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Toxicity Assessment of Chlorate to Zoospore Germination and Gametophyte Growth of the Kelp Ecklonia radiata Gunns Limited

DRAFT Test Report

July 2007

Toxicity Assessment of Chlorate to Zoospore Germination and Gametophyte Growth of the Kelp Ecklonia radiata Gunns Limited

DRAFT Test Report

July 2007

Ecotox Services Australasia Toxicity Assessment of Chlorate to a Ecklonia- DRAFT PR0262 1

Contents 1. Executive Summary ........................................................................................................ 2

Glossary of Terms ........................................................................................................... 4 2. Introduction...................................................................................................................... 6 3. Methodology.................................................................................................................... 7 4. Results and Discussion................................................................................................. 10

72-h Germination........................................................................................................... 10 14-d Gametophyte Growth............................................................................................ 12 21-day Gametophyte Growth ........................................................................................ 14

5. Quality Assurance ......................................................................................................... 16 6. References .................................................................................................................... 17 Appendix A: Statistical Print-outs for 72-h Germination ............................................................... 18 Appendix B: Statistical Print-outs for 14-d Gametophyte Growth ................................................ 19 Appendix C: Statistical Print-outs for 21-d Gametophyte Growth ................................................. 20 Appendix D: Analytical Test Reports for Chlorate Determinations................................................ 21 Appendix E: Analytical Test Reports for Nitrate Determinations.................................................. 22


In order to better understand the effect of chlorate on brown macro-alga in the receiving environment of the proposed Bell Bay pulp mill, and to provide data for the development of a site-specific water quality guideline value, a series of extended ecotoxicity tests were undertaken with the kelp Ecklonia radiata, and the results of which are reported herein. This follows on from a series of toxicity tests with the inter-tidal brown macro-alga Hormosira banksii that were initiated over October and November 2006 (ESA 2006a). Although H. banksii is common in parts of Bass Strait, it is an inter-tidal species and is less likely to come into contact with the pulp mill effluent plume than would a sub-tidal macroalgal species. Gunns Limited wished to assess the effect of chlorate on a sub-tidal macroalgal species. A germination success test using the zoospores of the kelp Ecklonia radiata was identified at the most suitable and readily adaptable tests for the assessment of chlorate toxicity. Further development of the test was undertaken by Ecotox Services Australasia to extend the assays to examine growth of the female gametophyte at 14 and 21-days, providing sub-chronic test end-points.

This report describes the results of each of the toxicity tests performed, with print-outs from the statistical analyses for the 72-h germination, 14-d gametophtye growth and 21-day gametophyte growth provided in Appendix A, B and C, respectively. The concentration of chlorate and nitrate was analytically confirmed from sub-samples taken throughout the tests, and the analytical reports and associated chain-of-custody documentation are provided in Appendix D and E.

Test results indicated the following:

72-h Germination: In the absence of added nitrate, chlorate concentrations up to 10,000 µg/L did not cause any significant reduction in the germination success of E. radiata. In the presence of 50 µg/L nitrate, germination success was significantly reduced by approximately 23% at a chlorate concentration of 10,000 µg/L. In each case the 72-h EC50 was >10,000 µg chlorate. These results agree with the earlier series of tests with Hormosira banksii germination (ESA 2006a, 2006b), which were also relatively insensitive to chlorate.

14-day Gametophyte Growth: For the chlorate only treatments, there was no significant reduction in gametophyte length at the 14-d endpoint. Consequently the 14-d EC50 was >10,000 µg chlorate/L., and the NOEC and LOEC estimates were 10,000 and >10,000 µg chlorate/L, respectively.

The addition of 50 and 500 µg nitrate/L appeared to enhance gametophyte length at 14-d. This increase in the length of the gametophytes in the respective control treatments may have resulted in significant inhibitory effects being detected in the 100 µg chlorate/L treatments in the 500 µg nitrate/L tests. The 14-d EC50 estimates were >10,000 µg chlorate/L for both 50 and 500 µg nitrate/L tests. The NOEC and LOEC for the tests spiked with 50 µg nitrate/L was 200 and 1000 µg chlorate/L, respectively. The NOEC and LOEC for the tests spiked with 500 µg nitrate/L was 50 and 100 µg chlorate/L, respectively.

21-day Gametophyte Growth: With respect to the chlorate only test, there was no significant reduction in gametophyte length at the 21-d endpoint at and below the 1,000 µg chlorate/L. The 21-d EC50 was >10,000 µg chlorate/L., and the NOEC and LOEC estimates were 1,000 and 10,000 µg chlorate/L, respectively. There was a significant increase in gametophyte growth at 50 and 1000 µg chlorate/L.

1. Executive Summary


With the addition of 50 µg nitrate/L, there appeared to be an enhancement of gametophyte length at 14-d. This increase in the length of the gametophytes in the respective control treatments may have resulted in significant inhibitory effects being detected at and above the 100 µg chlorate/L treatments. Therefore the NOEC and LOEC for chlorate in the presence of 50 µg nitrate/L was 50 and 100 µg chlorate/L, respectively.

The addition of 500 µg nitrate/L has a relatively smaller enhancing effect on gametophyte length compared with the 50 µg nitrate/L. Significant inhibition in gametophyte length was detected only in the 10,000 µg chlorate/L. The NOEC and LOEC for the tests spiked with 500 µg nitrate/L was 1,000 and 10,000 µg chlorate/L, respectively.

Table 1.1. Summary of toxicity test data for chlorate, with and without the addition of nitrate, to Ecklonia radiata.

Toxicity Test End-point

Value Background Nitrate

50µg/L Nitrate 500µg/L Nitrate

72-h E. radiata macroalgal germination

72-h EC50

>10000

>10000

>10000

NOEC 10000 1000 10000

LOEC >10000 10000 >10000

14-d E. radiata macroalgal gametophyte growth 14-d EC50

>10000

>10000

>10000

NOEC 1000 200 50

LOEC >10000 1000 100

21-d E. radiata macroalgal gametophyte growth 21-d EC50 >10000 >10000 >10000

NOEC 1000 50 1000

LOEC 10000 100 10000


Glossary of Terms

The following glossary is based on that provided by Environment Canada (1999)

Acute toxicity is an adverse effect (lethal or sub-lethal) induced in the test organisms within a short period of exposure to a test material, usually a few days.

Bioassay is a test (=assay) in which the strength or potency of a substance is measured by the response of living organisms or living system. Toxicity test is a more specific and preferred term for environmental work.

Chronic toxicity implies long-term effects that are related to changes in metabolism, growth, reproduction, or ability to survive

Control is a treatment in an investigation that duplicates all the factors that might effect results, except the specific condition being studied. In toxicity tests, the control must duplicate all the conditions in the exposure treatment(s) but must contain no test material. The control is used as a check for toxicity due to basic conditions such as quality of dilution water or health and handling of the test organisms. Control is synonymous with negative control. See also positive control.

EC50 is the median effective concentration. That is the concentration of material in water (eg., mg/L) that is estimated to cause some defined toxic effect to 50% of the test organisms. In most instances the EC50 and its 95% confidence limits are statistically derived by analysing the percentages of organisms affected at various test concentrations, after a fixed period of exposure. The duration of exposure must be specified (eg. 48h).

Endpoint means the measurement(s) or value(s) that characterise the results of a test (LC50, EC50, IC50). It also means the reaction of the organism to show the effect which is intended to mark completion of the test (eg., death, number of shell abnormalities).

ICp is the inhibiting concentration for a specified percent effect (eg., IC50). It represents a point estimate of a concentration of test material that causes a designated percent inhibition (p) compared to the control, in a quantitative biological measurement such as micro-algal cell yield attained at the end of a test.

LC50 is the median effective concentration. That is the concentration of material in water (eg., mg/L) that is estimated to be lethal to 50% of the test organisms. In most instances the EC50 and its 95% confidence limits are statistically derived by analysing the percentage mortality at various test concentrations, after a fixed period of exposure. The duration of exposure must be specified (eg. 96h).

LOEC is the lowest-observed-effect concentration. This represents the lowest concentration of a test material for which a statistically significant effect on the test organisms was observed, relative to the control.

NOEC is the no-observed-effect concentration. This represents the highest test concentration of a test material for which no statistically significant effect on the test organisms was observed, relative to the control.


Positive Control is a toxicity test with a reference toxicant, used to assess the sensitivity of the organisms at the time of the test material is evaluated and the precision of the results obtained by the laboratory for that chemical.

Reference toxicant is a standard chemical used to measure the sensitivity of the test organisms to establish confidence in the toxicity data obtained for a test material. In most instances, a toxicity test with a reference toxicant is performed to assess the sensitivity of the organisms at the time the test material is evaluated and the precision of the results obtained by the laboratory for that chemical.

Replicate is a single test chamber containing a prescribed number of test organisms in either one concentration of test solution or in dilution water as a control. In a toxicity test comprising five test concentrations and a control, and using four replicates, 18 test chambers would be used. For each concentration or control, there would be 4 test chambers or replicates. A replicate must be an independent unit, and therefore, any transfer of test material or organisms from one replicate to another would invalidate a statistical analysis based on replication.

Static describes toxicity tests in which test solutions are not renewed during the test.

Sub-lethal means detrimental to the organism, but below the level that directly causes death within the test period.

Toxic means poisonous. A toxic material can cause adverse effects on living organisms, if present in sufficient amount at the right location.

Toxicant is a toxic material.


In order to better understand the effect of chlorate on brown macro-alga in the receiving environment of the proposed Bell Bay pulp mill, and to provide data for the development of a site-specific water quality guideline value, a series of extended ecotoxicity tests were undertaken with the kelp Ecklonia radiata, and the results of which are reported herein. This follows on from a series of toxicity tests with the inter-tidal brown macro-alga Hormosira banksii that were initiated over October and November 2006 (ESA 2006a). These tests with H. banksii utilised the 72-h germination test as well as extensions to the assay where growth of the rhizoid were assessed at 14 and 21-days post fertilisation of eggs. These tests were run with sodium chlorate alone and with the addition of 50, 500 and 2000 µg/L nitrate to assess whether the production of nitrogen reductase (the enzyme responsible for converting chlorate to the toxic metabolite chlorite) could mediate chlorate toxicity. The results of these tests confirmed earlier germination success test result demonstrating that H. banksii germination was insensitive to chlorate at concentrations up to 10,000 µg/L (ESA 2006b). The NOEC and LOEC for chlorate for the 14 and 21-d exposures to H. banksii was 100 and 200 µg/L. The addition of nitrate resulted in an increase in the NOEC for chlorate to 200 µg/L.

Although H. banksii is common in parts of Bass Strait, it is an inter-tidal species and is less likely to come into contact with the pulp mill effluent plume than would a sub-tidal macroalgal species. Gunns Limited wished to assess the effect of chlorate on a sub-tidal macroalgal species. The availability of test procedures using macro-algae is limited, particularly using species endemic to temperate Australian waters. A germination success test using the zoospores of the kelp Ecklonia radiata has been assessed (Bidwell et al., 1998; Ross and Bidwell, 1999; Burridge et al., 1999), and given its limited presence in the vicinity of the proposed Bell Bay discharge (Mr Daniel Elson Aquenal, personal communication), it was decided to use E. radiata for the assessment of chlorate toxicity. Further development of the test was undertaken by Ecotox Services Australasia to extend the assays to examine growth of the female gametophyte at 14 and 21-days, providing a sub-chronic test end-point.

The bioassay assessed by Bidwell et al., 1998, Ross and Bidwell, 1999, Burridge et al., 1999 utilised germination of the zoospores at 48-h following release by the sporophyte (the full-sized kelp plant). The test protocol on which these researchers based their test method was that developed by the USEPA (1995) for the kelp Macrocystis pyifera. Ecklonia radiata is a common kelp species occurring sub-tidally throughout temperate coastal waters in Australia. It belongs to the Order Laminariales, the reproductive cycles of which are characterised by heteromorphic alternation of generations. The large-leafy kelp plant (which may grow up to 2m in height) is the sporophyte, and is made up of a hold-fast, the stype (stem) and the thallus (leaf-like structures). The sporophyte is diploid (2n) The thallus contains patches of productive cells, called sori. The reproductive cells release motile zoospores from the sori that are haploid (n), which settle on suitable available substrate in approximately 1-hour. The spores germinate, mostly within 1-2 days of settlement, and develop a rhizoid. These germinated spores develop into either male of female gametophytes, which are the sexual and microscopic stage of the life cycle. The female gametophytes develop a filamentous branch with one or more eggs. The male gametophytes develop many branched filaments with lateral antheridia. The antheridia fertilise the eggs, and develop to form the large-leafy sprorophyte. The tests described in this report assess the effects of sodium chlorate alone, and sodium chlorate with 50 or 500µg nitrate /L, on germination success at 72-h, and on the growth of the female gametophyte following 14 and 21-days exposure.

2. Introduction


The methodology for the macro-algal germination test using the kelp Ecklonia radiata was based on procedures described by Bidwell et al. (1998), Burridge et al. (1999) and Ross and Bidwell (1999). However, the test duration was extended from 48 hours, as used in the above-mentioned studies, to 72 hours. It was observed over several trial experiments that differentiation between germinated and non-germinated zoospores was easier and more reproducible following 72 hours post-release rather than 48 hours.

In addition to the germination test, additional tests were conducted to examine the potential effects of chlorate on germ tube length of E. radiata. Germ tube length has been used as an endpoint in previous experiments with E. radiata (Bidwell et al. 1998, Burridge et al. 1999), however, these studies have only assessed germ tube length 48 hours after zoospore release. The present study assessed germ tube length (which mostly included the terminal eggs) at both 14- and 21-day post-zoospore release following exposure to chlorate.

Clean seawater was collected from Lurline Bay, Sydney and filtered to 0.45µm on return to the laboratory. This filtered seawater was used for spawning the broodstock and as the diluent water in the toxicity tests

Sporophyte E. radiata were collected from the sub-tidal zone (8-10m water) at Bellerive Bluff, Hobart Tasmania, sealed in clear polyethylene bags and placed on a 1-inch thick layer of moist paper overlying frozen ice-bricks in a 44L esky and transported by same-day airfreight service to the Ecotox Services Australasia laboratory in Lane Cove, NSW. Upon arrival, fertile blades were identified by the presence of sori – the spore-producing area of the sporophyte visible in distinct patches. The sori were cut from the blades and washed with filtered seawater. The sori were then cut into segments of approximately 4 x 4 cm and air-dried at room temperature (approximately 20ºC) for 30 minutes.

Once dried, the segments of sori were placed into 1 L beakers containing approximately 400 mL of chilled (<15 ºC) filtered seawater. During this soaking, gamete release usually occurs and, depending on the density of spores released, the water may go cloudy from the release. The suspensions were assessed every 5-10 minutes for the presence of spores by pipetting a few drops of the suspension on to a glass slide and examining under 400x magnification. The suspensions which showed the highest densities of motile zoospores were used in the subsequent experiments.

Once sufficient zoospore release had taken place, usually 30-45 minutes following the initial soaking, the segmented sori were removed from each of the selected beakers and the suspensions were combined. A 1 mL aliquot of the zoospore suspension was drawn-off, fixed using a single drop of 50% glacial acetic, and zoospore density determined using a hemocytometer. The zoospore suspension was adjusted to a density of 65,000 spores/mL prior to addition to the test vessels.

The 72-h germination tests were undertaken in borosilicate glass Petri dishes of 60mm diameter. A 24 x 40mm cover-slip was placed on the bottom surface of each Petri dish to provide a site of attachment and to permit easy recovery of the spores at the end of the test. Five mL of the zoospore suspension was added to each Petri dish and left for 2 hours to allow for adhesion of the spores on to the cover slip on the bottom of the Petri dish.

The 14- and 21-day growth tests were undertaken in boroscillate glass Petri dishes of 60mm diameter. Two cover slips were placed on the bottom surface of the Petri dishes,

3. Methodology


one cover slip for each time period. Five mL of the spore suspension was added to each Petri dish and left for 2 hours to allow for adhesion of the spores to the cover slips.

Seven concentrations of sodium chlorate (Sigma-Aldrich ACS Reagent, >99% purity) were prepared. The test concentrations were 10000, 1000, 200, 100, 50, 10 and 2 µg ClO3/L. This concentration series was repeated with diluent seawater containing three different nitrate levels using sodium nitrate (APS Univar AR grade reagent, minimum 99% purity) – Background, 50, and 500µg NO3/L. A filtered seawater (FSW) control, consisting of seawater collected from the Sydney Region, was tested concurrently with the chlorate treatments. Each test was conducted using four replicates per chlorate/nitrate treatment.

The temperature, pH, salinity and dissolved oxygen concentration of a representative sample from each concentration/treatment was measured. Salinity and conductivity were measured using a WTW LF330 salinity/conductivity meter with a WTW Tetracon 325 probe. The pH and temperature were measured using a WTW pH330 meter, with a WTW SenTix 41 electrode. Dissolved oxygen was measured using a WTW Oxi 330 Oximeter, with a WTW CellOx 325 probe.

Approximately 2 hours following the addition of the spores to the Petri dishes, the spore suspensions were gently decanted off and replaced with 5 mL of the appropriate test solutions. The test vessels were then incubated in a constant temperature chamber of 18±1°C with a 12:12 hr light:dark photoperiod for the duration of the exposure periods.

Test solutions were renewed with fresh test solution every 48 or 72 hours. A sub-sample of the highest chlorate treatment of 10 mg/L at each nitrate treatment was taken at each renewal period for the determination of chlorate and nitrate concentrations. Samples for the verification of chlorate concentrations were preserved by the addition of EDA preservative, while those for nitrate were preserved by freezing. All samples were shipped to the National Measurement Institute, Pymble NSW, for analysis.

At 72-h exposure, germination was assessed by removing the cover slip from each Petri dish and inverting it onto a clean glass slide for examination under a compound microscope at 400x magnification. The first 100 spores observed for each replicate test vessel were scored as being germinated or un-germinated, based on the presence of a germ tube.

Germ tube length was assessed at 14- and 21-d exposure by removing a cover slip from each Petri dish and inverting it onto a clean glass slide. The slides were then placed under a compound microscope at 400x magnification and the germination rhizoids of ten randomly selected germinated spores were photographed using a Leica digital microscope camera. Germ tube length was recorded to nearest 1 µm using the Leica IM500 image analysis software.

The concentrations of chlorate inhibiting germination or germ tube length, respectively, in 50% of the test population (the EC50) were determined by the trimmed Spearman-Karber Method using TOXCALC v5.0 (Tidepool Scientific Software) for each endpoint measured. The concentrations of chlorate causing no significant toxicity (No Observed Effect Concentration, NOEC) and the lowest concentration of test materials causing significant toxicity (Lowest Observed Effect Concentration, LOEC) were determined by performing Dunnetts or a non-parametric test, depending on the data being normally distributed and homoscedastic.


Table 3.1. Summary of test conditions for the Ecklonia radiata macroalgal germination test

Test species Ecklonia radiata

Test type Static, non-renewal

Test duration 72 hours

Test end-point Germination

Test temperature 18 ± 1oC

Test salinity 35 ± 1 ‰

Test chamber size / volume 5mL in Petri dishes

Test Concentrations 10000, 1000, 200, 100, 50, 10, 2 µg/L

Source of test organisms Field collection, Tasmania

Test acceptability criteria ≥70% germination in controls, reference toxicant results within prescribed range

Table 3.2. Summary of test conditions for the Ecklonia radiata macroalgal female gametophyte growth test

Test species Ecklonia radiata

Test type Static, 48 to 72 h renewal

Test duration 21-days, with measurement also made at 14-days

Test end-point Germ tube Length (including spore and eggs) (µm)

Test temperature 18 ± 1oC

Test salinity 35 ± 1 ‰

Test chamber size / volume 15mL in Petri dishes

Test Concentrations 10000, 1000, 200, 100, 50, 10, 2 µg/L

Source of test organisms Field collection, Belleive Bluff, Hobart, Tasmania

Test acceptability criteria ≥70% germination in controls, reference toxicant results within prescribed range

To test the relative sensitivity of the test organisms and the proficiency of the Laboratory Technicians, a separate positive (toxic) control test was conducted using copper chloride. The test was performed in the same manner as the chlorate test treatments. The results of these tests were compared with results from previous testing from the same laboratory using a control chart.


72-h Germination

The results of the E. radiata macroalgal germination test are summarised in Tables 4.1 and 4.2 below. Statistical print-outs for each of the toxicity tests are provided in Appendix A.

Table 4.1. Summary of toxicity data for chlorate using the 72-h Ecklonia radiata macroalgal germination test

Sample Concentration (µg/L)

Background (%Germinated)

50 µg/L Nitrate (%Germinated)

500 µg/L Nitrate (%Germinated)

Control 0 92.8 ± 2.5

(Filtered seawater) 95.3 ± 1.7

(filtered seawater + 50 µg NO3/L)

82.0 ± 1.8 (filtered seawater + 500

µg NO3/L)

Chlorate 2 91.0 ± 3.9 93.0 ± 2.5 81.0 ± 1.8

10 92.3 ± 2.2 91.5 ± 6.0 84.5 ± 4.2

50 94.3 ± 2.9 93.5 ± 2.7 90.0 ± 2.2

100 91.0 ± 3.2 92.5 ± 1.3 89.0 ± 6.6

200 92.3 ± 2.5 93.3 ± 2.8 82.5 ± 2.7

1000 93.3 ± 2.8 92.3 ± 1.7 91.8 ± 1.3

10000 92.5 ± 2.1 72.3 ± 14.8* 92.8 ± 1.9 Data represent Mean % Germinated ± S.D. (n=4)

* Indicates significant difference to respective Filtered Seawater Control treatment (Dunnetts Test, 1 tailed, P=0.05, df 7,24).

Table 4.2. The 72-h EC50, NOEC and LOEC for chlorate with the 72-h Ecklonia radiata macroalgal germination test end-point initiated on 10th October, 2006

Sample 72-h EC50 (µg/L) NOEC (µg/L) LOEC (µg/L)

Chlorate Only >10000 10000 >10000

Chlorate with 50 µg/L Nitrate >10000 1000 10000

Chlorate with 500 µg/L Nitrate >10000 10000 >10000

Chlorate concentrations up to 10,000 µg/L did not cause any significant reduction in the germination success of E. radiata at the background nitrate concentration. In the presence of 50 µg/L nitrate, germination success was significantly reduced by approximately 23% at a chlorate concentration of 10,000 µg/L. In contrast, in the presence of 500 µg/L nitrate, chlorate did not cause any significant reduction in germination compared with the nitrate control. However the addition of nitrate of 500 µg/L to filtered seawater diluent had an inhibitory effect on E. radiata germination. Germination success was reduced by approximately 10% at 500 µg/L nitrate compared with the Background nitrate treatment. There was no significant difference in germination success between the Background nitrate treatment and the 50 µg/L treatment. Germination success in the 500 µg/L nitrate treatment did appear to

4. Results and Discussion


improve with increasing chlorate concentrations such that at 10,000 µg/L chlorate, there was no significant difference in germination success between the background and 500 µg/L nitrate treatments.


14-d Gametophyte Growth

The test results for the 14-day E. radiata macroalgal gametophyte growth inhibition test are summarised in Tables 4.3 and 4.4 below. Statistical print-outs for each of the toxicity tests are provided in Appendix B.

Table 4.3. Summary of gametophyte length (µm) using the 14-d Ecklonia radiata gametophyte growth test.


Background (Filtered

seawater)

50 µg/L Nitrate

500 µg/L Nitrate

Control 0 14.3 ± 0.9 (Filtered

seawater)

17.6 ± 0.7 (filtered seawater + 50 µg NO3/L)

17.5 ± 1.8 (filtered seawater + 500 µg NO3/L)

Chlorate 2 17.4 ± 1.0 17.2 ± 0.5 16.3 ± 0.5

10 15.6 ± 1.1 17.6 ± 1.0 17.4 ± 1.8

50 16.3 ± 1.1 17.0 ± 2.6 17.0 ± 2.6

100 16.3 ± 1.4 15.9 ± 1.5 14.3 ± 0.9*

200 17.2 ± 1.9 15.9 ± 2.0 14.1 ± 1.1*

1,000 16.0 ± 2.2 13.7 ± 1.7* 20.3 ± 1.2

10,000 13.3 ± 0.9 11.3 ± 1.2* 8.9 ± 1.4* Data represent Mean Gametophyte Length (µm) ± SD; n=4 * Indicates significant difference to respective Filtered Seawater Control treatment (Dunnetts Test, 1 tailed, P=0.05, df 7,24). Table 4.4. The 14-d EC50, NOEC and LOEC for chlorate with the 14-d Ecklonia radiata gametophyte growth test end-point, initiated on 12th June, 2007. Each treatment was statistically compared with respective nitrate control treatment.

Sample 14-d EC50 (µg/L) NOEC (µg/L) LOEC (µg/L)

Chlorate Only >10000 10000 >10000



For the chlorate only treatments, there was no significant reduction in gametophyte length at the 14-d endpoint. Consequently the 14-d EC50 was >10,000 µg chlorate/L., and the NOEC and LOEC estimates were 10,000 and >10,000 µg chlorate/L, respectively. These analyses were based on a 1-tailed Dunnetts test, and are provided in Appendix B. A 2-tailed Dunnetts test was also performed (Appendix B) given that there appeared to be a significant enhancement in growth in some treatment, and this additional analysis indicated that there was a significant increase in gametophyte growth at 2 and 200 µg chlorate/L. The addition of 50 and 500 µg nitrate/L appeared to enhance gametophyte length at 14-d. This increase in the length of the gametophytes in the respective control treatments may have resulted in significant inhibitory effects being detected in the 100 µg chlorate/L treatments in the 500 µg nitrate/L tests. The 14-d EC50 estimates were >10,000 µg chlorate/L for both 50 and 500 µg nitrate/L tests. The NOEC and LOEC for the tests spiked with 50 µg nitrate/L was 200 and 1000 µg chlorate/L, respectively. The


NOEC and LOEC for the tests spiked with 500 µg nitrate/L was 50 and 100 µg chlorate/L, respectively.


21-day Gametophyte Growth

The results for the 21-day E. radiata gametophyte growth test are summarised in Tables 4.5and 4.6 below. Statistical print-outs for each of the toxicity tests are provided in Appendix C.

Table 4.5. Summary of gametophyte length (µm) using the 21-d Ecklonia radiata gametophyte growth test


Background

50 µg/L Nitrate

500 µg/L Nitrate

FSW Control 0 14.1 ± 1.0 20.0 ± 1.4 15.3 ± 0.8

Chlorate 2 16.0 ± 1.1 18.5 ± 0.1 15.5 ± 0.3

10 16.1 ± 1.0 18.8 ± 1.5 16.5 ± 1.0

50 16.9 ± 1.3 22.1 ± 1.2 15.6 ± 0.7

100 15.9 ± 1.3 14.7 ± 1.1* 14.8 ± 1.1

200 14.7 ± 1.2 14.7 ± 1.2* 15.4 ± 1.2

1,000 17.1 ± 2.0 13.8 ± 1.3* 19.3 ± 1.4

10,000 11.1 ± 0.8* 10.6 ± 1.4* 9.1 ± 1.5* Data represent Mean Gametophyte Length (µm) ± SD; n=4. * Indicates significant difference to the Filtered Seawater Control treatment (Dunnetts Test, 1 tailed, P=0.05, df 7,24). Table 4.6. The 21-d EC50, NOEC and LOEC for chlorate with the 21-d Ecklonia radiata gametophyte growth test initiated on 10th October, 2006

Sample 14-d EC50 (µg/L) NOEC (µg/L) LOEC (µg/L)

Chlorate Only >10000 1000 10000



With respect to the chlorate only test, there was no significant reduction in gametophyte length at the 21-d endpoint at and below the 1,000 µg chlorate/L. The 14-d EC50 was >10,000 µg chlorate/L., and the NOEC and LOEC estimates were 1,000 and 10,000 µg chlorate/L, respectively. These analyses were based on a 1-tailed Dunnetts test, and are provided in Appendix C. A 2-tailed Dunnetts test was also performed (also see Appendix C) given that there appeared to be a significant enhancement in growth in the 1000 µg chlorate/L treatment. This additional analysis indicated that there was a significant increase in gametophyte growth at 50 and 1000 µg chlorate/L. With the addition of 50 µg nitrate/L, there appeared to be an enhancement of gametophyte length at 21-d. This increase in the length of the gametophytes in the respective control treatments may have resulted in significant inhibitory effects being detected at and above the 100 µg chlorate/L treatments. Therefore the NOEC and LOEC for chlorate in the presence of 50 µg nitrate/L was 50 and 100 µg chlorate/L, respectively.


The addition of 500 µg nitrate/L has a relatively smaller enhancing effect on gametophyte length compared with the 50 µg nitrate/L. Significant inhibition in gametophyte length was detected only in the 10,000 µg chlorate/L. The NOEC and LOEC for the tests spiked with 500 µg nitrate/L was 1,000 and 10,000 µg chlorate/L, respectively.


The E. radiata germination tests at 72-h and gametophyte growth at 14-d undertaken concurrently with the chlorate treatments met all quality assurance criteria for the test. The mean percent germination at 72-h in the filtered seawater control treatment exceeded the minimum control germination of 70%. Water quality parameters for control samples were also within test acceptability ranges.

The 72-h EC50 estimate for the copper chloride reference toxicant run concurrently with the 10 October and 16 November tests fell within the reference toxicant cusum chart control limits (Table 5.1 and 5.2). This indicated that all toxicity tests were within the expected range with respect to performance and sensitivity at 72-h.

Table 5.6 The Quality Assurance limits for the Ecklonia radiata macroalgal germination test.

QA Measure QA Limit Observed value Within Limits?

Control germination minimum ≥70% 92.8 Yes

Test temperature Limits 18±1oC 18.0-18.5°C Yes

Reference toxicant within Cusum limits

178-645 µg Cu2+/L

382.9 µg Cu2+/L

Yes

Table 5.7 The Quality Assurance limits for the Ecklonia radiata gametophyte growth at 14-d.

QA Measure QA Limit Observed value Within Limits?

Test temperature Limits 18±1oC 18.0-19.0°C Yes

Reference toxicant within Cusum limits

295-623 µg Cu2+/L

623 µg Cu2+/L

Yes

5. Quality Assurance


Bidwell, J.R., Wheeler, K.W., and Burridge, T.R. (1998) Toxicant effects on the zoospore stage of the marine macroalga Ecklonia radiata (Phaeophyta: Laminariales). Marine Ecology Progress Series 163: 259-265.

Burridge, T., Campbell, S. and Bidwell, J. (1999) Use of the kelp Ecklonia radiata (Laminariales: Phaephyta) in routine toxicity testing of sewage effluents. Australasian Journal of Ecotoxicology 5:133-140.

Ross, K.E. and Bidwell, J.R. (1999) Comparative response of Ecklonia radiata zoospores and other marine species to complex effluents. Australasian Journal of Ecotoxicology 5:113-122.

ESA (2006a) Toxicity assessment of chlorate to the brown macro alga Hormosira banksii. Draft Test Report for Gunns Limited, December 2006.

ESA (2006b) Toxicity assessment of chlorate to the brown macro alga Hormosira banksii. Test Report TR0177/1, for Gunns Limited, June 2006.

USEPA (1995) Short term methods for estimating the chronic toxicity of effluents and receiving waters to West-coast marine and estuarine organisms. First Edition. United States Environmental Protection Agency, Environmental Monitoring Systems Laboratory, Cincinatti, OH. EPA/600/R-95-136.

6. References


Appendix A: Statistical Print-outs for 72-h Germination

Macroalgal Germination Test-GerminationStart Date: 12/06/07 Test ID: PR0177/03 Sample ID: 500ug/L NitrateEnd Date: 15/06/07 Lab ID: Sample Type: ChlorateSample Date: Protocol: ESASOP116 Test Species: ER-Ecklonia radiataComments: Chlorate concentrations prepared in 500 ug/L Nitrate

Conc-ug/L 1 2 3 4ontrol (500ug/L) 0.8100 0.8400 0.8000 0.8300

2 0.8300 0.8000 0.8200 0.790010 0.9000 0.8500 0.8000 0.830050 0.8900 0.9300 0.9000 0.8800

100 0.9500 0.8200 0.9000200 0.8000 0.8100 0.8600 0.8300

1000 0.9200 0.9300 0.9200 0.900010000 0.9400 0.9300 0.9400 0.9000

Transform: Arcsin Square Root 1-TailedConc-ug/L Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD

ontrol (500ug/L) 0.8200 1.0000 1.1330 1.1071 1.1593 2.100 42 0.8100 0.9878 1.1201 1.0948 1.1458 2.080 4 0.392 2.651 0.0874

10 0.8450 1.0305 1.1688 1.1071 1.2490 5.131 4 -1.086 2.651 0.087450 0.9000 1.0976 1.2505 1.2171 1.3030 2.991 4 -3.564 2.651 0.0874

100 0.8900 1.0854 1.2423 1.1326 1.3453 8.571 3 -3.071 2.651 0.0944200 0.8250 1.0061 1.1400 1.1071 1.1873 3.105 4 -0.213 2.651 0.0874

1000 0.9175 1.1189 1.2800 1.2490 1.3030 1.759 4 -4.462 2.651 0.087410000 0.9275 1.1311 1.2997 1.2490 1.3233 2.700 4 -5.058 2.651 0.0874

Auxiliary Tests Statistic Critical SkewShapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.967655 0.902 0.046365Bartlett's Test indicates equal variances (p = 0.14) 10.96026 18.47531Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-ProbBonferroni t Test 10000 >10000 0.071617 0.087309 0.020348 0.002172 1.8E-05

Dose-Response Plot

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Auxiliary Data SummaryConc-ug/L Parameter Mean Min Max SD CV% N

ontrol (500ug/L) % Germinated 82.00 80.00 84.00 1.83 1.65 42 81.00 79.00 83.00 1.83 1.67 4

10 84.50 80.00 90.00 4.20 2.43 450 90.00 88.00 93.00 2.16 1.63 4

100 89.00 82.00 95.00 6.56 2.88 3200 82.50 80.00 86.00 2.65 1.97 4

1000 91.75 90.00 93.00 1.26 1.22 410000 92.75 90.00 94.00 1.89 1.48 4

ontrol (500ug/L) Temp C 18.00 18.00 18.00 0.00 0.00 12 18.00 18.00 18.00 0.00 0.00 1

10 18.00 18.00 18.00 0.00 0.00 150 18.00 18.00 18.00 0.00 0.00 1

100 18.00 18.00 18.00 0.00 0.00 1200 18.00 18.00 18.00 0.00 0.00 1

1000 18.00 18.00 18.00 0.00 0.00 110000 18.00 18.00 18.00 0.00 0.00 1

ontrol (500ug/L) pH 8.20 8.20 8.20 0.00 0.00 12 8.10 8.10 8.10 0.00 0.00 1

10 8.10 8.10 8.10 0.00 0.00 150 8.10 8.10 8.10 0.00 0.00 1

100 8.10 8.10 8.10 0.00 0.00 1200 8.10 8.10 8.10 0.00 0.00 1

1000 8.10 8.10 8.10 0.00 0.00 110000 8.20 8.20 8.20 0.00 0.00 1

ontrol (500ug/L) Salinity ppt 37.60 37.60 37.60 0.00 0.00 12 38.00 38.00 38.00 0.00 0.00 1

10 38.00 38.00 38.00 0.00 0.00 150 37.90 37.90 37.90 0.00 0.00 1

100 37.90 37.90 37.90 0.00 0.00 1200 37.80 37.80 37.80 0.00 0.00 1

1000 37.80 37.80 37.80 0.00 0.00 110000 37.30 37.30 37.30 0.00 0.00 1

ontrol (500ug/L) DO (%sat) 99.00 99.00 99.00 0.00 0.00 12 98.20 98.20 98.20 0.00 0.00 1

10 98.40 98.40 98.40 0.00 0.00 150 98.20 98.20 98.20 0.00 0.00 1

100 98.60 98.60 98.60 0.00 0.00 1200 98.30 98.30 98.30 0.00 0.00 1

1000 98.80 98.80 98.80 0.00 0.00 110000 97.90 97.90 97.90 0.00 0.00 1



Conc-ug/L 1 2 3 4Control (50ug/L) 0.9500 0.9700 0.9300 0.9600

2 0.9300 0.9000 0.9600 0.930010 0.9400 0.8300 0.9200 0.970050 0.9500 0.9000 0.9300 0.9600

100 0.9200 0.9400 0.9100 0.9300200 0.9600 0.9200 0.9500 0.9000

1000 0.9200 0.9000 0.9300 0.940010000 0.6300 0.5700 0.8900 0.8000


Control (50ug/L) 0.9525 1.0000 1.3536 1.3030 1.3967 2.935 42 0.9300 0.9764 1.3061 1.2490 1.3694 3.773 4 0.828 2.480 0.1422

10 0.9150 0.9606 1.2875 1.1458 1.3967 8.183 4 1.153 2.480 0.142250 0.9350 0.9816 1.3167 1.2490 1.3694 4.010 4 0.644 2.480 0.1422

100 0.9250 0.9711 1.2941 1.2661 1.3233 1.903 4 1.037 2.480 0.1422200 0.9325 0.9790 1.3120 1.2490 1.3694 4.210 4 0.726 2.480 0.1422

1000 0.9225 0.9685 1.2899 1.2490 1.3233 2.449 4 1.112 2.480 0.1422*10000 0.7225 0.7585 1.0281 0.8556 1.2327 16.869 4 5.676 2.480 0.1422

Auxiliary Tests Statistic Critical SkewShapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.962391 0.904 0.082628Bartlett's Test indicates equal variances (p = 0.02) 16.74052 18.47531Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-ProbDunnett's Test 1000 10000 3162.278 0.077279 0.081042 0.041106 0.006578 3.1E-04

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Control (50ug/L) % Germinated 95.25 93.00 97.00 1.71 1.37 42 93.00 90.00 96.00 2.45 1.68 4

10 91.50 83.00 97.00 6.03 2.68 450 93.50 90.00 96.00 2.65 1.74 4

100 92.50 91.00 94.00 1.29 1.23 4200 93.25 90.00 96.00 2.75 1.78 4

1000 92.25 90.00 94.00 1.71 1.42 410000 72.25 57.00 89.00 14.82 5.33 4

Control (50ug/L) Temp C 18.00 18.00 18.00 0.00 0.00 12 18.00 18.00 18.00 0.00 0.00 1

10 18.00 18.00 18.00 0.00 0.00 150 18.00 18.00 18.00 0.00 0.00 1

100 18.00 18.00 18.00 0.00 0.00 1200 18.00 18.00 18.00 0.00 0.00 1

1000 18.00 18.00 18.00 0.00 0.00 110000 18.00 18.00 18.00 0.00 0.00 1

Control (50ug/L) pH 8.10 8.10 8.10 0.00 0.00 12 8.10 8.10 8.10 0.00 0.00 1

10 8.10 8.10 8.10 0.00 0.00 150 8.10 8.10 8.10 0.00 0.00 1

100 8.10 8.10 8.10 0.00 0.00 1200 8.10 8.10 8.10 0.00 0.00 1

1000 8.10 8.10 8.10 0.00 0.00 110000 8.10 8.10 8.10 0.00 0.00 1

Control (50ug/L) Salinity ppt 38.10 38.10 38.10 0.00 0.00 12 38.00 38.00 38.00 0.00 0.00 1

10 38.00 38.00 38.00 0.00 0.00 150 38.10 38.10 38.10 0.00 0.00 1

100 38.00 38.00 38.00 0.00 0.00 1200 37.90 37.90 37.90 0.00 0.00 1

1000 37.90 37.90 37.90 0.00 0.00 110000 37.70 37.70 37.70 0.00 0.00 1

Control (50ug/L) DO (%sat) 98.50 98.50 98.50 0.00 0.00 12 97.40 97.40 97.40 0.00 0.00 1

10 97.80 97.80 97.80 0.00 0.00 150 99.10 99.10 99.10 0.00 0.00 1

100 98.20 98.20 98.20 0.00 0.00 1200 98.60 98.60 98.60 0.00 0.00 1

1000 98.70 98.70 98.70 0.00 0.00 110000 98.10 98.10 98.10 0.00 0.00 1


Macroalgal Germination Test-GerminationStart Date: 12/06/07 Test ID: PR0177/01 Sample ID: 0 NitrateEnd Date: 15/06/07 Lab ID: Sample Type: ChlorateSample Date: Protocol: ESASOP116 Test Species: ER-Ecklonia radiataComments: Chlorate concentrations prepared in 0 ug/L Nitrate (Background)

Conc-ug/L 1 2 3 4NO3 Control (0) 0.9300 0.9600 0.9200 0.9000

2 0.9500 0.9300 0.9000 0.860010 0.9100 0.9000 0.9300 0.950050 0.9200 0.9800 0.9200 0.9500

100 0.9000 0.8700 0.9300 0.9400200 0.8900 0.9300 0.9500 0.9200

1000 0.9600 0.9000 0.9500 0.920010000 0.9200 0.9500 0.9000 0.9300


NO3 Control (0) 0.9275 1.0000 1.3014 1.2490 1.3694 3.886 42 0.9100 0.9811 1.2712 1.1873 1.3453 5.380 4 0.787 2.480 0.0953

10 0.9225 0.9946 1.2909 1.2490 1.3453 3.308 4 0.274 2.480 0.095350 0.9425 1.0162 1.3356 1.2840 1.4289 5.136 4 -0.890 2.480 0.0953

100 0.9100 0.9811 1.2693 1.2019 1.3233 4.316 4 0.835 2.480 0.0953200 0.9225 0.9946 1.2913 1.2327 1.3453 3.614 4 0.263 2.480 0.0953

1000 0.9325 1.0054 1.3120 1.2490 1.3694 4.210 4 -0.275 2.480 0.095310000 0.9250 0.9973 1.2954 1.2490 1.3453 3.096 4 0.157 2.480 0.0953

Auxiliary Tests Statistic Critical SkewShapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.959775 0.904 0.138734Bartlett's Test indicates equal variances (p = 0.98) 1.482114 18.47531Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-ProbDunnett's Test 10000 >10000 0.056345 0.060641 0.001843 0.00295 0.730547

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Macroalgal Germination Test-GerminationStart Date: 12/06/07 Test ID: PR0177/01 Sample ID: 0 NitrateEnd Date: 15/06/07 Lab ID: Sample Type: ChlorateSample Date: Protocol: ESASOP116 Test Species: ER-Ecklonia radiataComments: Chlorate concentrations prepared in 0 ug/L Nitrate (Background)


NO3 Control (0) % Germinated 92.75 90.00 96.00 2.50 1.70 42 91.00 86.00 95.00 3.92 2.17 4

10 92.25 90.00 95.00 2.22 1.61 450 94.25 92.00 98.00 2.87 1.80 4

100 91.00 87.00 94.00 3.16 1.95 4200 92.25 89.00 95.00 2.50 1.71 4

1000 93.25 90.00 96.00 2.75 1.78 410000 92.50 90.00 95.00 2.08 1.56 4

NO3 Control (0) Temp C 18.00 18.00 18.00 0.00 0.00 12 18.00 18.00 18.00 0.00 0.00 1

10 18.00 18.00 18.00 0.00 0.00 150 18.00 18.00 18.00 0.00 0.00 1

100 18.00 18.00 18.00 0.00 0.00 1200 18.00 18.00 18.00 0.00 0.00 1

1000 18.00 18.00 18.00 0.00 0.00 110000 18.00 18.00 18.00 0.00 0.00 1

NO3 Control (0) pH 8.10 8.10 8.10 0.00 0.00 12 8.10 8.10 8.10 0.00 0.00 1

10 8.10 8.10 8.10 0.00 0.00 150 8.10 8.10 8.10 0.00 0.00 1

100 8.10 8.10 8.10 0.00 0.00 1200 8.10 8.10 8.10 0.00 0.00 1

1000 8.10 8.10 8.10 0.00 0.00 110000 8.10 8.10 8.10 0.00 0.00 1

NO3 Control (0) Salinity ppt 37.50 37.50 37.50 0.00 0.00 12 38.20 38.20 38.20 0.00 0.00 1

10 38.20 38.20 38.20 0.00 0.00 150 38.20 38.20 38.20 0.00 0.00 1

100 38.20 38.20 38.20 0.00 0.00 1200 38.10 38.10 38.10 0.00 0.00 1

1000 38.10 38.10 38.10 0.00 0.00 110000 37.80 37.80 37.80 0.00 0.00 1

NO3 Control (0) DO (%sat) 97.10 97.10 97.10 0.00 0.00 12 98.50 98.50 98.50 0.00 0.00 1

10 97.70 97.70 97.70 0.00 0.00 150 97.90 97.90 97.90 0.00 0.00 1

100 98.30 98.30 98.30 0.00 0.00 1200 98.50 98.50 98.50 0.00 0.00 1

1000 97.10 97.10 97.10 0.00 0.00 110000 98.30 98.30 98.30 0.00 0.00 1


Macroalgal Germination Test-GerminationStart Date: 12/06/07 Test ID: PR0177/04 Sample ID: Nitrate OnlyEnd Date: 15/06/07 Lab ID: Sample Type: Nitrate ControlsSample Date: Protocol: ESASOP116 Test Species: ER-Ecklonia radiataComments:

Conc-ug/L 1 2 3 4Background 0.9300 0.9600 0.9200 0.9000

50 0.9500 0.9700 0.9300 0.9600500 0.8100 0.8400 0.8000 0.8300

Transform: Arcsin Square Root 1-TailedConc-ug/L Mean N-Mean Mean Min Max CV% N t-Stat Critical MSDBackground 0.9275 1.0000 1.3014 1.2490 1.3694 3.886 4

50 0.9525 1.0270 1.3536 1.3030 1.3967 2.935 4 -1.866 2.180 0.0610*500 0.8200 0.8841 1.1330 1.1071 1.1593 2.100 4 6.015 2.180 0.0610


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Macroalgal Germination Test-GerminationStart Date: 12/06/07 Test ID: PR0177/04 Sample ID: Nitrate OnlyEnd Date: 15/06/07 Lab ID: Sample Type: Nitrate ControlsSample Date: Protocol: ESASOP116 Test Species: ER-Ecklonia radiataComments:

Auxiliary Data SummaryConc-ug/L Parameter Mean Min Max SD CV% NBackground % Germinated 92.75 90.00 96.00 2.50 1.70 4

50 95.25 93.00 97.00 1.71 1.37 4500 82.00 80.00 84.00 1.83 1.65 4

Background Temp C 18.00 18.00 18.00 0.00 0.00 150 18.00 18.00 18.00 0.00 0.00 1

500 18.00 18.00 18.00 0.00 0.00 1Background pH 8.10 8.10 8.10 0.00 0.00 1

50 8.10 8.10 8.10 0.00 0.00 1500 8.20 8.20 8.20 0.00 0.00 1

Background Salinity ppt 37.50 37.50 37.50 0.00 0.00 150 38.10 38.10 38.10 0.00 0.00 1

500 37.60 37.60 37.60 0.00 0.00 1Background DO (%sat) 97.10 97.10 97.10 0.00 0.00 1

50 98.50 98.50 98.50 0.00 0.00 1500 99.00 99.00 99.00 0.00 0.00 1



Appendix B: Statistical Print-outs for 14-d Gametophyte Growth

Macroalgal Growth-GrowthStart Date: 12/06/07 Test ID: PR0177/07 Sample ID: 500UG/L NIEnd Date: 26/06/07 Lab ID: Sample Type: CHLORATESample Date: Protocol: ESASOP116 Test Species: ER-Ecklonia radiataComments: Chlorate concentrations prepared in 500 ug/L Nitrate

Conc-ug/L 1 2 3 4Control (500) 15.000 17.500 18.100 19.200

2 16.500 15.900 15.900 17.00010 20.000 17.500 16.200 16.00050 15.600 16.300 16.000 16.000

100 16.500 14.600 15.200 14.200200 15.600 14.000 13.900 13.000

1000 19.700 21.900 19.200 20.50010000 7.100 9.900 10.200 8.400

Transform: Untransformed 1-Tailed IsotoConc-ug/L Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD MeanControl (500) 17.450 1.0000 17.450 15.000 19.200 10.192 4 17.450

2 16.325 0.9355 16.325 15.900 17.000 3.256 4 1.271 2.480 2.196 16.87510 17.425 0.9986 17.425 16.000 20.000 10.565 4 0.028 2.480 2.196 16.87550 15.975 0.9155 15.975 15.600 16.300 1.798 4 1.666 2.480 2.196 16.388

*100 15.125 0.8668 15.125 14.200 16.500 6.642 4 2.626 2.480 2.196 16.388*200 14.125 0.8095 14.125 13.000 15.600 7.655 4 3.755 2.480 2.196 16.3881000 20.325 1.1648 20.325 19.200 21.900 5.799 4 -3.247 2.480 2.196 16.388

*10000 8.900 0.5100 8.900 7.100 10.200 16.127 4 9.656 2.480 2.196 8.900


Linear Interpolation (200 Resamples)Point ug/L SD 95% CL(Exp) SkewIC05 34.41026 715.4004 0 3063.767 0.8750IC10 1820.367 876.4291 0 3541.043 -0.6357IC15 2869.115 661.3967 882.2654 4631.602 -0.0565IC20 3917.863 651.3713 2024.843 5749.162 0.0028IC25 4966.611 655.8297 2958.476 6864.728 0.0529IC40 8112.855IC50 >10000

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Auxiliary Data SummaryConc-ug/L Parameter Mean Min Max SD CV% NControl (500) Gametophyte length 17.45 15.00 19.20 1.78 7.64 4

2 16.33 15.90 17.00 0.53 4.47 410 17.43 16.00 20.00 1.84 7.79 450 15.98 15.60 16.30 0.29 3.35 4

100 15.13 14.20 16.50 1.00 6.63 4200 14.13 13.00 15.60 1.08 7.36 4

1000 20.33 19.20 21.90 1.18 5.34 410000 8.90 7.10 10.20 1.44 13.46 4

Control (500) Temp C 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

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Control (500) pH 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

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Control (500) Salinity ppt 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

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Control (500) DO (%sat) 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

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Macroalgal Growth-GrowthStart Date: 12/06/07 Test ID: PR0177/06 Sample ID: 50UG/L NITEnd Date: 26/06/07 Lab ID: Sample Type: CHLORATESample Date: Protocol: ESASOP116 Test Species: ER-Ecklonia radiataComments: Chlorate concentrations prepared in 50 ug/L Nitrate

Conc-ug/L 1 2 3 4Control (50) 17.100 18.600 17.000 17.600

2 16.700 16.900 17.400 17.80010 17.800 16.700 19.000 17.00050 13.200 18.500 18.300 18.100

100 15.000 14.300 17.400 17.000200 14.300 14.000 17.500 17.700

1000 13.800 16.000 12.500 12.30010000 9.600 11.200 12.100 12.200

Transform: Untransformed 1-Tailed IsotoConc-ug/L Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Mean

Control (50) 17.575 1.0000 17.575 17.000 18.600 4.165 4 17.5752 17.200 0.9787 17.200 16.700 17.800 2.888 4 0.344 2.480 2.701 17.413

10 17.625 1.0028 17.625 16.700 19.000 5.830 4 -0.046 2.480 2.701 17.41350 17.025 0.9687 17.025 13.200 18.500 15.009 4 0.505 2.480 2.701 17.025

100 15.925 0.9061 15.925 14.300 17.400 9.473 4 1.515 2.480 2.701 15.925200 15.875 0.9033 15.875 14.000 17.700 12.581 4 1.561 2.480 2.701 15.875

*1000 13.650 0.7767 13.650 12.300 16.000 12.469 4 3.604 2.480 2.701 13.650*10000 11.275 0.6415 11.275 9.600 12.200 10.677 4 5.785 2.480 2.701 11.275

Auxiliary Tests Statistic Critical SkewShapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.964149 0.904 -0.58234Bartlett's Test indicates equal variances (p = 0.25) 8.969102 18.47531Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-ProbDunnett's Test 200 1000 447.2136 2.700976 0.153683 20.03054 2.372292 3.3E-05

Linear Interpolation (200 Resamples)Point ug/L SD 95% CL(Exp) SkewIC05 64.94318 49.41184 0 327.8912 2.5600IC10 220.6742 139.0996 0 660.7637 1.0544IC15 536.6292 354.8173 0 1975.301 2.4134IC20 852.5843 897.5015 0 5638.9 1.6874IC25 2776.316 1725.625 0 7927.129 0.4753IC40 >10000IC50 >10000

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Control (50) Gametophyte length 17.58 17.00 18.60 0.73 4.87 42 17.20 16.70 17.80 0.50 4.10 4

10 17.63 16.70 19.00 1.03 5.75 450 17.03 13.20 18.50 2.56 9.39 4

100 15.93 14.30 17.40 1.51 7.71 4200 15.88 14.00 17.70 2.00 8.90 4

1000 13.65 12.30 16.00 1.70 9.56 410000 11.28 9.60 12.20 1.20 9.73 4

Control (50) Temp C 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0

Control (50) pH 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0


10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0

Control (50) DO (%sat) 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0


Macroalgal Growth-GrowthStart Date: 12/06/07 Test ID: PR0177/05 Sample ID: 0 NitrateEnd Date: 26/06/07 Lab ID: Sample Type: ChlorateSample Date: Protocol: ESASOP116 Test Species: ER-Ecklonia radiataComments: Chlorate concentrations prepared in 0 ug/L Nitrate (Background)

Conc-ug/L 1 2 3 4Control (0) 13.700 13.300 15.300 14.700

2 16.100 18.200 18.100 17.30010 16.300 15.600 14.000 16.40050 16.800 14.600 17.100 16.500

100 14.200 17.200 16.500 17.100200 14.700 19.100 17.000 18.000

1000 13.000 15.900 18.000 16.90010000 13.900 12.300 12.900 14.100

Transform: Untransformed 1-TailedConc-ug/L Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD

Control (0) 14.250 1.0000 14.250 13.300 15.300 6.419 42 17.425 1.2228 17.425 16.100 18.200 5.571 4 -3.271 2.480 2.408

10 15.575 1.0930 15.575 14.000 16.400 7.118 4 -1.365 2.480 2.40850 16.250 1.1404 16.250 14.600 17.100 6.935 4 -2.060 2.480 2.408

100 16.250 1.1404 16.250 14.200 17.200 8.623 4 -2.060 2.480 2.408200 17.200 1.2070 17.200 14.700 19.100 10.898 4 -3.039 2.480 2.408

1000 15.950 1.1193 15.950 13.000 18.000 13.452 4 -1.751 2.480 2.40810000 13.300 0.9333 13.300 12.300 14.100 6.380 4 0.979 2.480 2.408

Auxiliary Tests Statistic Critical SkewShapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.924228 0.904 -0.7792Bartlett's Test indicates equal variances (p = 0.71) 4.621995 18.47531Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-ProbDunnett's Test 10000 >10000 2.407511 0.168948 7.843571 1.884792 0.003963

Dose-Response Plot


0

5

10

15

20

25

Con

trol (

0) 2 10 50 100

200

1000

1000

0

Gro

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10 15.58 14.00 16.40 1.11 6.76 450 16.25 14.60 17.10 1.13 6.53 4

100 16.25 14.20 17.20 1.40 7.28 4200 17.20 14.70 19.10 1.87 7.96 4

1000 15.95 13.00 18.00 2.15 9.18 410000 13.30 12.30 14.10 0.85 6.93 4

Control (0) Temp C 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0

Control (0) pH 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0


10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0

Control (0) DO (%sat) 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0



Conc-ug/L 1 2 3 4Control (0) 13.700 13.300 15.300 14.700

2 16.100 18.200 18.100 17.30010 16.300 15.600 14.000 16.40050 16.800 14.600 17.100 16.500

100 14.200 17.200 16.500 17.100200 14.700 19.100 17.000 18.000

1000 13.000 15.900 18.000 16.90010000 13.900 12.300 12.900 14.100


Control (0) 14.250 1.0000 14.250 13.300 15.300 6.419 4*2 17.425 1.2228 17.425 16.100 18.200 5.571 4 3.271 2.910 2.82510 15.575 1.0930 15.575 14.000 16.400 7.118 4 1.365 2.910 2.82550 16.250 1.1404 16.250 14.600 17.100 6.935 4 2.060 2.910 2.825

100 16.250 1.1404 16.250 14.200 17.200 8.623 4 2.060 2.910 2.825*200 17.200 1.2070 17.200 14.700 19.100 10.898 4 3.039 2.910 2.8251000 15.950 1.1193 15.950 13.000 18.000 13.452 4 1.751 2.910 2.825

10000 13.300 0.9333 13.300 12.300 14.100 6.380 4 0.979 2.910 2.825

Auxiliary Tests Statistic Critical SkewShapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.924228 0.904 -0.7792Bartlett's Test indicates equal variances (p = 0.71) 4.621995 18.47531Hypothesis Test (2-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-ProbDunnett's Test <2 2 2.824943 0.198242 7.843571 1.884792 0.003963

Dose-Response Plot


0

5

10

15

20

25

Con

trol (

0) *2 10 50 100

*200

1000

1000

0

Gro

wth





10 15.58 14.00 16.40 1.11 6.76 450 16.25 14.60 17.10 1.13 6.53 4

100 16.25 14.20 17.20 1.40 7.28 4200 17.20 14.70 19.10 1.87 7.96 4

1000 15.95 13.00 18.00 2.15 9.18 410000 13.30 12.30 14.10 0.85 6.93 4

Control (0) Temp C 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0

Control (0) pH 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0


10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0

Control (0) DO (%sat) 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0



Conc-ug/L 1 2 3 4Control (50) 17.100 18.600 17.000 17.600

2 16.700 16.900 17.400 17.80010 17.800 16.700 19.000 17.00050 13.200 18.500 18.300 18.100

100 15.000 14.300 17.400 17.000200 14.300 14.000 17.500 17.700

1000 13.800 16.000 12.500 12.30010000 9.600 11.200 12.100 12.200

Transform: Untransformed 2-Tailed IsotoConc-ug/L Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Mean

Control (50) 17.575 1.0000 17.575 17.000 18.600 4.165 4 17.5752 17.200 0.9787 17.200 16.700 17.800 2.888 4 0.344 2.910 3.169 17.413

10 17.625 1.0028 17.625 16.700 19.000 5.830 4 0.046 2.910 3.169 17.41350 17.025 0.9687 17.025 13.200 18.500 15.009 4 0.505 2.910 3.169 17.025

100 15.925 0.9061 15.925 14.300 17.400 9.473 4 1.515 2.910 3.169 15.925200 15.875 0.9033 15.875 14.000 17.700 12.581 4 1.561 2.910 3.169 15.875

*1000 13.650 0.7767 13.650 12.300 16.000 12.469 4 3.604 2.910 3.169 13.650*10000 11.275 0.6415 11.275 9.600 12.200 10.677 4 5.785 2.910 3.169 11.275

Auxiliary Tests Statistic Critical SkewShapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.964149 0.904 -0.58234Bartlett's Test indicates equal variances (p = 0.25) 8.969102 18.47531Hypothesis Test (2-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-ProbDunnett's Test 200 1000 447.2136 3.16929 0.180329 20.03054 2.372292 3.3E-05

Linear Interpolation (200 Resamples)Point ug/L SD 95% CL(Exp) SkewIC05 64.94318 49.27168 0 317.7808 2.6385IC10 220.6742 184.8367 0 803.8326 3.1073IC15 536.6292 485.657 0 3269.127 3.3122IC20 852.5843 1012.069 21.77946 6245.967 1.8909IC25 2776.316 1774.3 0 8386.497 0.4783IC40 >10000IC50 >10000

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 5000 10000 15000

Dose ug/L

Res

pons

e



Dose-Response Plot


0

2

4

6

8

10

12

14

16

18

20

Con

trol (

50) 2 10 50 100

200

*100

0

*100

00

Gro

wth





10 17.63 16.70 19.00 1.03 5.75 450 17.03 13.20 18.50 2.56 9.39 4

100 15.93 14.30 17.40 1.51 7.71 4200 15.88 14.00 17.70 2.00 8.90 4

1000 13.65 12.30 16.00 1.70 9.56 410000 11.28 9.60 12.20 1.20 9.73 4

Control (50) Temp C 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0

Control (50) pH 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0


10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0

Control (50) DO (%sat) 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0



Conc-ug/L 1 2 3 4Control (500) 15.000 17.500 18.100 19.200

2 16.500 15.900 15.900 17.00010 20.000 17.500 16.200 16.00050 15.600 16.300 16.000 16.000

100 16.500 14.600 15.200 14.200200 15.600 14.000 13.900 13.000

1000 19.700 21.900 19.200 20.50010000 7.100 9.900 10.200 8.400

Transform: Untransformed 2-Tailed IsotoConc-ug/L Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD MeanControl (500) 17.450 1.0000 17.450 15.000 19.200 10.192 4 17.450

2 16.325 0.9355 16.325 15.900 17.000 3.256 4 1.271 2.910 2.577 16.87510 17.425 0.9986 17.425 16.000 20.000 10.565 4 0.028 2.910 2.577 16.87550 15.975 0.9155 15.975 15.600 16.300 1.798 4 1.666 2.910 2.577 16.388

100 15.125 0.8668 15.125 14.200 16.500 6.642 4 2.626 2.910 2.577 16.388*200 14.125 0.8095 14.125 13.000 15.600 7.655 4 3.755 2.910 2.577 16.388

*1000 20.325 1.1648 20.325 19.200 21.900 5.799 4 3.247 2.910 2.577 16.388*10000 8.900 0.5100 8.900 7.100 10.200 16.127 4 9.656 2.910 2.577 8.900


Linear Interpolation (200 Resamples)Point ug/L SD 95% CL(Exp) SkewIC05 34.41026 758.0376 0 3140.326 0.6342IC10 1820.367 955.1368 0 3662.982 -0.5617IC15 2869.115 768.6402 633.987 4807.63 -0.4649IC20 3917.863 721.1212 1793.902 5934.512 -0.0976IC25 4966.611 714.5075 2903.458 7141.622 -0.0089IC40 8112.855IC50 >10000

-0.3-0.2-0.10.00.10.20.30.40.50.60.70.80.91.0

0 5000 10000 15000

Dose ug/L

Res

pons

e



Dose-Response Plot


0

5

10

15

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25

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trol (

500) 2 10 50 100

*200

*100

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*100

00

Gro

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2 16.33 15.90 17.00 0.53 4.47 410 17.43 16.00 20.00 1.84 7.79 450 15.98 15.60 16.30 0.29 3.35 4

100 15.13 14.20 16.50 1.00 6.63 4200 14.13 13.00 15.60 1.08 7.36 4

1000 20.33 19.20 21.90 1.18 5.34 410000 8.90 7.10 10.20 1.44 13.46 4

Control (500) Temp C 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0

Control (500) pH 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0


10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0

Control (500) DO (%sat) 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 010000 0.00 0.00 0.00 0.00 0



Appendix C: Statistical Print-outs for 21-d Gametophyte Growth

Macroalgal Growth-GrowthStart Date: 12/06/07 Test ID: PR0177/11 Sample ID: 500ug/L NitrateEnd Date: 03/07/07 Lab ID: Sample Type: ChlorateSample Date: Protocol: ESASOP116 Test Species: ER-Ecklonia radiataComments: Chlorate concentrations prepared in 500 ug/L Nitrate

Conc-ug/L 1 2 3 4Control (500) 14.200 15.500 16.100 15.400

2 15.900 15.300 15.500 15.30010 15.000 16.900 16.700 17.20050 15.400 14.700 16.300 15.800

100 14.900 14.500 16.200 13.500200 15.100 16.400 16.400 13.800

1000 18.200 17.900 20.200 20.800100000 11.200 9.000 7.700 8.400

Transform: Untransformed 1-TailedConc-ug/L Mean N-Mean Mean Min Max CV% N t-Stat Critical MSDControl (500) 15.300 1.0000 15.300 14.200 16.100 5.201 4

2 15.500 1.0131 15.500 15.300 15.900 1.825 4 -0.262 2.480 1.89210 16.450 1.0752 16.450 15.000 17.200 6.008 4 -1.508 2.480 1.89250 15.550 1.0163 15.550 14.700 16.300 4.346 4 -0.328 2.480 1.892

100 14.775 0.9657 14.775 13.500 16.200 7.565 4 0.688 2.480 1.892200 15.425 1.0082 15.425 13.800 16.400 8.069 4 -0.164 2.480 1.892

1000 19.275 1.2598 19.275 17.900 20.800 7.475 4 -5.211 2.480 1.892*100000 9.075 0.5931 9.075 7.700 11.200 16.672 4 8.161 2.480 1.892

Auxiliary Tests Statistic Critical SkewShapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.969415 0.904 0.082592Bartlett's Test indicates equal variances (p = 0.39) 7.424892 18.47531Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-ProbDunnett's Test 1000 100000 10000 1.891762 0.123645 32.07411 1.16375 5.4E-10

Dose-Response Plot


0

5

10

15

20

25

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500) 2 10 50 100

200

1000

*100

000

Gro

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2 15.50 15.30 15.90 0.28 3.43 410 16.45 15.00 17.20 0.99 6.04 450 15.55 14.70 16.30 0.68 5.29 4

100 14.78 13.50 16.20 1.12 7.16 4200 15.43 13.80 16.40 1.24 7.23 4

1000 19.28 17.90 20.80 1.44 6.23 4100000 9.08 7.70 11.20 1.51 13.55 4

Control (500) Temp C 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0

Control (500) pH 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0


10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0

Control (500) DO (%sat) 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0



Conc-ug/L 1 2 3 4Control (0) 14.700 12.600 14.700 14.300

2 14.300 16.500 16.700 16.30010 14.600 16.400 16.200 17.00050 18.000 15.800 18.100 15.800

100 14.700 15.200 17.600 16.200200 16.200 15.000 13.700 13.900

1000 20.100 16.100 16.600 15.700100000 12.000 11.300 10.200 10.831


Control (0) 14.075 1.0000 14.075 12.600 14.700 7.114 42 15.950 1.1332 15.950 14.300 16.700 6.972 4 2.114 2.910 2.581

10 16.050 1.1403 16.050 14.600 17.000 6.384 4 2.227 2.910 2.581*50 16.925 1.2025 16.925 15.800 18.100 7.679 4 3.213 2.910 2.581100 15.925 1.1314 15.925 14.700 17.600 8.031 4 2.086 2.910 2.581200 14.700 1.0444 14.700 13.700 16.200 7.835 4 0.705 2.910 2.581

*1000 17.125 1.2167 17.125 15.700 20.100 11.779 4 3.439 2.910 2.581*100000 11.083 0.7874 11.083 10.200 12.000 6.855 4 3.374 2.910 2.581


Dose-Response Plot


0

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10 16.05 14.60 17.00 1.02 6.31 450 16.93 15.80 18.10 1.30 6.74 4

100 15.93 14.70 17.60 1.28 7.10 4200 14.70 13.70 16.20 1.15 7.30 4

1000 17.13 15.70 20.10 2.02 8.29 4100000 11.08 10.20 12.00 0.76 7.86 4

Control (0) Temp C 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0

Control (0) pH 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0


10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0

Control (0) DO (%sat) 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0



Conc-ug/L 1 2 3 4Control (50) 19.100 22.000 19.100 19.800

2 18.400 18.400 18.600 18.40010 20.600 19.400 17.100 18.00050 21.900 21.300 23.900 21.400

100 15.800 13.400 14.200 15.400200 15.500 14.400 15.600 13.100

1000 12.800 12.700 14.400 15.300100000 8.600 11.500 11.400 10.900


Control (50) 20.000 1.0000 20.000 19.100 22.000 6.868 42 18.450 0.9225 18.450 18.400 18.600 0.542 4 1.807 2.910 2.496

10 18.775 0.9388 18.775 17.100 20.600 8.210 4 1.428 2.910 2.49650 22.125 1.1063 22.125 21.300 23.900 5.478 4 2.477 2.910 2.496

*100 14.700 0.7350 14.700 13.400 15.800 7.493 4 6.179 2.910 2.496*200 14.650 0.7325 14.650 13.100 15.600 7.970 4 6.237 2.910 2.496

*1000 13.800 0.6900 13.800 12.700 15.300 9.185 4 7.228 2.910 2.496*100000 10.600 0.5300 10.600 8.600 11.500 12.820 4 10.959 2.910 2.496


Dose-Response Plot


0

5

10

15

20

25

30

Con

trol (

50) 2 10 50

*100

*200

*100

0

*100

000

Gro

wth





10 18.78 17.10 20.60 1.54 6.61 450 22.13 21.30 23.90 1.21 4.98 4

100 14.70 13.40 15.80 1.10 7.14 4200 14.65 13.10 15.60 1.17 7.38 4

1000 13.80 12.70 15.30 1.27 8.16 4100000 10.60 8.60 11.50 1.36 11.00 4

Control (50) Temp C 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0

Control (50) pH 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0


10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0

Control (50) DO (%sat) 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0



Conc-ug/L 1 2 3 4Control (50) 19.100 22.000 19.100 19.800

2 18.400 18.400 18.600 18.40010 20.600 19.400 17.100 18.00050 21.900 21.300 23.900 21.400

100 15.800 13.400 14.200 15.400200 15.500 14.400 15.600 13.100

1000 12.800 12.700 14.400 15.300100000 8.600 11.500 11.400 10.900


Control (50) 20.000 1.0000 20.000 19.100 22.000 6.868 4 202 18.450 0.9225 18.450 18.400 18.600 0.542 4 1.807 2.480 2.127 18.45

10 18.775 0.9388 18.775 17.100 20.600 8.210 4 1.428 2.480 2.127 18.77550 22.125 1.1063 22.125 21.300 23.900 5.478 4 -2.477 2.480 2.127 22.125

*100 14.700 0.7350 14.700 13.400 15.800 7.493 4 6.179 2.480 2.127 14.7*200 14.650 0.7325 14.650 13.100 15.600 7.970 4 6.237 2.480 2.127 14.65

*1000 13.800 0.6900 13.800 12.700 15.300 9.185 4 7.228 2.480 2.127 13.8*100000 10.600 0.5300 10.600 8.600 11.500 12.820 4 10.959 2.480 2.127 10.6


Dose-Response Plot


0

5

10

15

20

25

30

Con

trol (

50) 2 10 50

*100

*200

*100

0

*100

000

Gro

wth





10 18.78 17.10 20.60 1.54 6.61 450 22.13 21.30 23.90 1.21 4.98 4

100 14.70 13.40 15.80 1.10 7.14 4200 14.65 13.10 15.60 1.17 7.38 4

1000 13.80 12.70 15.30 1.27 8.16 4100000 10.60 8.60 11.50 1.36 11.00 4

Control (50) Temp C 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0

Control (50) pH 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0


10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0

Control (50) DO (%sat) 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0



Conc-ug/L 1 2 3 4Control (500) 14.200 15.500 16.100 15.400

2 15.900 15.300 15.500 15.30010 15.000 16.900 16.700 17.20050 15.400 14.700 16.300 15.800

100 14.900 14.500 16.200 13.500200 15.100 16.400 16.400 13.800

1000 18.200 17.900 20.200 20.800100000 11.200 9.000 7.700 8.400

Transform: Untransformed 2-TailedConc-ug/L Mean N-Mean Mean Min Max CV% N t-Stat Critical MSDControl (500) 15.300 1.0000 15.300 14.200 16.100 5.201 4

2 15.500 1.0131 15.500 15.300 15.900 1.825 4 0.262 2.910 2.22010 16.450 1.0752 16.450 15.000 17.200 6.008 4 1.508 2.910 2.22050 15.550 1.0163 15.550 14.700 16.300 4.346 4 0.328 2.910 2.220

100 14.775 0.9657 14.775 13.500 16.200 7.565 4 0.688 2.910 2.220200 15.425 1.0082 15.425 13.800 16.400 8.069 4 0.164 2.910 2.220

*1000 19.275 1.2598 19.275 17.900 20.800 7.475 4 5.211 2.910 2.220*100000 9.075 0.5931 9.075 7.700 11.200 16.672 4 8.161 2.910 2.220


Dose-Response Plot


0

5

10

15

20

25

Con

trol (

500) 2 10 50 100

200

*100

0

*100

000

Gro

wth




2 15.50 15.30 15.90 0.28 3.43 410 16.45 15.00 17.20 0.99 6.04 450 15.55 14.70 16.30 0.68 5.29 4

100 14.78 13.50 16.20 1.12 7.16 4200 15.43 13.80 16.40 1.24 7.23 4

1000 19.28 17.90 20.80 1.44 6.23 4100000 9.08 7.70 11.20 1.51 13.55 4

Control (500) Temp C 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0

Control (500) pH 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0


10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0

Control (500) DO (%sat) 0.00 0.00 0.00 0.00 02 0.00 0.00 0.00 0.00 0

10 0.00 0.00 0.00 0.00 050 0.00 0.00 0.00 0.00 0

100 0.00 0.00 0.00 0.00 0200 0.00 0.00 0.00 0.00 0

1000 0.00 0.00 0.00 0.00 0100000 0.00 0.00 0.00 0.00 0



Table F.1 Summary of the chlorate analysis results for the Ecklonia radiata macroalgal tests initiated on 12 June 2007. These solutions were nominally 10 mg ClO3/L, the highest concentration tested.

Measured chlorate concentration (mg ClO3/L) Sample

Day 0 12-6-07

Day 2 14-6-07

Day 6 18-6-07

Day 8 20-6-07

Day 10 22-6-07

Day 13 25-6-07

Seawater Blank <0.010 <0.010 <0.010 <0.010 <0.010 <0.010

Background 9.6 10 9.6 9.7 9.7 9.9

50 µg/L Nitrate 10 9.9 9.8 9.1 9.9 9.4

500 µg/L Nitrate 11 9.6 10 9.5 9.3 9.9

Measured chlorate concentration (mg ClO3/L) Sample

Day 15 27-6-07

Day 17 29-6-07

Day 20* 2-7-07

Seawater Blank <0.010 <0.010 <0.010

Background 8.9 9.0 10

50 µg/L Nitrate 9.7 9.3 9.6

500 µg/L Nitrate 9.3 9.8 9.6 Data is chlorate concentration of sample in mg/L * These chlorate results may have error associated with degradation of sample as the EDA preservative added to all other chlorate samples was not available for this batch of samples

Appendix D: Analytical Test Reports for Chlorate Determinations


Table G.1 Summary of the nitrate analysis results for the Ecklonia radiata macroalgal tests initiated on 12 June 2007.

Measured nitrate concentration (µg NO3/L) Sample

Day 0 12-6-07

Day 2 14-6-07

Day 6 18-6-07

Day 8 20-6-07

Day 10 22-6-07

Day 13 25-6-07

Background 40.8 <17 125.8 200.6 312.8 119

50 µg/L Nitrate 64.6 51 302.6 554 265.2 197.2

500 µg/L Nitrate 408 374 544 299.2 510 408

Measured nitrate concentration (µg NO3/L) Sample

Day 15 27-6-07

Day 17 29-6-07

Day 20* 2-7-07

Background 146.2 166.6 159.8

50 µg/L Nitrate 374 510 183.6

500 µg/L Nitrate 850 646 646 Data is nitrate concentration of sample in µg/L. They have been calculated based on mg N/L data reported herein by NMI.

Appendix E: Analytical Test Reports for Nitrate Determinations

Australian Government____________________________________________National Measurement Institute

REPORT OF ANALYSISPage: 1 of 6

Report No. RN626696Client : GUNNS LIMITED Job No. : ECOT02/070703

C/- ECOTOX SERVICES ASUTRALIA Quote No. : QT-00047 27 / 2 CHAPLIN DRIVE Order No. : 0177 LANE COVE NSW 2066 Date Sampled : Date Received : 3-JUL-2007

Attention : RICHARD FAWKES Sampled By : CLIENTProject Name : Your Client Services Manager : BRIAN WOODWARD Phone : (02) 94490151

Lab Reg No. Sample Ref Sample DescriptionN07/024405 DAY 8 FSW BLANK 20/06/07N07/024410 DAY 8 CHLORATE +50 N A B 20/06/07N07/024411 DAY 8 CHLORATE +500 N A C 22/06/07N07/024412 DAY 10 FSW BLANK 22/06/07

Lab Reg No. N07/024405 N07/024410 N07/024411 N07/024412Sample Reference DAY 8 DAY 8 DAY 8 DAY 10

Units MethodMiscellaneous Nitrate-N mg/L 0.059 0.52 0.88 0.92 NW_B19

Wei Huang, AnalystInorganics - NSWAccreditation No. 198

16-JUL-2007


Units MethodInorganics Chlorate mg/L <0.010 9.1 9.5 <0.010 WL119

David Lynch, Section ManagerInorganics - WAAccreditation No. 2474

16-JUL-2007

This report is issued in accordance with NATA’s accreditation requirements1 Suakin Street, Pymble NSW 2073 Tel: +61 2 9449 0111 Fax: +61 2 9449 1653 www.measurement.gov.au_______________________________________________________________________________________

N a t i o n a l M e a s u r e m e n t I n s t i t u t e





Lab Reg No. Sample Ref Sample DescriptionN07/024417 DAY 10 CHLORATE +50 N A B 22/06/07N07/024418 DAY 10 CHLORATE +500 N A C 22/06/07N07/024419 DAY 13 FSW BLANK 25/06/07N07/024424 DAY 13 CHLORATE +50 N A B 25/06/07




16-JUL-2007


Units MethodInorganics Chlorate mg/L 9.9 9.3 <0.010 9.4 WL119


16-JUL-2007

1 Suakin Street, Pymble NSW 2073 Tel: +61 2 9449 0111 Fax: +61 2 9449 1653 www.measurement.gov.au_______________________________________________________________________________________






Lab Reg No. Sample Ref Sample DescriptionN07/024425 DAY 13 CHLORATE +500 N A C 25/06/07N07/024426 DAY 15 FSW BLANK 27/06/07N07/024431 DAY 15 CHLORATE +50 N A B 27/06/07N07/024432 DAY 15 CHLORATE +500 N A C 27/06/07




16-JUL-2007


Units MethodInorganics Chlorate mg/L 9.9 <0.010 9.7 9.3 WL119


16-JUL-2007







Lab Reg No. Sample Ref Sample DescriptionN07/024433 DAY 17 FSW BLANK 29/06/07N07/024438 DAY 17 CHLORATE +50 N A B 29/06/07N07/024439 DAY 17 CHLORATE +500 N A C 29/06/07N07/024440 DAY 20 FSW BLANK 02/07/07




16-JUL-2007


Units MethodInorganics Chlorate mg/L <0.010 9.3 9.8 <0.010 WL119


16-JUL-2007







Lab Reg No. Sample Ref Sample DescriptionN07/024445 DAY 20 CHLORATE +50 N A B 02/07/07N07/024446 DAY 20 CHLORATE +500 N A C 02/07/07

Lab Reg No. N07/024445 N07/024446Sample Reference DAY 20 DAY 20

Units MethodMiscellaneous Nitrate-N mg/L 0.054 0.19 NW_B19


16-JUL-2007

Lab Reg No. N07/024445 N07/024446Sample Reference DAY 20 DAY 20

Units MethodInorganics Chlorate mg/L 9.6 9.6 WL119


16-JUL-2007




Report No. RN626696

This report is issued in accordance with NATA’saccreditation requirements.Accreditated for compliance with ISO/IEC 17025.This report shall not be reproduced except in full.Results relate only to the sample(s) tested.

This Report supersedes reports: RN626170 RN626171 RN626637






C/- ECOTOX SERVICES ASUTRALIA Quote No. : QT-00782 27 / 2 CHAPLIN DRIVE Order No. : LANE COVE NSW 2066 Date Sampled : Date Received : 20-JUN-2007

Attention : RICK KRASSOI Sampled By : CLIENTProject Name : Your Client Services Manager : BRIAN WOODWARD Phone : (02) 94490151

Lab Reg No. Sample Ref Sample DescriptionN07/023098 DAY 6 SALINE WATER CHLORATE 35PPT A 18/06/07N07/023099 DAY 6 SALINE WATER CHLORATE 25PPT A 18/06/07N07/023100 DAY 6 SALINE WATER CHLORATE 20PPT A 18/06/07N07/023101 DAY 6 SALINE WATER CHLORATE 7PPT A 18/06/07


Units MethodInorganics Chlorate mg/L 9.6 10 10 11 WL119

Peter Anstis, AnalystInorganics - WAAccreditation No. 2474

6-JUL-2007









Units MethodInorganics Chlorate mg/L 10 9.6 10 10 WL119


6-JUL-2007









Units MethodInorganics Chlorate mg/L 9.6 10 10 10 WL119


6-JUL-2007


This Report supersedes reports: RN624853








Lab Reg No. Sample Ref Sample DescriptionN07/023097 DAY 6 SALINE WATER FSW BLANK 18/06/07N07/023102 DAY 6 SALINE WATER CHLORATE + 50 N A B 18/06/07N07/023103 DAY 6 SALINE WATER CHLORATE + 500 N A C 18/06/07N07/023104 DAY 2 SALINE WATER FSW BLANK 14/06/07


Units MethodMiscellaneous Nitrate-N mg/L 0.037 0.089 0.16 <0.005 NW_B19

Nina Ilina, AnalystInorganics - NSWAccreditation No. 198

6-JUL-2007


Units MethodInorganics Chlorate mg/L <0.010 9.8 10 <0.010 WL119


6-JUL-2007







Lab Reg No. Sample Ref Sample DescriptionN07/023109 DAY 2 SALINE WATER CHLORATE + 50 N A B 14/06/07N07/023110 DAY 2 SALINE WATER CHLORATE + 500 N A C 14/06/07N07/023111 DAY 0 SALINE WATER FSW BLANK 12/06/07N07/023116 DAY 0 SALINE WATER CHLORATE + 50 N A B 12/06/07




6-JUL-2007


Units MethodInorganics Chlorate mg/L 9.9 9.6 <0.010 10 WL119


6-JUL-2007







Lab Reg No. Sample Ref Sample DescriptionN07/023117 DAY 0 SALINE WATER CHLORATE + 500 N A C 12/06/07

Lab Reg No. N07/023117Sample Reference DAY 0

Units MethodMiscellaneous Nitrate-N mg/L 0.12 NW_B19


6-JUL-2007

Lab Reg No. N07/023117Sample Reference DAY 0

Units MethodInorganics Chlorate mg/L 11 WL119


6-JUL-2007




Report No. RN624905


This Report supersedes reports: RN624438 RN624853








Lab Reg No. Sample Ref Sample DescriptionN07/024406 DAY 8 CHLORATE 35 PPT A 20/06/07N07/024407 DAY 8 CHLORATE 25 PPT A 20/06/07N07/024408 DAY 8 CHLORATE 20 PPT A 20/06/07N07/024409 DAY 8 CHLORATE 7 PPT A 20/06/07


Units MethodInorganics Chlorate mg/L 9.7 9.5 9.7 9.7 WL119


16-JUL-2007











16-JUL-2007











16-JUL-2007











16-JUL-2007











16-JUL-2007











16-JUL-2007


This Report supersedes reports: RN626637



page 1 of 1Australian Government______________________________National Measurement Institute

QUALITY ASSURANCE REPORT

NMI Job No. ECOT02/070620

Sample Matrix: Water

Analyte Laboratory LOR Blank Result Duplicate Recovery Acceptability Identification Result criteria

mg/L mg/L %

Chlorate N07/023100 0.01 <0.01 10 10 - -Chlorate N07/023110 0.01 <0.01 9.6 9.6 101% 80 - 120

LOR: Limit Of ReportingNA: Not Applicable

Signed:

David LynchSenior Environmental ChemistNMI WA, Inorganic Section

Date:

THIS REPORT SHALL NOT BE REPRODUCED EXCEPT IN FULL

PO Box 1246 Bentley DC WA 6983. Tel: +61 8 9368 8400 Fax: +61 8 9368 8499 www.measurement.gov.au

National Measurement Institute

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