595 7.56401 6.94675 8.06927 EC05 -2.944 9.23234 8.74941 9.62004 0.8 EC10 -2.197 10.104 9.70841 10.4213 EC15 -1.735 10.6845 10.3505 10.9544 0.7 EC20 -1.386 11.1434 10.8578 11.3778 0.6 EC25 -1.099 11.5373 11.2907 11.7448 EC40 -0.405 12.5445 12.3624 12.7232 0.5 EC50 0.000 13.1739 12.9845 13.3855 0.4 EC60 0.405 13.8349 13.6007 14.121 Response EC75 1.099 15.0427 14.6767 15.5214 0.3 EC80 1.386 15.5744 15.1401 16.1509 0.2 EC85 1.735 16.2433 15.7177 16.9507 EC90 2.197 17.1765 16.5157 18.0783 0.1 EC95 2.944 18.7983 17.8861 20.0657 0.0 EC99 4.595 22.9445 21.3186 25.2785 1 10 100 Dose % Dose-Response Plot
1 1-tail, 0.05 level 0.9 of significance
0.8
0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
25 50
6.3
100 *12.5
B-Control
Page 8 of 11 Report Ref: ECX18-0801-4
MOLLUSC LARVAL DEVELOPMENT -Proportion Normal Start Date: Test ID: ECX18-0801 Sample ID: ECX0801-4 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent 2g/L Sample Date: Protocol: WIECX-23 Test Species: M. edulis Comments: Conc-% 1 2 3 B-Control 1.0000 1.0000 0.9699 0.8 1.0000 1.0000 1.0000 1.6 0.8760 0.9377 0.9046 3.1 0.2226 0.2139 6.3 0.0000 0.0000 0.0000 12.5 0.0000 0.0000 0.0000 25 0.0000 0.0000 0.0000 50 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9900 1.0000 1.4793 1.3963 1.5208 4.859 3 3 300 0.8 1.0000 1.0102 1.5208 1.5208 1.5208 0.000 3 -1.056 2.642 0.1038 0 300 *1.6 0.9061 0.9153 1.2621 1.2110 1.3186 4.279 3 5.524 2.642 0.1038 28 300 *3.1 0.2182 0.2204 0.4860 0.4807 0.4913 1.539 2 22.600 2.642 0.1161 157 200 6.3 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300 12.5 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300 25 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300 50 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.9012 0.792 -0.7164 0.73767 Equality of variance cannot be confirmed Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Bonferroni t Test 0.8 1.6 1.13137 125 0.03414 0.03443 0.51513 0.00232 2.7E-07 3, 7
Maximum Likelihood-Logit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 13.3031 0.44999 12.4211 14.1851 0.01 3.87368 11.0705 0.57 6 Intercept -5.1543 0.18729 -5.5214 -4.7872 TSCR 0.00524 0.0032 -0.001 0.0115 1.0 Point Logits % 95% Fiducial Limits 0.9 EC01 -4.595 1.10163 1.03527 1.16393 EC05 -2.944 1.46595 1.40267 1.52505 0.8 EC10 -2.197 1.66835 1.60815 1.72483 EC15 -1.735 1.80743 1.74941 1.86226 0.7 EC20 -1.386 1.91975 1.86327 1.97357 0.6 EC25 -1.099 2.01776 1.96234 2.07107 EC40 -0.405 2.27496 2.22017 2.32944 0.5 EC50 0.000 2.44036 2.38376 2.49806 0.4 EC60 0.405 2.61777 2.55704 2.68136 Response EC75 1.099 2.95146 2.87719 3.03243 0.3 EC80 1.386 3.10214 3.01972 3.19326 0.2 EC85 1.735 3.29491 3.20061 3.40067 EC90 2.197 3.5696 3.45605 3.69892 0.1 EC95 2.944 4.06245 3.90929 4.24024 0.0 EC99 4.595 5.40593 5.12288 5.74427 0.1 1 10 100 Dose % Dose-Response Plot
1 1-tail, 0.05 level of significance 0.9
0.8
0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
25 50
0.8 6.3
*1.6 *3.1 12.5
B-Control
Page 9 of 11 Report Ref: ECX18-0801-4
COPEPOD LARVAL DEVELOPMENT -Proportion Normal Start Date: Test ID: ECX18-0801 Sample ID: ECX18-0801-4 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent + 2.0g/L Sample Date: Protocol: WIECX-26 Test Species: GI-Gladioferens Imparipes Comments: Conc-% 1 2 3 B-Control 0.9615 0.8846 1.0000 1.6 1.0000 1.0000 1.0000 3.1 1.0000 0.9615 6.3 1.0000 1.0000 12.5 1.0000 1.0000 25 0.0100 0.0000 0.0000 50 0.0000 0.0000 0.0000 100 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Isotonic Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Mean N-Mean B-Control 0.9487 1.0000 1.3728 1.2242 1.5208 10.801 3 0.9853 1.0000 1.6 1.0000 1.0541 1.5208 1.5208 1.5208 0.000 3 -2.287 2.821 0.1825 0.9853 1.0000 3.1 0.9808 1.0338 1.4471 1.3734 1.5208 7.201 2 -1.027 2.821 0.2041 0.9853 1.0000 6.3 1.0000 1.0541 1.5208 1.5208 1.5208 0.000 2 -2.046 2.821 0.2041 0.9853 1.0000 12.5 1.0000 1.0541 1.5208 1.5208 1.5208 0.000 2 -2.046 2.821 0.2041 0.9853 1.0000 *25 0.0033 0.0035 0.0667 0.0500 0.1002 43.383 3 20.187 2.821 0.1825 0.0033 0.0034 50 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 0.0000 0.0000 100 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 0.0000 0.0000
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates non-normal distribution (p <= 0.01) 0.83493 0.835 -0.0038 3.15912 Equality of variance cannot be confirmed Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Bonferroni t Test 12.5 25 17.6777 8 0.09926 0.10326 0.9567 0.00628 2.0E-08 5, 9
Log-Logit Interpolation (200 Resamples) Point % SD 95% CL(Exp) Skew IC05 13.932 0.449 12.585 16.262 1.9520 IC10 14.562 0.520 12.758 16.964 1.2611 IC15 14.999 0.566 12.899 17.390 0.8896 1.0 IC20 15.347 0.603 13.018 17.711 0.6366 0.9 IC25 15.646 0.636 13.124 17.978 0.4467 IC40 16.402 0.726 13.397 18.625 0.0661 0.8 IC50 16.867 0.786 13.565 19.108 -0.1053 0.7 0.6 0.5 0.4
0.3 Response 0.2 0.1 0.0 -0.1 -0.2 1 10 100 Dose % Dose-Response Plot
1
0.9 1-tail, 0.05 level 0.8 of significance
0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
50
1.6 3.1 6.3
*25
100 12.5
B-Control
Page 10 of 11 Report Ref: ECX18-0801-4
FISH LARVAL DEVELOPMENT -Proportion Normal Start Date: Test ID: ECX18-0805 Sample ID: ECX18-0805-4 End Date: Lab ID: FREO-Geotech Fremantle LabSample Type: Whole Effluent + 2 g/L Sample Date: Protocol: WIECX-16 Test Species: S. lalandi Comments: Conc-% 1 2 3 B-Control 1.0000 0.9412 1.0000 1.6 1.0000 0.8824 0.9412 3.1 0.8824 1.0000 1.0000 6.3 0.9412 1.0000 0.8235 12.5 0.7059 0.5882 0.8235 25 0.0100 0.0000 0.0000 50 0.0000 0.0000 0.0000 100 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9804 1.0000 1.4558 1.3258 1.5208 7.732 3 6 300 1.6 0.9412 0.9600 1.3558 1.2207 1.5208 11.231 3 0.863 2.500 0.2897 18 300 3.1 0.9608 0.9800 1.4207 1.2207 1.5208 12.195 3 0.302 2.500 0.2897 12 300 6.3 0.9216 0.9400 1.3280 1.1373 1.5208 14.441 3 1.103 2.500 0.2897 24 300 *12.5 0.7059 0.7200 1.0030 0.8741 1.1373 13.127 3 3.908 2.500 0.2897 88 300 *25 0.0033 0.0034 0.0667 0.0500 0.1002 43.383 3 11.989 2.500 0.2897 299 300 50 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300 100 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.954 0.858 -0.1994 -0.9135 Bartlett's Test indicates equal variances (p = 0.49) 4.43669 15.0863 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 6.3 12.5 8.87412 15.873 0.14184 0.14373 0.85424 0.02014 3.2E-07 5, 12
Maximum Likelihood-Probit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 11.1213 1.13797 8.89083 13.3517 0.02 6.94047 11.0705 0.23 1.15579 0.08992 9 Intercept -7.8539 1.27266 -10.348 -5.3594 TSCR 0.04998 0.00629 0.03765 0.06231 1.0 Point Probits % 95% Fiducial Limits 0.9 EC01 2.674 8.84324 7.95975 9.50805 EC05 3.355 10.1833 9.46341 10.7309 0.8 EC10 3.718 10.9789 10.3615 11.4639 0.7 EC15 3.964 11.5504 11.0013 12.0017 EC20 4.158 12.0258 11.5243 12.4616 0.6 EC25 4.326 12.4492 11.9791 12.8849 EC40 4.747 13.5835 13.1257 14.1023 0.5 EC50 5.000 14.315 13.809 14.9527 0.4 EC60 5.253 15.0859 14.494 15.8915 Response EC75 5.674 16.4604 15.66 17.639 0.3 EC80 5.842 17.0399 16.138 18.3963 0.2 EC85 6.036 17.7413 16.7088 19.3258 EC90 6.282 18.6649 17.4503 20.5688 0.1 EC95 6.645 20.123 18.6021 22.5696 0.0 EC99 7.326 23.1724 20.9542 26.8847 1 10 100 Dose % Dose-Response Plot
1
0.9 1-tail, 0.05 level 0.8 of significance
0.7
0.6
0.5
0.4
ProportionNormal 0.3
0.2
0.1
0
50
1.6 3.1 6.3
*25
100
*12.5 B-Control
Page 11 of 11 Report Ref: ECX18-0801-4
Appendix B Intertek Ecotoxicology Laboratory Report 15/05/2020
120WAU-0008/ R200092 Water Corporation 28 Onslow Seawater Desalination Plant: Desalination Brine Toxicity Assessment
ECOTOXICOLOGY LABORATORY TEST REPORT
CLIENT ATTN: JOSH ABBOTT O2 MARINE WA MARINE PTY LTD PO BOX 1370 DUNSBOROUGH WA 6281
JOB INFORMATION JOB REFERENCE : 66984 INTERTEK REFERENCE : 905112 CLIENT ORDER NO. : 0033 PROJECT : BEADON BAY DESALINATION PLANT DATE SAMPLED : 30/03/2020 DATE RECEIVED : 01/04/2020 DATE REPORTED : 15/05/2020
REPORT NOTES Sample received in good condition
TESTED BY COMPANY APPROVED SIGNATORY
Intertek Ecotoxicology Laboratory Intertek Geotech 41-45 Furnace Road Welshpool, Western Australia 6106
Tel: +61 8 9263 0263 DR TRISTAN STRINGER [email protected] PRINCIPAL ECOTOXICOLOGIST
This analytical report has been prepared for the titled project or named part thereof and should not be relied upon or used for any other project without an independent check being carried out as to its suitability and prior written authority of Intertek being obtained. This report relates specifically to the sample(s) tested that were drawn and/or provided by the client or their nominated third party to Intertek. Intertek accepts no responsibility or liability for the consequences of this document being used for a purpose other than the purposes for which it was commissioned. Any person using or relying on the document for such other purposes agrees and will by such use or reliance be taken to confirm his agreement to indemnify Intertek for all loss or damage resulting therefrom. Intertek accepts no responsibility or liability for this document to any party other than the person by whom it was commissioned.
NATA Accreditation Number: 5646 Accredited for compliance with ISO/IEC 17025 – Testing This report shall not be reproduced, except in full.
Page 1 of 8 Report Ref: 66984 - 905112
List of Revisions REV. DATE REVISION DETAILS AUTHOR ISSUE 0 15/05/2020 Document prepared Dr Tristan Stringer Dr Tristan Stringer This document replaces and supersedes all previous versions.
Sample Information SAMPLE REFERENCE SAMPLE DATE MATRIX PRESERVATION INTERTEK REFERENCE Desalination Brine 30/03/2020 Water Chilled 905112
Additional Sample Information
One (1) Desalination Brine sample was received on 01/04/2020 for testing as per quote QU20036
Methodology Following the protocol outlined in ANZECC/ARMCANZ (2000) and ANZG (2018), a suite of bioassays was used to assess the toxicity of one Desalination Brine. For quality assurance purposes reference toxicant) bioassays were tested simultaneously. Statistically calculated effect concentrations (EC10, EC50, NOEC and LOEC) have been reported the samples and reference toxicants.
Bioassay Details BIOASSAY PROTOCOL REFERENCE TEST SPECIES TEMPERATURE Microalgae Growth WIECX-06 Stauber et al. 1994 N. closterium 22°C Sea Urchin Development WIECX-25 ASTM E1563 E. mathaei 25°C Milky Oyster Larval Development [1] ESA SOP 106 ESA 2016 S. echinate 29°C Sea Anemone Pedal Lacerate ESA SOP 128 ESA 2014 A. pulchella 25°C Development [1] Fish Imbalance and Biomass [1] ESA SOP 122 ESA 2017 L. calcarifer 25°C Note [1] – Subcontracted test
Page 2 of 8 Report Ref: 66984 - 905112
RESULTS
Sample Information SAMPLE REFERENCE INTERTEK REFERENCE Desalination Brine 905112
Physicochemistry PARAMETER CONTROL 3.1% 6.3% 12.5% pH [2] 8.2 8.2 8.2 8.2 Salinity (‰) [2] 36.1 37.5 38.3 40.1 DO (%) [2] 100 100 100 100
PARAMETER 25% 50% 100% pH [2] 8.2 8.2 8.1 Salinity (‰) [2] 43.5 50.2 64.0 DO (%) [2] 100 100 100
Note [2] – Non-NATA Accredited Method
Page 3 of 8 Report Ref: 66984 - 905112
Concentration-Response Data
BIOASSAY (N-MEAN) CONTROL 3.1% 6.3% 12.5% 25% 50% 100% Microalgae Growth 100 ± 11 95 ± 9 78 ± 10 85 ± 5 74 ± 5 45 ±9 7 ± 1 Sea Urchin Development 100 ± 3 91 ± 3 90 ± 2 87 ± 1 5 ± 1 0 ± 0 0 ± 0 Milky Oyster Larval Development [1] 100 ± 3 101 ± 4 100 ± 3 71 ± 4 0 ± 0 0 ± 0 0 ± 0 Sea Anemone Pedal Lacerate Development [1] 100 ± 11 100 ± 11 100 ± 11 101 ± 9 0 ± 0 0 ± 0 0 ± 0 Fish Imbalance [1] 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 0 ± 0 0 ± 0 Fish Biomass [1] 100 ± 3 103 ± 4 102 ± 2 100 ± 1 89 ± 8 0 ± 0 0 ± 0 Note [1] – Subcontracted test
Page 4 of 8 Report Ref: 66984
Statistical Effects Data
BIOASSAY EC10 (%) EC50 (%) NOEC (%) LOEC (%) Microalgae Growth 21.3 46.6 3.1 6.3 Sea Urchin Development 13.0 17.2 <3.1 3.1 Milky Oyster Larval Development [1] 8.3 14.5 6.3 12.5 Sea Anemone Pedal Lacerate 13.0 17.5 12.5 25 Development [1] Fish Imbalance [1] 32.6 35.4 25 50 Fish Biomass [1] 22.6 35.8 12.5 25 Note [1] – Subcontracted test
Quality Assurance Limits REFERENCE OBSERVED CUSUM CHART COEFFICIENT OF CONTROL TEST BIOASSAY TOXICANT EC50 LIMITS VARIANCE RESPONSE ACCEPTABILITY > 3 x cell Microalgae Growth Copper 44.9 µg/L 18.7 – 72.0 µg/L 34.2% PASS divisions >80% Sea Urchin Development Copper 16.1 µg/L 15.0 – 33.1 µg/L 18.8% PASS Development Milky Oyster Larval >70% Copper 14.5 µg/L 13.1 – 18.3 µg/L NA PASS Development [1] Development Sea Anemone Pedal >90% Copper 6.9 µg/L 8.4 – 24.6 µg/L NA PASS Lacerate Development [1] Development Fish Imbalance and >80% Ammonium 19.0 mg/L 9.2 – 118 mg/L NA PASS Biomass [1] Unaffected Note [1] – Subcontracted test
Page 5 of 8 Report Ref: 66984
APPENDIX
Statistical Data
Page 6 of 8 Report Ref: 66984
MICROALGAL GROWTH
Start Date: Test ID: 66984 Sample ID: 905112 End Date: Lab ID: ECOTOX-Intertek Geotech Sample Type: Brine Sample Date: Protocol: WIECX-06-Marine MicroalgaeTest Growth Species: NC-Nitzschia closterium Comments: Conc-% 1 2 3 B-Control 1.0000 0.9957 0.8936 3.1 0.9191 1.0000 0.8809 6.3 0.8043 0.6766 0.8681 12.5 0.7915 0.8809 0.8681 25 0.6894 0.7787 0.7532 50 0.4213 0.5489 0.3830 100 0.0638 0.0638 0.0766
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9631 1.0000 1.4216 1.2386 1.5208 11.165 3 11 300 3.1 0.9333 0.9691 1.3405 1.2184 1.5208 11.887 3 0.933 2.530 0.2199 20 300 *6.3 0.7830 0.8130 1.0925 0.9659 1.1991 10.790 3 3.786 2.530 0.2199 65 300 *12.5 0.8468 0.8792 1.1714 1.0966 1.2184 5.588 3 2.879 2.530 0.2199 46 300 *25 0.7404 0.7688 1.0372 0.9796 1.0811 5.023 3 4.423 2.530 0.2199 78 300 *50 0.4511 0.4683 0.7360 0.6673 0.8344 11.878 3 7.888 2.530 0.2199 165 300 *100 0.0681 0.0707 0.2637 0.2554 0.2804 5.474 3 13.321 2.530 0.2199 280 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.98718 0.873 -0.1043 -0.1673 Bartlett's Test indicates equal variances (p = 0.21) 8.37026 16.8119 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 3.1 6.3 4.41928 32.2581 0.10806 0.1105 0.47211 0.01133 3.9E-08 6, 14
Maximum Likelihood-Logit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 6.44149 0.83245 4.13023 8.75276 0.03667 44.3696 9.48773 5.4E-09 22 Intercept -10.75 1.42879 -14.717 -6.7828 TSCR 0.11096 0.03304 0.01923 0.20269 1.0 Point Logits % 95% Fiducial Limits 0.9 EC01 -4.595 9.02537 3.25963 14.7634 EC05 -2.944 16.2825 8.09787 23.0267 0.8 EC10 -2.197 21.2678 12.1788 28.2671 0.7 EC15 -1.735 25.0924 15.6404 32.1736 EC20 -1.386 28.4193 18.8428 35.5409 0.6 EC25 -1.099 31.4974 21.9327 38.6635 0.5 EC40 -0.405 40.3535 31.2195 47.9714 0.4
EC50 0.000 46.6474 37.8282 55.2188 Response EC60 0.405 53.9229 45.0679 64.6443 0.3 EC75 1.099 69.0844 58.2272 88.3662 0.2 EC80 1.386 76.5668 63.957 101.87 EC85 1.735 86.7187 71.2177 121.75 0.1 EC90 2.197 102.313 81.6038 155.314 0.0 EC95 2.944 133.639 100.763 232.223 1 10 100 1000 EC99 4.595 241.096 157.947 574.039 Dose % Significant heterogeneity detected (p = 5.38E-09)
Dose-Response Plot
1
0.9 1-tail, 0.05 level 0.8 of significance
0.7
0.6
0.5
0.4
0.3 ProportionNormal 0.2
0.1
0
3.1
*25 *50
*6.3
*100 *12.5
B-Control
Page 7 of 8 Report Ref: 66984
SEA URCHIN LARVAL DEVELOPMENT
Start Date: Test ID: 66984 Sample ID: 905112 End Date: Lab ID: ECOTOX-Intertek Geotech Sample Type: Brine Sample Date: Protocol: WIECX-25-Sea Urchin Larval TestDevelopment Species: EM-Echinometra mathaei Comments: Conc-% 1 2 3 B-Control 1.0000 0.9734 1.0000 3.1 0.9004 0.9493 0.8817 6.3 0.9170 0.8753 0.8940 12.5 0.8636 0.8578 0.8737 25 0.0576 0.0464 0.0411 50 0.0000 0.0000 0.0000 100 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number B-Control 0.9911 1.0000 1.4828 1.4070 1.5208 4.431 3 3 300 *3.1 0.9105 0.9186 1.2710 1.2197 1.3437 5.088 3 5.742 2.470 0.0911 27 300 *6.3 0.8954 0.9034 1.2425 1.2099 1.2786 2.775 3 6.513 2.470 0.0911 31 300 *12.5 0.8650 0.8728 1.1947 1.1841 1.2075 0.992 3 7.810 2.470 0.0911 41 300 *25 0.0484 0.0488 0.2213 0.2042 0.2424 8.790 3 34.196 2.470 0.0911 285 300 50 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300 100 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 3 300 300
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.98356 0.835 -0.1062 0.12819 Bartlett's Test indicates equal variances (p = 0.22) 5.72769 13.2767 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test <3.1 3.1 0.02401 0.0242 0.73179 0.00204 9.5E-11 4, 10
Maximum Likelihood-Logit Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter Slope 17.9212 0.69458 16.5599 19.2826 0.01 8.42536 9.48773 0.08 16 Intercept -22.177 0.88868 -23.919 -20.435 TSCR 0.06754 0.0084 0.05108 0.084 1.0 Point Logits % 95% Fiducial Limits 0.9 EC01 -4.595 9.57329 8.9877 10.1154 EC05 -2.944 11.835 11.2807 12.3475 0.8 EC10 -2.197 13.0276 12.4956 13.5217 0.7 EC15 -1.735 13.8254 13.3087 14.3081 EC20 -1.386 14.4581 13.9528 14.9332 0.6 EC25 -1.099 15.0026 14.506 15.4724 0.5 EC40 -0.405 16.4 15.919 16.8653 0.4
EC50 0.000 17.277 16.7985 17.748 Response EC60 0.405 18.2009 17.7173 18.6869 0.3 EC75 1.099 19.8962 19.3796 20.4359 0.2 EC80 1.386 20.6453 20.1042 21.2198 EC85 1.735 21.5902 21.0101 22.2176 0.1 EC90 2.197 22.9125 22.2641 23.6292 0.0 EC95 2.944 25.2212 24.4226 26.1297 1 10 100 EC99 4.595 31.1798 29.8716 32.7304 Dose %
Dose-Response Plot
1 1-tail, 0.05 level of significance 0.9
0.8
0.7
0.6
0.5
0.4
0.3 ProportionNormal 0.2
0.1
0
50
*25
100
*3.1 *6.3 *12.5
B-Control
Page 8 of 8 Report Ref: 66984
Toxicity Assessment of a Brine Sample
Intertek
Test Report
April 2020
Toxicity Test Report: TR1915/1 (Page 1 of 2)
Accredited for compliance with ISO/IEC 17025 - Testing
Client: Intertek ESA Job #: PR1915 1 Fleet Street Date Sampled: 30 March 2020 Fremantle WA 6160 Date Received: 03 April 2020 Attention: Tristan Stringer Sampled By: Client Client Ref: Not Supplied ESA Quote #: PL1915_Q01
Lab ID No.: Sample Name: Sample Description: 9516 Brine Aqueous sample, pH 8.1*, salinity 64.0‰*, total ammonia <2.0 mg/L*. Sample received at 9ºC* in apparent good condition *NATA accreditation does not cover the performance of this service
Test Performed: 48-hr larval development test using the milky oyster Saccostrea echinata Test Protocol: ESA SOP 106 (ESA 2016), based on APHA (1998) and Krassoi (1995) Test Temperature: The test was performed at 29±1°C. Deviations from Protocol: Nil Comments on Solution The sample was serially diluted with filtered seawater (FSW) to Preparation: achieve the test concentrations. A FSW control was tested concurrently with the sample. Three additional salinity controls were also tested concurrently with the sample. Source of Test Organisms: Field collected from Mackay, QLD. Test Initiated: 03 April 2020 at 1730h
Controls Sample 9516: Brine Vacant Concentration % Normal Concentration % Normal (%) larvae (%) larvae (Mean SD) (Mean SD) FSW Control 77.0 2.5 FSW Control 77.0 2.5 12.5% Sal Ctrl 50.3 5.4 * 3.1 78.0 3.9 25% Sal Ctrl 0.0 0.0 6.3 77.5 2.4 50% Sal Ctrl 0.0 0.0 12.5 55.0 3.4 ** 25 0.0 0.0 50 0.0 0.0 100 0.0 0.0
48-hr IC10 = 8.3 (7.22-8.61)% 48-hr EC50 = 14.5 (14.03-14.93)% NOEC = 6.3% LOEC = 12.5% *Significantly lower percentage of normal larvae compared with the FSW Control (Homoscedastic Test, 1-tailed, P=0.05) **Significantly lower percentage of normal larvae compared with the FSW Control (Dunnett’s Test, 1-tailed, P=0.05)
QA/QC Parameter Criterion This Test Criterion met? FSW Control mean % normal ≥70% 77.0% Yes Reference Toxicant within cusum chart limits 13.1-18.3µg Cu/L 14.5µg Cu/L Yes
Toxicity Test Report: TR1915/1 (Page 2 of 2)
Test Report Authorised by: Dr Rick Krassoi, Director on 23 April 2020
Results are based on the samples in the condition as received by ESA.
NATA Accredited Laboratory Number: 14709 This document shall not be reproduced except in full.
Citations:
APHA (1998) Standard Methods for the Examination of Water and Wastewater. 20th Ed. American Public Health Association, American Water Works Association and the Water Environment Federation, Washington, DC.
ESA (2016) SOP 106 – Bivalve Larval Development Test. Issue No. 15. Ecotox Services Australasia, Sydney, NSW.
Krassoi, R (1995) Salinity adjustment of effluents for use with marine bioassays: effects on the larvae of the doughboy scallop Chlamys asperrimus and the Sydney rock oyster Saccostrea commercialis. Australasian Journal of Ecotoxicology, 1: 143-148.
Toxicity Test Report: TR1915/2 (Page 1 of 2)
Client: Intertek ESA Job #: PR1915 1 Fleet Street Date Sampled: 30 March 2020 Fremantle WA 6160 Date Received: 03 April 2020 Attention: Tristan Stringer Sampled By: Client Client Ref: Not Supplied ESA Quote #: PL1915_Q01
Lab ID No.: Sample Name: Sample Description: 9516 Brine Aqueous sample, pH 8.1, salinity 64.0‰, total ammonia <2.0 mg/L. Sample received at 9ºC in apparent good condition
Test Performed: 8-day Sea anemone pedal lacerate development test using Aiptasia pulchella Test Protocol: ESA SOP 128 (ESA 2014), based on Howe et al (2014) Test Temperature: The test was performed at 25±1°C. Deviations from Protocol: Nil Comments on Solution The sample was serially diluted with filtered seawater (FSW) to Preparation: achieve the test concentrations. A FSW control was tested concurrently with the sample. Three additional salinity controls were also tested concurrently with the sample. Source of Test Organisms: In-house culture, originally sourced from Marine Ecology Research Centre at Southern Cross University, Lismore, NSW Test Initiated: 03 April 2020 at 1800h
Controls Sample 9516: Brine Vacant Concentration % Normal Concentration % Normal (%) (Mean SD) (%) (Mean SD) FSW Control 90.0 11.6 FSW Control 90.0 11.6 12.5% Sal Ctrl 85.0 10.0 3.1 90.0 11.6 25% Sal Ctrl 0.0 0.0 6.3 95.0 10.0 50% Sal Ctrl 0.0 0.0 12.5 90.0 11.6 25 0.0 0.0 50 0.0 0.0 100 0.0 0.0
8-d IC10 = 13.0%* 8-d IC50 = 17.5 (16.75-18.19)% NOEC = 12.5% LOEC = 25% *95% Confidence Limits are not reliable.
QA/QC Parameter Criterion This Test Criterion met? Control mean % unaffected ≥90.0% 90.0% Yes Reference Toxicant within cusum chart limits 8.4-24.6 µg Cu/L 9.6µg Cu/L Yes
Toxicity Test Report: TR1915/2 (Page 2 of 2)
Test Report Authorised by: Dr Rick Krassoi, Director on 23 April 2020
Results are based on the samples in the condition as received by ESA.This document shall not be reproduced except in full.
Citations:
ESA (2014) SOP 128 – Sea Anemone Pedal Lacerate Development Toxicity Test. Issue No 1. Ecotox Services Australasia, Sydney, NSW.
Howe, Pelli L., Reichelt-Brushett, Amanda J. and Clark, Malcolm W (2014) Development of a chronic, early life-stage sub-lethal toxicity test and recovery assessment for the tropical zooxanthellate sea anemone Aiptasia pulchella. Ecotoxicology and Environmental Safety 100: 138-147.
Toxicity Test Report: TR1915/3 (Page 1 of 2)
Client: Intertek ESA Job #: PR1915 1 Fleet Street Date Sampled: 30 March 2020 Fremantle WA 6160 Date Received: 03 April 2020 Attention: Tristan Stringer Sampled By: Client Client Ref: Not Supplied ESA Quote #: PL1915_Q01
Lab ID No.: Sample Name: Sample Description: 9516 Brine Aqueous sample, pH 8.1, salinity 64.0‰, total ammonia <2.0 mg/L. Sample received at 9ºC in apparent good condition
Test Performed: 7-day fish imbalance and biomass toxicity test using barramundi Lates calcarifer Test Protocol: ESA SOP 122 (ESA 2017), based on USEPA (2002) Test Temperature: The test was performed at 25±2°C. Deviations from Protocol: Nil Comments on Solution The sample was serially diluted with filtered seawater (FSW) to Preparation: achieve the test concentrations. A FSW control was tested concurrently with the sample. Three additional salinity controls were also tested concurrently with the sample. Source of Test Organisms: Hatchery reared, SA Size of Test Organisms: 12mm Test Initiated: 03 April 2020 at 1900h
Controls Controls Concentration % Unaffected Concentration Biomass, mg (%) (Mean SD) (%) (Mean SD) FSW Control 100 0.0 FSW Control 18.4 0.6 12.5% Sal Ctrl 100 0.0 12.5% Sal Ctrl 18.1 1.2 25% Sal Ctrl 100 0.0 25% Sal Ctrl 17.0 0.7 * 50% Sal Ctrl 0.0 0.0 50% Sal Ctrl 0.0 0.0
*Significantly lower fish biomass compared with the FSW Control (Homoscedastic Test, 1-tailed, P=0.05)
Toxicity Test Report: TR1915/3 (Page 2 of 2)
Sample 9516: Brine Sample 9516: Brine Concentration % Unaffected Concentration Biomass, mg (%) (Mean SD) (%) (Mean SD) FSW Control 100 0.0 FSW Control 18.1 0.6 3.1 100 0.0 3.1 18.7 0.7 6.3 100 0.0 6.3 15.8 0.4 12.5 100 0.0 12.5 18.2 0.3 25 100 0.0 25 16.2 1.3 * 50 0.0 0.0 50 0.0 0.0 100 0.0 0.0 100 0.0 0.0
7 day IC10 (unaffected) = 32.6(25-50)% 7 day IC10 (biomass) =22.6 (16.60-28.06) % 7 day IC50 (unaffected) = 35.4(25-50)% 7 day IC50 (biomass) =35.8 (34.24-37.21) % NOEC = 25% NOEC = 12.5% LOEC = 50% LOEC = 25% *Significantly lower fish biomass compared with the FSW Control (Dunnett's t Test, 1-tailed, P=0.05)
QA/QC Parameter Criterion This Test Criterion met? Control mean % unaffected >80.0% 100% Yes Control mean growth >20% of initial weight 19.6% No + + Reference Toxicant within cusum chart limits 9.2-118.4mg NH4 /L 19.0mg NH4 /L Yes
Test Report Authorised by: Dr Rick Krassoi, Director on 23 April 2020
Results are based on the samples in the condition as received by ESA. This document shall not be reproduced except in full.
Citations:
ESA (2017) SOP 122 –7-day Fish Imbalance and Growth Test. Issue No 6. Ecotox Services Australasia, Sydney, NSW
USEPA (2002) Short-term methods for estimating the chronic toxicity of effluents and receiving waters to marine and estuarine organisms. Third edition EPA-821-R-02-014. United States Environmental Protection Agency, Office of Research and Development, Washington FC, USA
Chain-of-Custody Documentation
Datasheet ID: 601.2 Last Revised: 21 September 2018
Sample Receipt Notification
Attention : Tristan Stringer
Client : Intertek 1 Fleet Street Fremantle WA 6160
Email : [email protected] Telephone : 08 9336 5071 Facsimile :
Date : 3/04/2020
Re : Receipt of Samples Pages : 2 FALSE
ESA Project : PR1915 For Review Additional Documentation Required - Please Respond
Sample Delivery Details
Completed Chain of Custody accompanied samples: YES Samples received in apparent good condition and correctly bottled: YES Security seals on sample bottles and esky intact: YES
Date samples received : 3/04/2020 Time samples received : 8:30 No. of samples received : 1 Sample matrix : Aqueous Sample temperature : 6-10°C
Comments : 2 x 5L sample received at 9oC in apparent good condition
Contact Details
Projects Manager : Dr Rick Krassoi Telephone : 61 2 9420 9481 Facsimile : 61 2 9420 9484 Email : [email protected]
Please contact customer services officer for all queries or issues regarding samples
Note that the chain-of-custody provides definitive information on the tests to be performed
Ecotox Services Australia ABN 95619426201 Phone : 61 2 9420 9481 Unit 27, 2 Chaplin Drive Fax : 61 2 9420 9484 Lane Cove NSW 2066 Australia Email : [email protected]
Statistical Printouts for the Milky Oyster Larval Development Tests
Bivalve Acute Toxicity Tests-Proportion Normal Start Date: 3/04/2020 17:30 Test ID: PR1915/03 Sample ID: Controls End Date: 5/04/2020 17:30 Lab ID: Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 106 Test Species: SE-Saccostrea echinata Comments: Conc- 1 2 3 4 FSW Control 0.7400 0.7900 0.7900 0.7600 12.5% Sal 0.4400 0.5100 0.5700 0.4900 25% Sal 0.0000 0.0000 0.0000 0.0000 50% Sal 0.0000 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Conc- Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD FSW Control 0.7700 1.0000 1.0710 1.0357 1.0948 2.707 4 *12.5% Sal 0.5025 0.6526 0.7879 0.7253 0.8556 6.844 4 9.248 1.943 0.0595 25% Sal 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 4 50% Sal 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 4
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.05) 0.98047 0.818 0.129476 0.237459 F-Test indicates equal variances (p = 0.34) 3.459488 47.46723 Hypothesis Test (1-tail, 0.05) MSDu MSDp MSB MSE F-Prob df Homoscedastic t Test indicates significant differences 0.051836 0.06729 0.16029 0.001874 9.0E-05 1, 6 Treatments vs FSW Control Dose-Response Plot
1
0.9
0.8 1-tail, 0.05 level 0.7 of significance 0.6
0.5
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0 25%Sal 50%Sal *12.5%Sal FSW Control FSW
Page 1 ToxCalc v5.0.23 Reviewed by:_____ Bivalve Acute Toxicity Tests-Proportion Normal Start Date: 3/04/2020 17:30 Test ID: PR1915/03 Sample ID: Controls End Date: 5/04/2020 17:30 Lab ID: Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 106 Test Species: SE-Saccostrea echinata Comments: Auxiliary Data Summary Conc- Parameter Mean Min Max SD CV% N FSW Control % Normal 77.00 74.00 79.00 2.45 2.03 4 12.5% Sal 50.25 44.00 57.00 5.38 4.61 4 25% Sal 0.00 0.00 0.00 0.00 4 50% Sal 0.00 0.00 0.00 0.00 4 FSW Control pH 8.20 8.20 8.20 0.00 0.00 1 12.5% Sal 8.20 8.20 8.20 0.00 0.00 1 25% Sal 8.20 8.20 8.20 0.00 0.00 1 50% Sal 8.30 8.30 8.30 0.00 0.00 1 FSW Control Salinity ppt 36.10 36.10 36.10 0.00 0.00 1 12.5% Sal 40.40 40.40 40.40 0.00 0.00 1 25% Sal 43.20 43.20 43.20 0.00 0.00 1 50% Sal 50.60 50.60 50.60 0.00 0.00 1 FSW Control DO % 102.40 102.40 102.40 0.00 0.00 1 12.5% Sal 100.60 100.60 100.60 0.00 0.00 1 25% Sal 98.90 98.90 98.90 0.00 0.00 1 50% Sal 99.70 99.70 99.70 0.00 0.00 1
Page 2 ToxCalc v5.0.23 Reviewed by:_____ Bivalve Acute Toxicity Tests-Proportion Normal Start Date: 3/04/2020 17:30 Test ID: PR1915/04 Sample ID: Brine End Date: 5/04/2020 17:30 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 106 Test Species: SE-Saccostrea echinata Comments: Conc-% 1 2 3 4 FSW Control 0.7400 0.7900 0.7900 0.7600 3.1 0.7600 0.7400 0.7900 0.8300 6.3 0.8000 0.7600 0.7900 0.7500 12.5 0.5600 0.5900 0.5100 0.5400 25 0.0000 0.0000 0.0000 0.0000 50 0.0000 0.0000 0.0000 0.0000 100 0.0000 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number FSW Control 0.7700 1.0000 1.0710 1.0357 1.0948 2.707 4 92 400 3.1 0.7800 1.0130 1.0838 1.0357 1.1458 4.425 4 -0.505 2.290 0.0578 88 400 6.3 0.7750 1.0065 1.0770 1.0472 1.1071 2.650 4 -0.236 2.290 0.0578 90 400 *12.5 0.5500 0.7143 0.8356 0.7954 0.8759 4.054 4 9.323 2.290 0.0578 180 400 25 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 4 400 400 50 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 4 400 400 100 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 4 400 400
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.05) 0.963617 0.887 0.22395 -0.85375 Bartlett's Test indicates equal variances (p = 0.80) 1.006896 11.34487 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 6.3 12.5 8.87412 15.87302 0.050348 0.065358 0.058524 0.001276 7.5E-07 3, 12 Treatments vs FSW Control Trimmed Spearman-Karber Trim Level EC50 95% CL 0.0% 14.472 14.026 14.932 5.0% 14.675 14.168 15.200 10.0% 14.868 14.270 15.490 1.0 20.0% 15.197 14.279 16.175 0.9 Auto-0.0% 14.472 14.026 14.932 0.8 0.7 0.6 0.5 0.4
Response 0.3 0.2 0.1 0.0 -0.1 1 10 100 Dose %
Page 1 ToxCalc v5.0.23 Reviewed by:_____ Bivalve Acute Toxicity Tests-Proportion Normal Start Date: 3/04/2020 17:30 Test ID: PR1915/04 Sample ID: Brine End Date: 5/04/2020 17:30 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 106 Test Species: SE-Saccostrea echinata Comments: Dose-Response Plot
1
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0.1
0 25 50 3.1 6.3 100 *12.5 FSW Control FSW
Page 2 ToxCalc v5.0.23 Reviewed by:_____ Bivalve Acute Toxicity Tests-Proportion Normal Start Date: 3/04/2020 17:30 Test ID: PR1915/04 Sample ID: Brine End Date: 5/04/2020 17:30 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 106 Test Species: SE-Saccostrea echinata Comments: Auxiliary Data Summary Conc-% Parameter Mean Min Max SD CV% N FSW Control % Normal 77.00 74.00 79.00 2.45 2.03 4 3.1 78.00 74.00 83.00 3.92 2.54 4 6.3 77.50 75.00 80.00 2.38 1.99 4 12.5 55.00 51.00 59.00 3.37 3.34 4 25 0.00 0.00 0.00 0.00 4 50 0.00 0.00 0.00 0.00 4 100 0.00 0.00 0.00 0.00 4 FSW Control pH 8.20 8.20 8.20 0.00 0.00 1 3.1 8.20 8.20 8.20 0.00 0.00 1 6.3 8.20 8.20 8.20 0.00 0.00 1 12.5 8.20 8.20 8.20 0.00 0.00 1 25 8.20 8.20 8.20 0.00 0.00 1 50 8.20 8.20 8.20 0.00 0.00 1 100 8.10 8.10 8.10 0.00 0.00 1 FSW Control Salinity ppt 36.10 36.10 36.10 0.00 0.00 1 3.1 37.50 37.50 37.50 0.00 0.00 1 6.3 38.30 38.30 38.30 0.00 0.00 1 12.5 40.10 40.10 40.10 0.00 0.00 1 25 43.50 43.50 43.50 0.00 0.00 1 50 50.20 50.20 50.20 0.00 0.00 1 100 64.00 64.00 64.00 0.00 0.00 1 FSW Control DO % 102.40 102.40 102.40 0.00 0.00 1 3.1 106.40 106.40 106.40 0.00 0.00 1 6.3 102.80 102.80 102.80 0.00 0.00 1 12.5 102.40 102.40 102.40 0.00 0.00 1 25 102.50 102.50 102.50 0.00 0.00 1 50 104.80 104.80 104.80 0.00 0.00 1 100 103.00 103.00 103.00 0.00 0.00 1
Page 3 ToxCalc v5.0.23 Reviewed by:_____ Bivalve Acute Toxicity Tests-Proportion Normal Start Date: 3/04/2020 17:30 Test ID: PR1915/04 Sample ID: Brine End Date: 5/04/2020 17:30 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 106 Test Species: SE-Saccostrea echinata Comments: Conc-% 1 2 3 4 FSW Control 0.7400 0.7900 0.7900 0.7600 3.1 0.7600 0.7400 0.7900 0.8300 6.3 0.8000 0.7600 0.7900 0.7500 12.5 0.5600 0.5900 0.5100 0.5400 25 0.0000 0.0000 0.0000 0.0000 50 0.0000 0.0000 0.0000 0.0000 100 0.0000 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Isotonic Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Mean N-Mean FSW Control 0.7700 1.0000 1.0710 1.0357 1.0948 2.707 4 0.7750 1.0000 3.1 0.7800 1.0130 1.0838 1.0357 1.1458 4.425 4 -0.505 2.290 0.0578 0.7750 1.0000 6.3 0.7750 1.0065 1.0770 1.0472 1.1071 2.650 4 -0.236 2.290 0.0578 0.7750 1.0000 *12.5 0.5500 0.7143 0.8356 0.7954 0.8759 4.054 4 9.323 2.290 0.0578 0.5500 0.7097 25 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 4 0.0000 0.0000 50 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 4 0.0000 0.0000 100 0.0000 0.0000 0.0500 0.0500 0.0500 0.000 4 0.0000 0.0000
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.05) 0.963617 0.887 0.22395 -0.85375 Bartlett's Test indicates equal variances (p = 0.80) 1.006896 11.34487 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 6.3 12.5 8.87412 15.87302 0.050348 0.065358 0.058524 0.001276 7.5E-07 3, 12 Treatments vs FSW Control Log-Logit Interpolation (200 Resamples) Point % SD 95% CL(Exp) Skew IC05 7.270 0.206 6.210 7.430 -1.0176 IC10 8.263 0.227 7.223 8.614 -0.5754 IC15 9.293 0.277 8.303 9.899 -0.0403 1.0 IC20 10.373 0.355 9.302 11.345 0.3321 0.9 IC25 11.518 0.438 10.235 12.972 0.2239 IC40 12.824 0.059 12.630 12.998 0.0327 0.8 IC50 13.134 0.058 12.948 13.304 0.0248 0.7 0.6 0.5 0.4
Response 0.3 0.2 0.1 0.0 -0.1 1 10 100 Dose %
Page 1 ToxCalc v5.0.23 Reviewed by:_____ Bivalve Acute Toxicity Tests-Proportion Normal Start Date: 3/04/2020 17:30 Test ID: PR1915/04 Sample ID: Brine End Date: 5/04/2020 17:30 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 106 Test Species: SE-Saccostrea echinata Comments: Dose-Response Plot
1
0.9
0.8 1-tail, 0.05 level 0.7 of significance 0.6
0.5
0.4
0.3 Proportion Proportion Normal 0.2
0.1
0 25 50 3.1 6.3 100 *12.5 FSW Control FSW
Page 2 ToxCalc v5.0.23 Reviewed by:_____ Bivalve Acute Toxicity Tests-Proportion Normal Start Date: 3/04/2020 17:30 Test ID: PR1915/04 Sample ID: Brine End Date: 5/04/2020 17:30 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 106 Test Species: SE-Saccostrea echinata Comments: Auxiliary Data Summary Conc-% Parameter Mean Min Max SD CV% N FSW Control % Normal 77.00 74.00 79.00 2.45 2.03 4 3.1 78.00 74.00 83.00 3.92 2.54 4 6.3 77.50 75.00 80.00 2.38 1.99 4 12.5 55.00 51.00 59.00 3.37 3.34 4 25 0.00 0.00 0.00 0.00 4 50 0.00 0.00 0.00 0.00 4 100 0.00 0.00 0.00 0.00 4 FSW Control pH 8.20 8.20 8.20 0.00 0.00 1 3.1 8.20 8.20 8.20 0.00 0.00 1 6.3 8.20 8.20 8.20 0.00 0.00 1 12.5 8.20 8.20 8.20 0.00 0.00 1 25 8.20 8.20 8.20 0.00 0.00 1 50 8.20 8.20 8.20 0.00 0.00 1 100 8.10 8.10 8.10 0.00 0.00 1 FSW Control Salinity ppt 36.10 36.10 36.10 0.00 0.00 1 3.1 37.50 37.50 37.50 0.00 0.00 1 6.3 38.30 38.30 38.30 0.00 0.00 1 12.5 40.10 40.10 40.10 0.00 0.00 1 25 43.50 43.50 43.50 0.00 0.00 1 50 50.20 50.20 50.20 0.00 0.00 1 100 64.00 64.00 64.00 0.00 0.00 1 FSW Control DO % 102.40 102.40 102.40 0.00 0.00 1 3.1 106.40 106.40 106.40 0.00 0.00 1 6.3 102.80 102.80 102.80 0.00 0.00 1 12.5 102.40 102.40 102.40 0.00 0.00 1 25 102.50 102.50 102.50 0.00 0.00 1 50 104.80 104.80 104.80 0.00 0.00 1 100 103.00 103.00 103.00 0.00 0.00 1
Page 3 ToxCalc v5.0.23 Reviewed by:_____
Statistical Printouts for the Sea Anemone Pedal Lacerate Development Test
Anemone Larval Toxicity Test-% Unaffected Start Date: 3/04/2020 18:00 Test ID: PR1915/05 Sample ID: Controls End Date: 11/04/2020 18:00 Lab ID: Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 128 Test Species: AI-Aiptasia pulchella Comments: Conc- 1 2 3 4 FSW Control 0.8000 0.8000 1.0000 1.0000 12.5% Sal 0.8000 0.8000 0.8000 1.0000 25% Sal 0.0000 0.0000 0.0000 0.0000 50% Sal 0.0000 0.0000 0.0000 0.0000
Transform: Arcsin Square Root 1-Tailed Conc- Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD FSW Control 0.9000 1.0000 1.2262 1.1071 1.3453 11.212 4 12.5% Sal 0.8500 0.9444 1.1667 1.1071 1.3453 10.206 4 0.655 1.943 0.1767 25% Sal 0.0000 0.0000 0.2255 0.2255 0.2255 0.000 4 50% Sal 0.0000 0.0000 0.2255 0.2255 0.2255 0.000 4
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.05) 0.827836 0.818 0.571429 -1.72857 F-Test indicates equal variances (p = 0.82) 1.333333 47.46723 Hypothesis Test (1-tail, 0.05) MSDu MSDp MSB MSE F-Prob df Homoscedastic t Test indicates no significant differences 0.133894 0.151141 0.007088 0.01654 0.536963 1, 6 Treatments vs FSW Control Dose-Response Plot
1
0.9
0.8 1-tail, 0.05 level 0.7 of significance
0.6
0.5
0.4
%Unaffected 0.3
0.2
0.1
0 25%Sal 50%Sal 12.5%Sal FSW Control FSW
Page 1 ToxCalc v5.0.23 Reviewed by:_____ Anemone Larval Toxicity Test-% Unaffected Start Date: 3/04/2020 18:00 Test ID: PR1915/05 Sample ID: Controls End Date: 11/04/2020 18:00 Lab ID: Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 128 Test Species: AI-Aiptasia pulchella Comments: Auxiliary Data Summary Conc- Parameter Mean Min Max SD CV% N FSW Control % unaffacted 90.00 80.00 100.00 11.55 3.78 4 12.5% Sal 85.00 80.00 100.00 10.00 3.72 4 25% Sal 0.00 0.00 0.00 0.00 4 50% Sal 0.00 0.00 0.00 0.00 4 FSW Control pH 8.20 8.20 8.20 0.00 0.00 1 12.5% Sal 8.20 8.20 8.20 0.00 0.00 1 25% Sal 8.20 8.20 8.20 0.00 0.00 1 50% Sal 8.30 8.30 8.30 0.00 0.00 1 FSW Control DO, % 102.40 102.40 102.40 0.00 0.00 1 12.5% Sal 100.60 100.60 100.60 0.00 0.00 1 25% Sal 98.90 98.90 98.90 0.00 0.00 1 50% Sal 99.70 99.70 99.70 0.00 0.00 1 FSW Control Salinity ppt 36.10 36.10 36.10 0.00 0.00 1 12.5% Sal 40.40 40.40 40.40 0.00 0.00 1 25% Sal 43.20 43.20 43.20 0.00 0.00 1 50% Sal 50.60 50.60 50.60 0.00 0.00 1
Page 2 ToxCalc v5.0.23 Reviewed by:_____ Anemone Larval Toxicity Test-% Unaffected Start Date: 3/04/2020 18:00 Test ID: PR1915/04 Sample ID: Brine End Date: 11/04/2020 18:00 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 128 Test Species: AI-Aiptasia pulchella Comments: Conc-% 1 2 3 4 FSW Control 0.8000 0.8000 1.0000 1.0000 3.1 0.8000 1.0000 0.8000 1.0000 6.3 1.0000 1.0000 1.0000 0.8000 12.5 0.8000 0.8000 1.0000 1.0000 25 0.0000 0.0000 0.0000 0.0000 50 0.0000 0.0000 0.0000 0.0000 100 0.0000 0.0000 0.0000 0.0000
Transform: Arcsin Square Root Rank 1-Tailed Number Total Conc-% Mean N-Mean Mean Min Max CV% N Sum Critical Resp Number FSW Control 0.9000 1.0000 1.2262 1.1071 1.3453 11.212 4 2 20 3.1 0.9000 1.0000 1.2262 1.1071 1.3453 11.212 4 18.00 10.00 2 20 6.3 0.9500 1.0556 1.2857 1.1071 1.3453 9.261 4 20.00 10.00 1 20 12.5 0.9000 1.0000 1.2262 1.1071 1.3453 11.212 4 18.00 10.00 2 20 25 0.0000 0.0000 0.2255 0.2255 0.2255 0.000 4 20 20 50 0.0000 0.0000 0.2255 0.2255 0.2255 0.000 4 20 20 100 0.0000 0.0000 0.2255 0.2255 0.2255 0.000 4 20 20
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates non-normal distribution (p <= 0.05) 0.767655 0.887 -0.22857 -1.99011 Bartlett's Test indicates equal variances (p = 0.99) 0.077781 11.34487 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU Steel's Many-One Rank Test 12.5 25 17.67767 8 Treatments vs FSW Control Trimmed Spearman-Karber Trim Level EC50 95% CL 0.0% 17.458 16.753 18.191 5.0% 17.565 17.191 17.946 10.0% 17.565 17.191 17.946 1.0 20.0% 17.565 17.191 17.946 0.9 Auto-0.0% 17.458 16.753 18.191 0.8 0.7 0.6 0.5 0.4 0.3
Response 0.2 0.1 0.0 -0.1 -0.2 1 10 100 Dose %
Page 1 ToxCalc v5.0.23 Reviewed by:_____ Anemone Larval Toxicity Test-% Unaffected Start Date: 3/04/2020 18:00 Test ID: PR1915/04 Sample ID: Brine End Date: 11/04/2020 18:00 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 128 Test Species: AI-Aiptasia pulchella Comments: Dose-Response Plot
1
0.9
0.8
0.7
0.6
0.5
0.4
%Unaffected 0.3
0.2
0.1
0 25 50 3.1 6.3 100 12.5 FSW Control FSW
Page 2 ToxCalc v5.0.23 Reviewed by:_____ Anemone Larval Toxicity Test-% Unaffected Start Date: 3/04/2020 18:00 Test ID: PR1915/04 Sample ID: Brine End Date: 11/04/2020 18:00 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 128 Test Species: AI-Aiptasia pulchella Comments: Auxiliary Data Summary Conc-% Parameter Mean Min Max SD CV% N FSW Control % unaffacted 90.00 80.00 100.00 11.55 3.78 4 3.1 90.00 80.00 100.00 11.55 3.78 4 6.3 95.00 80.00 100.00 10.00 3.33 4 12.5 90.00 80.00 100.00 11.55 3.78 4 25 0.00 0.00 0.00 0.00 4 50 0.00 0.00 0.00 0.00 4 100 0.00 0.00 0.00 0.00 4 FSW Control pH 8.20 8.20 8.20 0.00 0.00 1 3.1 8.20 8.20 8.20 0.00 0.00 1 6.3 8.20 8.20 8.20 0.00 0.00 1 12.5 8.20 8.20 8.20 0.00 0.00 1 25 8.20 8.20 8.20 0.00 0.00 1 50 8.20 8.20 8.20 0.00 0.00 1 100 8.10 8.10 8.10 0.00 0.00 1 FSW Control DO, % 102.40 102.40 102.40 0.00 0.00 1 3.1 106.40 106.40 106.40 0.00 0.00 1 6.3 102.80 102.80 102.80 0.00 0.00 1 12.5 102.40 102.40 102.40 0.00 0.00 1 25 102.50 102.50 102.50 0.00 0.00 1 50 104.80 104.80 104.80 0.00 0.00 1 100 103.00 103.00 103.00 0.00 0.00 1 FSW Control Salinity ppt 36.10 36.10 36.10 0.00 0.00 1 3.1 37.50 37.50 37.50 0.00 0.00 1 6.3 38.30 38.30 38.30 0.00 0.00 1 12.5 40.10 40.10 40.10 0.00 0.00 1 25 43.50 43.50 43.50 0.00 0.00 1 50 50.20 50.20 50.20 0.00 0.00 1 100 64.00 64.00 64.00 0.00 0.00 1
Page 3 ToxCalc v5.0.23 Reviewed by:_____ Anemone Larval Toxicity Test-% Unaffected Start Date: 3/04/2020 18:00 Test ID: PR1915/04 Sample ID: Brine End Date: 11/04/2020 18:00 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 128 Test Species: AI-Aiptasia pulchella Comments: Conc-% 1 2 3 4 FSW Control 0.8000 0.8000 1.0000 1.0000 3.1 0.8000 1.0000 0.8000 1.0000 6.3 1.0000 1.0000 1.0000 0.8000 12.5 0.8000 0.8000 1.0000 1.0000 25 0.0000 0.0000 0.0000 0.0000 50 0.0000 0.0000 0.0000 0.0000 100 0.0000 0.0000 0.0000 0.0000
Transform: Arcsin Square Root Rank 1-Tailed Isotonic Conc-% Mean N-Mean Mean Min Max CV% N Sum Critical Mean N-Mean FSW Control 0.9000 1.0000 1.2262 1.1071 1.3453 11.212 4 0.9167 1.0000 3.1 0.9000 1.0000 1.2262 1.1071 1.3453 11.212 4 18.00 10.00 0.9167 1.0000 6.3 0.9500 1.0556 1.2857 1.1071 1.3453 9.261 4 20.00 10.00 0.9167 1.0000 12.5 0.9000 1.0000 1.2262 1.1071 1.3453 11.212 4 18.00 10.00 0.9000 0.9818 25 0.0000 0.0000 0.2255 0.2255 0.2255 0.000 4 0.0000 0.0000 50 0.0000 0.0000 0.2255 0.2255 0.2255 0.000 4 0.0000 0.0000 100 0.0000 0.0000 0.2255 0.2255 0.2255 0.000 4 0.0000 0.0000
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates non-normal distribution (p <= 0.05) 0.767655 0.887 -0.22857 -1.99011 Bartlett's Test indicates equal variances (p = 0.99) 0.077781 11.34487 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU Steel's Many-One Rank Test 12.5 25 17.67767 8 Treatments vs FSW Control Log-Logit Interpolation (200 Resamples) Point % SD 95% CL(Exp) Skew IC05 12.726 3.665 0.000 13.218 -1.4628 IC10 13.011 2.052 0.000 13.600 -3.6874 IC15 13.246 0.585 12.052 13.885 -8.0820 1.0 IC20 13.452 0.302 12.384 14.120 -0.2519 0.9 IC25 13.638 0.297 12.630 14.325 -0.1658 0.8 IC40 14.141 0.288 13.158 14.850 -0.0207 0.7 IC50 14.464 0.283 13.499 15.175 0.0301 0.6 0.5 0.4 0.3
Response 0.2 0.1 0.0 -0.1 -0.2 1 10 100 Dose %
Page 1 ToxCalc v5.0.23 Reviewed by:_____ Anemone Larval Toxicity Test-% Unaffected Start Date: 3/04/2020 18:00 Test ID: PR1915/04 Sample ID: Brine End Date: 11/04/2020 18:00 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 128 Test Species: AI-Aiptasia pulchella Comments: Dose-Response Plot
1
0.9
0.8
0.7
0.6
0.5
0.4
%Unaffected 0.3
0.2
0.1
0 25 50 3.1 6.3 100 12.5 FSW Control FSW
Page 2 ToxCalc v5.0.23 Reviewed by:_____ Anemone Larval Toxicity Test-% Unaffected Start Date: 3/04/2020 18:00 Test ID: PR1915/04 Sample ID: Brine End Date: 11/04/2020 18:00 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 128 Test Species: AI-Aiptasia pulchella Comments: Auxiliary Data Summary Conc-% Parameter Mean Min Max SD CV% N FSW Control % unaffacted 90.00 80.00 100.00 11.55 3.78 4 3.1 90.00 80.00 100.00 11.55 3.78 4 6.3 95.00 80.00 100.00 10.00 3.33 4 12.5 90.00 80.00 100.00 11.55 3.78 4 25 0.00 0.00 0.00 0.00 4 50 0.00 0.00 0.00 0.00 4 100 0.00 0.00 0.00 0.00 4 FSW Control pH 8.20 8.20 8.20 0.00 0.00 1 3.1 8.20 8.20 8.20 0.00 0.00 1 6.3 8.20 8.20 8.20 0.00 0.00 1 12.5 8.20 8.20 8.20 0.00 0.00 1 25 8.20 8.20 8.20 0.00 0.00 1 50 8.20 8.20 8.20 0.00 0.00 1 100 8.10 8.10 8.10 0.00 0.00 1 FSW Control DO, % 102.40 102.40 102.40 0.00 0.00 1 3.1 106.40 106.40 106.40 0.00 0.00 1 6.3 102.80 102.80 102.80 0.00 0.00 1 12.5 102.40 102.40 102.40 0.00 0.00 1 25 102.50 102.50 102.50 0.00 0.00 1 50 104.80 104.80 104.80 0.00 0.00 1 100 103.00 103.00 103.00 0.00 0.00 1 FSW Control Salinity ppt 36.10 36.10 36.10 0.00 0.00 1 3.1 37.50 37.50 37.50 0.00 0.00 1 6.3 38.30 38.30 38.30 0.00 0.00 1 12.5 40.10 40.10 40.10 0.00 0.00 1 25 43.50 43.50 43.50 0.00 0.00 1 50 50.20 50.20 50.20 0.00 0.00 1 100 64.00 64.00 64.00 0.00 0.00 1
Page 3 ToxCalc v5.0.23 Reviewed by:_____
Statistical Printouts for the 7-d Larval Fish Growth Inhibition Tests
Fish Growth Test-7 Day Unaffected Start Date: 3/04/2020 19:00 Test ID: PR1915/01 Sample ID: Controls End Date: 10/04/2020 19:00 Lab ID: Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 122 Test Species: LT-Lates calcarifer Comments: Conc- 1 2 3 4 FSW Control 1.0000 1.0000 1.0000 1.0000 12.5% Sal 1.0000 1.0000 1.0000 1.0000 25% Sal 1.0000 1.0000 1.0000 1.0000 50% Sal 0.0000 0.0000 0.0000 0.0000
Transform: Arcsin Square Root Conc- Mean N-Mean Mean Min Max CV% N FSW Control 1.0000 1.0000 1.3453 1.3453 1.3453 0.000 4 12.5% Sal 1.0000 1.0000 1.3453 1.3453 1.3453 0.000 4 25% Sal 1.0000 1.0000 1.3453 1.3453 1.3453 0.000 4 50% Sal 0.0000 0.0000 0.2255 0.2255 0.2255 0.000 4
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.05) 1 0.859 Equality of variance cannot be confirmed Dose-Response Plot
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3 7 DayUnaffected 7 0.2
0.1
0 25%Sal 50%Sal 12.5%Sal FSW ControlFSW
Page 1 ToxCalc v5.0.23 Reviewed by:_____ Fish Growth Test-7 Day Unaffected Start Date: 3/04/2020 19:00 Test ID: PR1915/01 Sample ID: Controls End Date: 10/04/2020 19:00 Lab ID: Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 122 Test Species: LT-Lates calcarifer Comments: Auxiliary Data Summary Conc- Parameter Mean Min Max SD CV% N FSW Control % Un-affected 100.00 100.00 100.00 0.00 0.00 4 12.5% Sal 100.00 100.00 100.00 0.00 0.00 4 25% Sal 100.00 100.00 100.00 0.00 0.00 4 50% Sal 0.00 0.00 0.00 0.00 4 FSW Control Biomass 18.14 17.58 19.02 0.62 4.35 4 12.5% Sal 18.08 16.98 19.74 1.19 6.04 4 25% Sal 16.99 16.08 17.58 0.67 4.81 4 50% Sal 0.00 0.00 0.00 0.00 4 FSW Control pH 8.20 8.20 8.20 0.00 0.00 1 12.5% Sal 8.20 8.20 8.20 0.00 0.00 1 25% Sal 8.20 8.20 8.20 0.00 0.00 1 50% Sal 8.30 8.30 8.30 0.00 0.00 1 FSW Control Salinity 36.10 36.10 36.10 0.00 0.00 1 12.5% Sal 40.40 40.40 40.40 0.00 0.00 1 25% Sal 43.20 43.20 43.20 0.00 0.00 1 50% Sal 50.60 50.60 50.60 0.00 0.00 1 FSW Control % DO 102.40 102.40 102.40 0.00 0.00 1 12.5% Sal 100.60 100.60 100.60 0.00 0.00 1 25% Sal 98.90 98.90 98.90 0.00 0.00 1 50% Sal 99.70 99.70 99.70 0.00 0.00 1
Page 2 ToxCalc v5.0.23 Reviewed by:_____ Fish Growth Test-7 day Biomass Start Date: 3/04/2020 19:00 Test ID: PR1915/01 Sample ID: Controls End Date: 10/04/2020 19:00 Lab ID: Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 122 Test Species: LT-Lates calcarifer Comments: Conc- 1 2 3 4 FSW Control 18.080 17.880 19.020 17.580 12.5% Sal 19.740 16.980 18.080 17.520 25% Sal 16.080 16.920 17.380 17.580 50% Sal 0.000 0.000 0.000 0.000
Transform: Untransformed 1-Tailed Conc- Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD FSW Control 18.140 1.0000 18.140 17.580 19.020 3.427 4 12.5% Sal 18.080 0.9967 18.080 16.980 19.740 6.606 4 0.089 1.943 1.308 *25% Sal 16.990 0.9366 16.990 16.080 17.580 3.924 4 2.523 1.943 0.886 50% Sal 0.000 0.0000 0.000 0.000 0.000 0.000 4
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.05) 0.961108 0.859 0.692608 0.411016 Bartlett's Test indicates equal variances (p = 0.49) 1.445712 9.21034 Hypothesis Test (1-tail, 0.05) MSDu MSDp MSB MSE F-Prob df Homoscedastic t Test indicates significant differences 0.885587 0.04882 1.676133 0.7524 0.163701 2, 9 Treatments vs FSW Control Dose-Response Plot
25
20 1-tail, 0.05 level of significance 15
10 7 day 7 Biomass
5
0 50%Sal *25%Sal 12.5%Sal FSW Control FSW
Page 1 ToxCalc v5.0.23 Reviewed by:_____ Fish Growth Test-7 day Biomass Start Date: 3/04/2020 19:00 Test ID: PR1915/01 Sample ID: Controls End Date: 10/04/2020 19:00 Lab ID: Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 122 Test Species: LT-Lates calcarifer Comments: Auxiliary Data Summary Conc- Parameter Mean Min Max SD CV% N FSW Control % Un-affected 100.00 100.00 100.00 0.00 0.00 4 12.5% Sal 100.00 100.00 100.00 0.00 0.00 4 25% Sal 100.00 100.00 100.00 0.00 0.00 4 50% Sal 0.00 0.00 0.00 0.00 4 FSW Control Biomass 18.14 17.58 19.02 0.62 4.35 4 12.5% Sal 18.08 16.98 19.74 1.19 6.04 4 25% Sal 16.99 16.08 17.58 0.67 4.81 4 50% Sal 0.00 0.00 0.00 0.00 4 FSW Control pH 8.20 8.20 8.20 0.00 0.00 1 12.5% Sal 8.20 8.20 8.20 0.00 0.00 1 25% Sal 8.20 8.20 8.20 0.00 0.00 1 50% Sal 8.30 8.30 8.30 0.00 0.00 1 FSW Control Salinity 36.10 36.10 36.10 0.00 0.00 1 12.5% Sal 40.40 40.40 40.40 0.00 0.00 1 25% Sal 43.20 43.20 43.20 0.00 0.00 1 50% Sal 50.60 50.60 50.60 0.00 0.00 1 FSW Control % DO 102.40 102.40 102.40 0.00 0.00 1 12.5% Sal 100.60 100.60 100.60 0.00 0.00 1 25% Sal 98.90 98.90 98.90 0.00 0.00 1 50% Sal 99.70 99.70 99.70 0.00 0.00 1
Page 2 ToxCalc v5.0.23 Reviewed by:_____ Fish Growth Test-7 Day Unaffected Start Date: 3/04/2020 19:00 Test ID: PR1915/02 Sample ID: Brine End Date: 10/04/2020 19:00 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 122 Test Species: LT-Lates calcarifer Comments: Conc-% 1 2 3 4 FSW Control 1.0000 1.0000 1.0000 1.0000 3.1 1.0000 1.0000 1.0000 1.0000 6.3 1.0000 1.0000 1.0000 1.0000 12.5 1.0000 1.0000 1.0000 1.0000 25 1.0000 1.0000 1.0000 1.0000 50 0.0000 0.0000 0.0000 0.0000 100 0.0000 0.0000 0.0000 0.0000
Transform: Arcsin Square Root Rank 1-Tailed Isotonic Conc-% Mean N-Mean Mean Min Max CV% N Sum Critical Mean N-Mean FSW Control 1.0000 1.0000 1.3453 1.3453 1.3453 0.000 4 1.0000 1.0000 3.1 1.0000 1.0000 1.3453 1.3453 1.3453 0.000 4 18.00 10.00 1.0000 1.0000 6.3 1.0000 1.0000 1.3453 1.3453 1.3453 0.000 4 18.00 10.00 1.0000 1.0000 12.5 1.0000 1.0000 1.3453 1.3453 1.3453 0.000 4 18.00 10.00 1.0000 1.0000 25 1.0000 1.0000 1.3453 1.3453 1.3453 0.000 4 18.00 10.00 1.0000 1.0000 50 0.0000 0.0000 0.2255 0.2255 0.2255 0.000 4 0.0000 0.0000 100 0.0000 0.0000 0.2255 0.2255 0.2255 0.000 4 0.0000 0.0000
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.05) 1 0.905 Equality of variance cannot be confirmed Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU Steel's Many-One Rank Test 25 50 35.35534 4 Treatments vs FSW Control Log-Logit Interpolation (200 Resamples) Point % SD 95% CL(Exp) Skew IC05 31.697 0.000 31.697 31.697 -1.0076 IC10 32.602 0.000 32.602 32.602 -1.0076 IC15 33.176 0.000 33.176 33.176 1.0076 1.0 IC20 33.614 0.000 33.614 33.614 -1.0076 0.9 IC25 33.980 0.000 33.980 33.980 -1.0076 IC40 34.878 0.000 34.878 34.878 1.0076 0.8 IC50 35.414 0.000 35.414 35.414 -1.0076 0.7
0.6
0.5
0.4 Response 0.3
0.2
0.1
0.0 1 10 100 Dose %
Page 1 ToxCalc v5.0.23 Reviewed by:_____ Fish Growth Test-7 Day Unaffected Start Date: 3/04/2020 19:00 Test ID: PR1915/02 Sample ID: Brine End Date: 10/04/2020 19:00 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 122 Test Species: LT-Lates calcarifer Comments: Dose-Response Plot
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0.9
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Page 2 ToxCalc v5.0.23 Reviewed by:_____ Fish Growth Test-7 Day Unaffected Start Date: 3/04/2020 19:00 Test ID: PR1915/02 Sample ID: Brine End Date: 10/04/2020 19:00 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 122 Test Species: LT-Lates calcarifer Comments: Auxiliary Data Summary Conc-% Parameter Mean Min Max SD CV% N FSW Control % Un-affected 100.00 100.00 100.00 0.00 0.00 4 3.1 100.00 100.00 100.00 0.00 0.00 4 6.3 100.00 100.00 100.00 0.00 0.00 4 12.5 100.00 100.00 100.00 0.00 0.00 4 25 100.00 100.00 100.00 0.00 0.00 4 50 0.00 0.00 0.00 0.00 4 100 0.00 0.00 0.00 0.00 4 FSW Control Biomass 18.14 17.58 19.02 0.62 4.35 4 3.1 18.69 17.94 19.64 0.74 4.59 4 6.3 18.50 18.02 18.94 0.40 3.44 4 12.5 18.22 17.98 18.58 0.26 2.77 4 25 16.22 14.86 17.90 1.26 6.92 4 50 0.00 0.00 0.00 0.00 4 100 0.00 0.00 0.00 0.00 4 FSW Control pH 8.20 8.20 8.20 0.00 0.00 1 3.1 8.20 8.20 8.20 0.00 0.00 1 6.3 8.20 8.20 8.20 0.00 0.00 1 12.5 8.20 8.20 8.20 0.00 0.00 1 25 8.20 8.20 8.20 0.00 0.00 1 50 8.20 8.20 8.20 0.00 0.00 1 100 8.10 8.10 8.10 0.00 0.00 1 FSW Control Salinity 36.10 36.10 36.10 0.00 0.00 1 3.1 37.50 37.50 37.50 0.00 0.00 1 6.3 38.30 38.30 38.30 0.00 0.00 1 12.5 40.10 40.10 40.10 0.00 0.00 1 25 43.50 43.50 43.50 0.00 0.00 1 50 50.20 50.20 50.20 0.00 0.00 1 100 64.00 64.00 64.00 0.00 0.00 1 FSW Control % DO 102.40 102.40 102.40 0.00 0.00 1 3.1 106.40 106.40 106.40 0.00 0.00 1 6.3 102.80 102.80 102.80 0.00 0.00 1 12.5 102.10 102.10 102.10 0.00 0.00 1 25 102.50 102.50 102.50 0.00 0.00 1 50 104.80 104.80 104.80 0.00 0.00 1 100 103.00 103.00 103.00 0.00 0.00 1
Page 3 ToxCalc v5.0.23 Reviewed by:_____ Fish Growth Test-7 day Biomass Start Date: 3/04/2020 19:00 Test ID: PR1915/02 Sample ID: Brine End Date: 10/04/2020 19:00 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 122 Test Species: LT-Lates calcarifer Comments: Conc-% 1 2 3 4 FSW Control 18.080 17.880 19.020 17.580 3.1 19.640 18.320 17.940 18.860 6.3 18.340 18.940 18.020 18.700 12.5 18.180 18.140 18.580 17.980 25 14.860 17.900 16.140 15.960 50 0.000 0.000 0.000 0.000 100 0.000 0.000 0.000 0.000
Transform: Untransformed 1-Tailed Isotonic Conc-% Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Mean N-Mean FSW Control 18.140 1.0000 18.140 17.580 19.020 3.427 4 18.443 1.0000 3.1 18.690 1.0303 18.690 17.940 19.640 3.945 4 -1.051 2.360 1.235 18.443 1.0000 6.3 18.500 1.0198 18.500 18.020 18.940 2.184 4 -0.688 2.360 1.235 18.443 1.0000 12.5 18.220 1.0044 18.220 17.980 18.580 1.400 4 -0.153 2.360 1.235 18.220 0.9879 *25 16.215 0.8939 16.215 14.860 17.900 7.757 4 3.677 2.360 1.235 16.215 0.8792 50 0.000 0.0000 0.000 0.000 0.000 0.000 4 0.000 0.0000 100 0.000 0.0000 0.000 0.000 0.000 0.000 4 0.000 0.0000
Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribution (p > 0.05) 0.948378 0.905 0.650933 1.632693 Bartlett's Test indicates equal variances (p = 0.14) 6.987977 13.2767 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Dunnett's Test 12.5 25 17.67767 8 1.235395 0.068103 3.96928 0.548047 0.001866 4, 15 Treatments vs FSW Control Linear Interpolation (200 Resamples) Point % SD 95% CL(Exp) Skew IC05 16.857 2.016 12.011 23.895 1.6026 IC10 22.606 2.190 16.597 28.060 0.1145 IC15 25.830 1.077 20.927 28.257 -0.8356 1.0 IC20 27.252 0.766 24.777 29.536 0.2135 0.9 IC25 28.673 0.718 26.353 30.815 0.2135 IC40 32.939 0.575 31.083 34.652 0.2135 0.8 IC50 35.782 0.479 34.235 37.210 0.2135 0.7 0.6 0.5 0.4
Response 0.3 0.2 0.1 0.0 -0.1 0 50 100 150 Dose %
Page 1 ToxCalc v5.0.23 Reviewed by:_____ Fish Growth Test-7 day Biomass Start Date: 3/04/2020 19:00 Test ID: PR1915/02 Sample ID: Brine End Date: 10/04/2020 19:00 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 122 Test Species: LT-Lates calcarifer Comments: Dose-Response Plot
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20 1-tail, 0.05 level of significance 15
10 7 day 7 Biomass
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Page 2 ToxCalc v5.0.23 Reviewed by:_____ Fish Growth Test-7 day Biomass Start Date: 3/04/2020 19:00 Test ID: PR1915/02 Sample ID: Brine End Date: 10/04/2020 19:00 Lab ID: 9516 Sample Type: AQ-Aqueous Sample Date: Protocol: ESA 122 Test Species: LT-Lates calcarifer Comments: Auxiliary Data Summary Conc-% Parameter Mean Min Max SD CV% N FSW Control % Un-affected 100.00 100.00 100.00 0.00 0.00 4 3.1 100.00 100.00 100.00 0.00 0.00 4 6.3 100.00 100.00 100.00 0.00 0.00 4 12.5 100.00 100.00 100.00 0.00 0.00 4 25 100.00 100.00 100.00 0.00 0.00 4 50 0.00 0.00 0.00 0.00 4 100 0.00 0.00 0.00 0.00 4 FSW Control Biomass 18.14 17.58 19.02 0.62 4.35 4 3.1 18.69 17.94 19.64 0.74 4.59 4 6.3 18.50 18.02 18.94 0.40 3.44 4 12.5 18.22 17.98 18.58 0.26 2.77 4 25 16.22 14.86 17.90 1.26 6.92 4 50 0.00 0.00 0.00 0.00 4 100 0.00 0.00 0.00 0.00 4 FSW Control pH 8.20 8.20 8.20 0.00 0.00 1 3.1 8.20 8.20 8.20 0.00 0.00 1 6.3 8.20 8.20 8.20 0.00 0.00 1 12.5 8.20 8.20 8.20 0.00 0.00 1 25 8.20 8.20 8.20 0.00 0.00 1 50 8.20 8.20 8.20 0.00 0.00 1 100 8.10 8.10 8.10 0.00 0.00 1 FSW Control Salinity 36.10 36.10 36.10 0.00 0.00 1 3.1 37.50 37.50 37.50 0.00 0.00 1 6.3 38.30 38.30 38.30 0.00 0.00 1 12.5 40.10 40.10 40.10 0.00 0.00 1 25 43.50 43.50 43.50 0.00 0.00 1 50 50.20 50.20 50.20 0.00 0.00 1 100 64.00 64.00 64.00 0.00 0.00 1 FSW Control % DO 102.40 102.40 102.40 0.00 0.00 1 3.1 106.40 106.40 106.40 0.00 0.00 1 6.3 102.80 102.80 102.80 0.00 0.00 1 12.5 102.10 102.10 102.10 0.00 0.00 1 25 102.50 102.50 102.50 0.00 0.00 1 50 104.80 104.80 104.80 0.00 0.00 1 100 103.00 103.00 103.00 0.00 0.00 1
Page 3 ToxCalc v5.0.23 Reviewed by:_____
Appendix C Burrlioz 2.0 Statistical Report
120WAU-0008/ R200092 Water Corporation 29 Onslow Seawater Desalination Plant: Desalination Brine Toxicity Assessment Burrlioz 2.0 report
Toxicant: Input file: C:\Users\Travis\O2 Marine\OneDrive − Documents\DATA\PROJECTS\20WAU−0008_Water Corp SWRO Enviro Aprovals & Studies\4_Ecotox Assessment\4_Analysis\EC10ToxResults.csv Time read: Tue May 19 14:43:34 2020 Units: percent (%) Model: log logistic Protection level information Protect. level Guideline Value lower 95% CI upper 95% CI 99% 4.8 2.8 12 95% 7.5 4.9 15 90% 9.2 6.3 17 80% 12 7.9 19 notes: 100
L. calcarifer 80
L. calcarifer
60 N. closterium
A. pulchella 40
E. mathaei 20 S. echinata Percentage of species potentially affected 0
0 10 20 30 40
percent (%) Data:
EC10 species 21.3 N. closterium 13 E. mathaei 8.3 S. echinata 13 A. pulchella 32.6 L. calcarifer 22.6 L. calcarifer