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This document is issued in accordance with NATA’s accreditation requirements. Accredited for compliance with ISO/IEC 17025

Accreditation No. 12621

Report on the Acute Toxicity of Samples from West Cliff to the Cladoceran, Ceriodaphnia dubia Richard 1894

Office of Environment and Heritage Ecotoxicology and Environmental Contaminants Section

480 Weeroona Road, Lidcombe NSW 2141 Date of Issue of Report: 2 July 2012

Test Outline

The test was conducted to assess the potentially harmful effects of the sample to juveniles of the freshwater crustacean zooplankton species Ceriodaphnia dubia.

Following exposure for 48 hours to various concentrations of the sample/s, the number of C. dubia immobilised was counted. In this test immobilisation is considered similar to lethality.

Immobilisation data is statistically analysed to determine sample concentrations causing a significant adverse effect to C. dubia relative to a control group of animals.

If more than 50% of exposed animals are immobilised in any of the tested samples concentrations, a 48-hour EC50 (immobilisation) value is calculated, which is the Effective Concentration of the sample which causes immobilisation in 50% of exposed C. dubia.

The lower the concentration causing a significant adverse effect, or the lower the EC50 value, the greater the observed toxicity.

Results Summary See page 2 for sample identification details.

Samples 12082-12085 had no observable acute effect on C. dubia, in that there was no

immobilisation in exposed C. dubia during the 48 hour exposure period.

of 4 Ecotox 1 Test Report No. 12082

Sample Information EECS Sample Number

EFAS Sample Number Additional Sample Identification

12082 201201633 Inside Brennans Creek Dam

12083 201201638 LDP 10 Brennans Creek

12084 201201643 U/S LDP 11

12085 201201648 D/S LDP 12

Sample Details Collection Date: 28 May 2012 Sample/s Collected By: J. Byrne Date of delivery to EECS: 28 May 2012 Storage Conditions: 4oC Comments on Appearance: Clear Laboratory Accreditation does not extend to sample collection

Test Methods and Conditions Test Commencement Date: 29 May 2012 Test Conducted by: R. Patra, R. Sunderam, M. Julli, A. Rose Test Method Protocol No.: Test Method Protocol No.: ECOTOX 1. The test method is based on procedures published by the USEPA (2002), Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, 5th Edition. EPA-821-R-02-012. It differs from this guideline in that an Australian cladoceran species is used. Deviations from Protocol: None Test Type: Cladoceran, Acute, Static Duration of test: 48 Hours Test Species: Ceriodaphnia dubia Age:<24 hours Source: EECS Culture Location: Room No F. 27 Constant Temperature Room No.: F.29 Test Vessel Type: 100 mL beaker Test Volume: 50 mL Test Temperature: 25 oC Test Photoperiod: 16 h L: 8 h D Light intensity at surface of Test Vessels: <800 Lux Dilution Water Source: Filtered thiosulphate-treated Sydney mains water with 5% mineral water and conductivity adjusted to 500μS/cm with filtered seawater Conductivity: 500 μS/cm pH: 8.0 Hardness: 100 mg/L as CaCO3 Alkalinity: 50 mg/L as CaCO3

Test Design Concentrations tested: 30 and 100% Test Concentrations: Nominal Number of replicate test vessels per concentration and control/s: 4 Number of animals per replicate: 5 Statistical Methods Statistical Analysis Method: Probit for reference toxicant EC50


Results Table 1. Immobilisation of Ceriodaphnia dubia in test solutions

EECS Sample Number

Nominal Test Concentration

(% sample)

Percentage of animals

immobilised after 24 hours exposure



Control Diluent 0 0 30 0 0

12082 100 0 0 30 0 0

12083 100 0 0 30 0 0

12084 100 0 0 30 0 0

12085 100 0 0

A test validity criterion of Control group survival (greater than 90% ) was met. Samples 12082-12085 There was no immobilisation of C. dubia at any tested concentrations of the samples Table 2. Physico-chemical Variables in Test Solutions

Test Conc.

Temperature (0C)

pH Conductivity (µS/cm)

Dissolved Oxygen (% saturation)

(%) 0 h 48 h 0 h 48 h 0 h 48 h 0 h 48 h

Diluent 24.6 24.3 8.1 8.1 500 569 100 99 Sample 12082

30 24.4 24.2 8.6 8.7 870 886 100 98 100 24.4 24.3 8.8 9.1 1746 1784 100 98

Sample 12083 30 24.4 24.2 8.5 8.7 873 896 99 97 100 24.5 24.3 8.7 9.1 1732 1755 100 97

Sample 12084 30 24.4 24.1 7.9 8.0 402 416 100 99 100 24.5 24.1 7.6 7.9 159 171 100 98

Sample 12085 30 24.4 24.2 8.4 8.6 718 735 100 97 100 24.5 24.2 8.7 9.0 1224 1262 100 97


Reference toxicant test No. 293 A reference toxicity test using Cr (VI) run in parallel with the above test resulted in 48-h EC50 (immobilisation) value of 270 μg/L ( 240 μg/L lower and 300 μg/L upper 95% CL). This value is within the 95% confidence limits of previous reference toxicity test results conducted at this laboratory, and indicates that the test animals used in the current tests were of typical sensitivity. The current percentage coefficient of variation of the reference toxicity data is 4.3% This Report should only be reproduced in full and with the written approval of the Manager Ecotoxicology and Environmental Contaminants Section, OEH.

This report is issued with the approval of M Julli

Head, Ecotoxicology Testing Services

2 July 2012




Report on the acute toxicity of samples from West Cliff

investigation to Vibrio fischeri (Microtox®)


480 Weeroona Road, Lidcombe NSW 2141 Date of Issue of Report: 28 June 2012 Test Outline

The Microtox® test was conducted to assess the potentially harmful effects of the samples to the photoluminescent marine bacterium (Vibrio fischeri). Freshwater samples are salinity adjusted prior to testing.

Following exposure of up to 30 minutes to various concentrations of the sample/s, the light emitted by the bacteria is measured. Significant reduction in light output is considered to indicate an adverse effect on the bacteria.

If light output is reduced by more than 50% in any of the tested samples concentrations, an EC50 (luminescence inhibition) value is calculated which is the Effective Concentration of the sample which causes a 50% reduction in luminescence. The time period quoted with the EC50 indicates the exposure period of the bacteria to the sample.

The lower the EC50 value, the greater the observed toxicity, and the greater the amount of dilution required to eliminate the observed toxicity. Results Summary See page 2 for sample identification details.

Sample 12071 had minimal effect on bacterial luminescence, that is, it did not cause a

detectable adverse response in exposed bacteria. Samples 12072, 12073 and 12074 caused minimal reduction in luminescence in the

exposed bacteria following 30 min exposure, the effect was limited respectively to 7%, 14% and 13% reduction in light output at the highest testable sample concentration of 80%.

Page 1 of 3: Ecotox 4 Test Report No. 12071

of 3: Ecotox 4 Test Report No. 12071

Sample Information Submission no. 201200168

EECS Sample No. EFAS Sample No. Additional Sample Identification 12071 201201474 LDP 11 u/s 10:25 12072 201201469 LDP 12 d/s Georges River 10:45 12073 201201479 LDP 10 discharge 11:17 12074 201201484 Inside BCD 11:40

Sample Details Collection Date: 21 May 2012 Sample/s Collected By: J. Byrne Date of delivery to EECS: 21 May 2012 Storage Conditions: 4oC Comments on Appearance: Clear samples Laboratory Accreditation does not extend to sample collection.

Test Methods and Conditions Test Commencement Date: 22 May 2012 Test Conducted by : F. Pablo Test Method Protocol No.: ECOTOX4 - The test procedure is based on the

recommended tests described in the Microbics manual (1995) ‘Microtox® Acute Toxicity Basic Test Procedures’. The test conducted here has been modified in the aspects of: increased upper limit of sample concentration, maximized volume of test solutions used, and additional dilution of reconstituted bacteria to increase the accuracy & precision of bacterial transfer. These modifications result in greater accuracy and precision of the calculated EC50 values.

Test Type: Microtox® Acute, Basic Readings taken: 5, 15, 30 Minutes Test Species: Vibrio fischeri Test Vessel Type : 4 mL test tubes Test Volume: 1.0 mL Test Temperature: 15.0°C Dilution Water: Microbics Diluent Salinity: 20‰ Sample Treatment Salinity adjustment: Samples were adjusted to salinity of the diluent using solid NaCl. Filtration: Not required Colour correction required: Not required

Test Design Concentrations tested: 10, 20, 40, 80% Test Concentrations: Nominal Number of replicate test vessels per concentration and control: 2 per conc., 4 for control Volume of stock organism per test solution: 0.2 mL (containing approx 1x106 cells)

Results Maximum effect

(luminescence inhibition) at the highest tested concentration^ 5-min 15-min 30-min

EECS Sample No.

pH ConductivityμS/cm#

Dissolved Oxygen (mg/L)

12071 7.8 159 8.94 NT* NT NT

12072 8.4 1274 8.78 5 8 7

12073 8.4 1735 8.70 5 11 14

12074 8.6 1557 8.63 6 11 13

# conductivity of sample prior to salinity adjustment ^80% sample is the highest tested concentration *NT = not toxic Reference Toxicant Test No. 218 Reference toxicant (Phenol) test conducted with the same batch of organisms used for the tests gave 5min-EC50 value of 16 mg/L (16 mg/L lower and 17 mg/L upper 95% CL). This value is within the 95% confidence limits of previous reference toxicity test results conducted at this laboratory, indicating that the organisms used in the tests were of typical sensitivity. The percent coefficient of variation (%CV) for the previous twenty reference toxicant tests was 6.0%. This Report should only be reproduced in full and with the written approval of the Manager Ecotoxicology and Environmental Contaminants Section, OEH.

This report is issued with the approval of

M Julli


28 June 2012

Page 3 of 3: Ecotox 4 Test Report No. 12071


Report on the Acute Toxicity of Samples from West Cliff to the Glass Shrimp Paratya australiensis, (Kemp, 1917)


480 Weeroona Road, Lidcombe NSW 2141 Date of Issue of Report: 12 July 2012

Test Outline

The test was conducted to assess the potentially harmful effects of the samples to the native freshwater shrimp species Paratya australiensis. In this test mortality is used as the endpoint.

Following exposure for 72 hours to various concentrations of the sample/s, the number of P. australiensis affected was counted. This data is statistically analysed to determine sample concentrations causing a significant adverse effect to P. australiensis relative to a control group

If more than 50% of exposed animals die in any of the tested sample concentrations, a 72-hour LC50 value is calculated, which is the Lethal Concentration of the sample which causes 50% mortality of exposed P. australiensis.



Samples 12106, 12107 and 12108 had minimal observable effect on P. australiensis, in that

mortality in exposed P. australiensis was not statistically different to that occurring in the Control group.

Sample 12109 had no observable acute effect on P. australiensis, in that there was no mortality in exposed P. australiensis during the 72 hour exposure period.


Sample Information EECS Sample Number EFAS Sample Number Additional Sample Identification

12106 201202109 LDP 10 Discharge from Brennans Creek 11:20

12107 201202104 LDP 11 Upstream Georges River 10:40

12108 201202099 LDP 12 downstream Georges River 10:52

12109 201202114 Inside Brennans Creek Dam 11:36

Sample Details Collection Date: 25 June 2012 Sample/s Collected By: J. Byrne Date of delivery to EECS: 25 June 2012 Storage Conditions: 4oC Comments on Sample Appearance: Clear Laboratory Accreditation does not extend to sample collection

Test Methods and Conditions Test Commencement Date: 2 July 2012 Test Conducted by: R. Patra, M. Julli and A. Rose Test Method Protocol No ECOTOX 7. Test Type: Shrimp, Acute, Static Duration of test: 72 Hours Test Species: Paratya australiensis Age: Adult Source:EECS Culture, Shrimp Tank 1 Test Location:Room No. F.27 Test Room No.: F.29 Test Vessel Type: 100 mL beaker Test Volume: 50 mL Test Temperature: 25 oC Test Photoperiod: 16 h L: 8 h D Light intensity at surface of Test Vessels: <800 Lux Dilution Water Source: Filtered Sydney mains water with conductivity adjusted to 500 μS/cm with filtered seawater. Conductivity: 519 μS/cm pH: 7.7 Hardness: 78 mg/L as CaCO3 Alkalinity: 42 mg/L as CaCO3 Test Design Concentration tested: 100 % Test Conditions: Nominal Number of replicate test vessel per concentration and control/s: 10 Number of animals per replicate: 1 Statistical Methods Data Transformation for Lowest Observed Effect Concentration (LOEC): Untransformed Statistical Analysis Method: Fisher Exact Test for LOEC


Results Table 1. Mortality of Paratya australiensis in test solutions

EECS Sample Number


(% sample)


imbalanced after 24 hours exposure





Control Diluent 0 0 10 120106 100 0 10 20 120107 100 10 10 10 120108 100 0 10 10 120109 100 0 0 0

A test validity criterion of Control group survival (greater than 90%) was met. The tested samples did not elicit a significant acute toxic response in exposed P. australiensis. Table 2. Physico-chemical Variables in Test Solutions

Test Conc.

Temperature (0C)



(%) 0 h 72 Hrs 0 h 72 Hrs 0 h 72 Hrs 0 h 72 Hrs Control

Diluent 24.1 24.8 7.7 7.8 519 524 92 95 Sample 12106

100 24.5 24.9 8.7 9.1 1786 1780 111 98 Sample 12107

100 24.5 24.8 8.4 8.4 255 293 108 97 Sample 12108

100 24.7 24.8 8.6 9.0 1260 1255 114 110 Sample 12109

100 24.7 24.9 8.7 9.0 1607 1605 106 103 This Report should only be reproduced in full and with the written approval of the Manager Ecotoxicology and Environmental Contaminants Section, OEH.



12 July 2012

Report on the Chronic Toxicity of the Samples from West Cliff to the Cladoceran Ceriodaphnia dubia


480 Weeroona Road, Lidcombe 2141 NSW Date of Issue of Report: 7 August 2012

Test Outline

The test was conducted to assess the sub-lethal toxicity of the samples to the freshwater cladoceran zooplankton species Ceriodaphnia dubia.

The chronic reproduction impairment test subjects cladocerans less than 24 hours old, to a range of sample concentrations over the period of production of 3 broods. The end points of this test are parental mortality and young production. Following exposure for 7 days to various concentrations of the sample, the number of young produced by each parental C. dubia is assessed. Parental mortality and young production are statistically analyzed to determine sample concentrations causing a significant relative to a control group of animals.

The lower the value of inhibition concentration causing a significant effect, the greater the observed toxicity. Results Summary See page 2 for sample identification details.

Sample 12050/12071 caused significant chronic reproduction impairment in exposed C. dubia at a sample concentration of 100%. The sample would need to be diluted approximately 3.3 times to avoid these sub lethal toxic effects on C. dubia (based on the tested concentrations). The 7 day IC20 (reduction in producing young) concentration was calculated to be 55% sample.

Sample 12052/12072 caused significant chronic reproduction impairment in exposed

C. dubia at a concentration of 30% (lowest tested concentration). The 7 day IC20 (reduction in producing young) concentration was calculated to be 13% sample.


C. dubia at a concentration of 100%. The sample would need to be diluted approximately 3.3 times to avoid these sub lethal toxic effects on C. dubia (based on the tested concentrations). The 7 day IC20 (reduction in producing young) concentration was calculated to be 37% sample.


C. dubia at a concentration of 100%. The sample would need to be diluted approximately 3.3 times to avoid these sub lethal toxic effects on C. dubia (based on the tested concentrations). The 7 day IC20 (reduction in producing young) concentration was calculated to be 38% sample.

Page 1 of 5: ECOTOX 2 Test Report No.: 12050

of 5: ECOTOX 2 Test Report No.: 12050

Sample Information

EECS Sample Number EFAS Sample Number Additional sample identification

12050§/12071α 201201383/201201474 LDP11 Georges River U/S

12052§/12072 α 201201389/201201469 LDP 12 Georges River D/S

12054§/12073 α 201201395/201201479 LDP 10 Discharge Brennans Creek

12056§/12074 α 201201401/201201484 Inside Brennans Creek §= Tests started on 18 May 2012 using these samples; α = Test continued to the end with renewals using these samples on 23 May 2012 Collection Date: 14 May 2012(12050, 12052, 12054, 12056)

21 May 2012 (12071, 12072, 12073, 12074) Submitted By: M. Julli (14 May 2012) / J. Byrne (21 May 2012) Date of Delivery: 14 May 2012(12050, 12052, 12054, 12056) 21 May 2012 (12071, 12072, 12073, 12074) Storage Conditions: 40C Comments on Sample Appearance: Clear Liquid Test Methods and Conditions Date of Test Commencement: 18 May 2012 Test Conducted by: M. Julli, R. Patra, R. Sunderam and A. Rose Test Method Protocol No.: Ecotox 2 Deviations From Protocol: None Test Method: Cladoceran Reproduction Impairment Test Test Type: Static Renewal Renewal periods: 72 hours and then 48 hours Duration of test: 7 days Test Species: Ceriodaphnia dubia Age at test start: <24 hours Source: EECS Lab culture Location Test Room No. F27 Chamber No. F29 Test Vessel Type: 100 ml Glass beaker Test Volume: 50 mL Test Temperature: 25 oC Test Photoperiod: 16 : 8 L:D Light intensity at surface of Test Vessels: <800 Lux Dilution Water Source:Filtered Sydney mains+ Na2S2O3 + 5% mineral water+ Sea Water Conductivity: 504 μS/cm pH: 7.3 Hardness: 100 mg/L as CaCO3 Alkalinity: 50 mg /L as CaCO3


Test Design Concentrations tested: Control (Diluent water). Samples 12050/12071, 12052/12072: 30 and 100% Samples 12054/12073 and 12056/12074: 10, 30 and 100% Test Concentrations: Nominal Number of replicate test vessels per concentration and control/s: 10 Number of animals per replicate: 1 Statistical Methods:

Data transformation for LOEC: untransformed or log(x+1) Statistical Analysis Method: Dunnett’s Multiple Comparison Test/Steel Many-One Rank Test for LOEC

Linear Interpolation (bootstrap) for point estimates (IC20 and IC50) Results

Table 1. Young production and parental survival in the 7-day C. dubia reproduction test

% Concentration Sample No Test Endpoints

0 10 30 100

PMSD

#

Mean No. of Young / surviving adult (SE)

29.7 (0.9)

Control

Parental Mortalities 0

Mean No. of Young / surviving adult (SE) 26.7

(1.3) 20.9* (2.4)

12050/ 12071

Parental Mortalities 1 1 16

Mean No. of Young / surviving adult (SE) 21.9*

(2.8) 13.6* (3.3)

12052/ 12072



(2.4) 28.6 (3.4)

5.3* (1.7)

12054/ 12073

Parental Mortalities 1 1 1 17


(1.6) 27.2 (2.6)

13.7* (1.7)

12056/ 12074


*Significantly Different to Control (p<0.05) The test validity criteria were met: (a) the control group produced >15 young per adult on average after three broods; (b) parental mortalities were <20% in control treatment. # PMSD (Percent Minimum Significant Difference) is an estimation of the smallest % decrease in reproduction from the control that could be determined as statistically significant for this test.


Table 2. Estimated NOEC, LOEC, ICp 25 and ICp50 Values for Reproductive Impairment of C. dubia

Inhibition Concentrations (IC) of sample calculated to cause a 20% or 50%

reduction in reproduction in C.dubia (95% confidence intervals)

Sample Number

No Observed Effect

Concentration NOEC

(% Sample)

Lowest Observed Effect

Concentration LOEC

(% Sample) IC20 IC50

12050/ 12071 30 100 55

(47-NA)

Not Calculable (30% Effect at 100% Sample concentration)

12052/ 12072 NC 30 * 13

(3.4-40) 83

(45-NC)

12054/ 12073 30 100 37

(32-41) 60

(53-69)

12056/ 12074 30 100 38

(9-47) 90

(69-NA)

NC: Not calculable; *= Lowest Tested concentration for the sample Table 3. Physico-Chemical Variables in Test Solutions

pH Temperature

Conductivity (μS/cm)

Dissolved Oxygen (%Saturation)

Nominal Test Conc.

(% dilution) Fresh (0 hrs)

72 h old

Fresh (0 hrs)

48 h old

Fresh (0 hrs)

48 h old

Fresh (0 hrs)

48 h old

Control 7.3 8.6 25.4 25.1 504 530 96 1008.2 7.9 24.2 24.8 499 517 90 103Diluent 7.9 8.1 24.0 25.0 502 516 99 99

Sample 12050/12071 7.4 8.2 25.4 25.2 393 414 95 1007.8 7.9 25.3 24.9 403 411 100 10430 7.9 7.1 24.1 25.0 403 415 101 1007.6 8.2 26.2 24.9 160 172 99 1007.5 8.1 24.6 24.9 170 174 100 104100 7.9 7.3 24.3 25.0 164 175 101 100

Sample 12052/12072 7.9 8.9 25.2 25.0 696 721 96 1008.3 8.5 25.0 24.4 733 748 100 10230 8.3 8.2 24.1 24.9 746 765 101 998.7 8.6 26.2 25.1 1151 1200 99 1008.6 8.9 24.2 24.6 1295 1355 100 103100 8.6 9.0 24.2 24.8 1319 1323 103 98

Sample 12054/120738.3 8.7 24.8 25.1 612 631 97 1008.3 8.4 24.9 24.7 630 639 98 10310 8.1 8.5 24.6 24.7 636 658 102 978.1 8.9 25.5 25.3 848 862 98 1008.5 8.7 24.9 24.6 883 893 97 10530 8.4 8.7 24.0 24.7 893 905 102 988.7 9.2 25.6 25.2 1648 1707 100 1008.7 8.0 24.4 24.6 1769 1812 96 103100 8.6 9.1 24.0 25.0 1805 1857 103 98

Table 3. (Cont.) Physico-Chemical Variables in Test Solutions pH Temperature

Conductivity

(μS/cm) Dissolved Oxygen

(%Saturation) Nominal

Test Conc. (% dilution) Fresh

(0 hrs) 72 h old

Fresh (0 hrs)

48 h old

Fresh (0 hrs)

48 h old

Fresh (0 hrs)

48 h old

Sample 12056/12074 8.2 8.7 25.4 25.3 645 624 97 1008.5 8.5 24.8 24.6 712 728 98 10310 8.1 8.5 24.1 24.9 613 629 102 1008.4 8.9 25.4 25.2 827 848 98 1008.6 8.6 24.0 24.7 841 849 99 10330 8.4 8.6 24.0 24.9 824 841 101 1078.7 9.2 25.0 25.3 1599 1667 100 1058.8 8.9 23.9 24.7 1638 1657 100 101100 8.7 9.1 24.1 24.9 1597 1634 104 97

Reference toxicant test result: A Reference toxicity test using ZnSO4 run in parallel with the above test resulted ICp25 value of 15 μg/L (4μg/L lower and 60μg/L upper 95% CL). This value is within the 95% confidence limits of previous reference chronic toxicity test results conducted at this laboratory, and indicates that the test animals used in the current tests were of typical sensitivity. This Report should only be reproduced in full and with the written approval of the Manager Ecotoxicology and Environmental Contaminants Section, OEH.


M Julli


7 August 2012





Test Outline


The chronic reproduction impairment test subjects cladocerans less than 24 hours old, to a range of sample concentrations over the period of production of 3 broods. The end points of this test are parental mortality and young production. Following exposure for 7 days to various concentrations of the samples, the number of young produced by each parental C. dubia is assessed. Parental mortality and young production are statistically analyzed to determine sample concentrations causing a significant relative to a control group of animals.


Sample 12083/12096 caused significant reproduction impairment in exposed C. dubia at a concentration of 100%. The sample would need to be diluted approximately 3.3 times to avoid these effects on C. dubia (based on the tested concentrations). The 7 day IC20% and IC50% (reduction in producing young) was calculated to be 46% and 87% sample respectively.

Samples 12084/12099 and 12085/12100 had no observable reproduction impairment effect on C. dubia.

Page 1 of 5: ECOTOX 2 Test Report No.: 12083/12096

of 5: ECOTOX 2 Test Report No.: 12083/12096

Sample Information

EECS Sample Number EFAS Sample Number Additional sample identification

12083§/12096 α 201201638/201201753 LDP 10 Brennans Creek

12084§/12099 α 201201643/201201760 LDP 11 Georges U/S

12085§/12100 α 201201648/201201765 LDP 12 Georges River D/S §= Tests started on 1 June 2012 using these samples; α = Test continued to the end with renewals using these samples on 6 June 2012 Collection Date: 28 May 2012 (12083, 12084, 12085) 4 June 2012 (12096, 12099, 12100) Submitted By: J. Byrne Date of Delivery: 28 May 2012 (12083, 12084, 12085) 4 June 2012 (12096, 12099, 12100) Storage Conditions: 4oC Comments on Sample Appearance: Clear Liquid Test Methods and Conditions Date of Test Commencement: 1 June 2012 Test Conducted by: R. Patra, A. Rose, R. Sunderam and M. Julli Test Method Protocol No.: Ecotox 2 Deviations From Protocol: None Test Method: Cladoceran Reproduction Impairment Test Test Type: Static Renewal Renewal periods: 72 hours then 48 hours Duration of test: 7 days Test Species: Ceriodaphnia dubia Age at test start: <24 hours Source: EECS Lab culture Location Test Room No. F.27 Chamber No. F.29 Test Vessel Type: 100 ml Glass beaker Test Volume: 50 mL Test Temperature: 25 oC Test Photoperiod: 16: 8 L: D Light intensity at surface of Test Vessels: <800 Lux Dilution Water Source:Filtered Sydney mains+ Na2S2O3 + 5% mineral water+ Sea Water Conductivity: 506 μS/cm pH: 8.0 Hardness: 100 mg/L as CaCO3 Alkalinity: 50 mg /L as CaCO3


Test Design Concentrations tested: Control (Diluent water). Samples 12083/12096 and 12085/12100: 10, 30 and 100% Sample 12084/12099: 30 and 100% Test Concentrations: Nominal Number of replicate test vessels per concentration and control/s: 10 Number of animals per replicate: 1 Statistical Methods:

Data transformation for LOEC: untransformed or log(x+1) Statistical Analysis Method: Dunnett’s Multiple Comparison Test or Bonferroni Adjusted T- test for LOEC

Linear Interpolation (bootstrap) for point estimates (IC20 and IC50) Test Results of the 7-day Cladoceran Reproduction Impairment Test.


% Concentration Sample Number Test Endpoints

0 10 30 100 PMSD

# Mean No. of Young / surviving adult (SE)

21.4 (2.5)

Control (Diluent)



(3.9) 26.4 (2.6)

10.7* (4.6)

12083/ 12096

Parental Mortalities 0 0 0 22.5


(2.7) 21.5 (2.1)

12084/ 12099

Parental Mortalities 0 0 37.6


(3.6) 31.2 (4.0)

27.8 (1.9)

12085/ 12100

Parental Mortalities 0 0 0 49.7

*Significantly Different to Control (p<0.05) The test validity criteria were met: (a) the control group produced >15 young per adult on average after three broods; (b) parental mortalities was <20% in control treatment. # PMSD (Percent Minimum Significant Difference) is an estimation of the smallest % decrease in reproduction from the control that could be determined as statistically significant for this test.


Table 2. Estimated NOEC, LOEC, ICp20 and ICp50 Values for Reproductive Impairment of C.dubia

Inhibition Concentrations IC of sample calculated to cause a 20% or 50% reduction

in reproduction in C. dubia (95% confidence intervals)

Sample Number

No Observed Effect

Concentration NOEC

(% Sample)


Concentration LOEC

(% Sample) IC20 IC50

12083/ 12096 30 100 46

(43-51) 87

(73-NA)

12084/ 12099 100 - NC NC

12085/ 12100 100 - NC NC

NC or NA: Not calculable; LTC= Lowest Tested concentration for the sample Table 3. Physico-Chemical Variables in Test Solutions

pH Temperature

Conductivity (μS/cm)

Dissolved Oxygen (%Saturation)

Nominal Test Conc.

(% dilution) Fresh (0 hrs)

72 h old

Fresh (0 hrs)

48 h old

Fresh (0 hrs)

48 h old

Fresh (0 hrs)

48 h old

Control 8.3 8.1 25.0 25.2 506 520 97 978.1 8.2 24.8 24.8 509 521 96 98Diluent 8.2 8.2 24.4 24.5 500 519 96 97

Sample 12083/120968.3 8.3 24.7 24.8 628 643 96 968.2 8.5 24.4 24.8 631 644 96 9810 8.4 8.1 24.3 24.4 644 701 98 968.5 8.5 25.6 24.8 874 887 97 9.58.5 8.8 24.7 24.8 873 885 95 9730 8.6 8.1 24.3 24.6 933 971 97 968.7 9.1 24.7 24.7 1739 1768 100 968.8 9.2 24.8 24.4 1724 1702 96 96100 8.8 9.2 24.3 24.1 1921 1951 96 94

Sample 12084/120998.3 8.2 24.8 24.8 402 414 97 968.4 8.2 24.7 24.8 407 416 97 9730 8.1 7.5 24.3 24.3 406 431 97 968.3 8.1 24.7 24.7 163 174 100 988.2 8.3 24.4 24.9 170 186 97 88100 8.1 7.7 24.4 24.2 186 201 97 96

Sample 12085/121007.9 8.4 24.9 24.8 574 593 96 968.1 8.4 24.7 24.8 577 588 95 9610 8.1 7.7 24.4 24.3 516 538 97 958.2 8.5 25.0 24.7 716 729 98 978.3 8.7 24.7 24.9 716 731 96 9630 8.3 7.7 24.4 24.9 549 584 97 958.6 9.0 25.0 24.8 1225 1242 100 958.7 9.1 24.6 24.9 1209 1242 96 97100 8.5 8.7 24.5 24.1 671 696 96 93

Reference toxicant test result: A Reference toxicity test using ZnSO4 run in parallel with the above test resulted IC25 value of 70 µg/L (50 mg/L lower and 95 µg/L upper 95% CL). This value is within the 95% confidence limitsµ of previous reference chronic toxicity test results conducted at this laboratory, and indicates that the test animals used in the current tests were of typical sensitivity. This Report should only be reproduced in full and with the written approval of the Manager Ecotoxicology and Environmental Contaminants Section, OEH.


M Julli


7 August 2012

Page 5 of 5: ECOTOX 2 Test Report No.: 12083/12096




Test Outline


The chronic reproduction impairment test subjects cladocerans less than 24 hours old, to a range of sample concentrations over the period of production of 3 broods. The end points of this test are parental mortality and young production. Following exposure for 7 days to various concentrations of the sample, the number of young produced by each parental C. dubia is assessed. Parental mortality and young production are statistically analyzed to determine sample concentrations causing a significant relative to a control group of animals.


Sample 12102/12106 caused significant reproduction impairment in exposed C. dubia at

a concentration of 100%. The sample would need to be diluted approximately 3.3 times to avoid these sub lethal toxic effects on C. dubia (based on the tested concentrations). The 7 day IC20 (reduction in young production) was calculated to be 24% sample. Samples 12103/12107 and 12104/12108 had no observable reproduction impairment effect on C. dubia.



Sample Information EECS Sample Number EFAS Sample Number Additional sample identification

12102§/12106 α 201201943/201202109 LDP 10 Discharge from Brennans Creek

12103§/12107 α 201201948/201202104 LDP11 Georges River U/S

12104§/12108 α 201201953/20120299 LDP 12 Georges River D/S §= Tests started on 22 June 2012 using these samples; α = Test continued to the end with renewals using these samples on 27 June 2012 Collection Date: 18 June 2012 (12102, 12103, 12104)/ 25 June 2012 (12106, 12107, 12108) Submitted By: J. Byrne Date of Delivery: 18 June 2012(12102, 12103, 12104)/ 25 June 2012 (12106, 12107, 12108) Storage Conditions: 40C Comments on Sample Appearance: Clear Liquid Test Methods and Conditions Date of Test Commencement: 22 June 2012 Test Conducted by: R. Patra, R. Sunderam and A. Rose Test Method Protocol No.: Ecotox 2 Deviations From Protocol: None Test Method: Cladoceran Reproduction Impairment Test Test Type: Static Renewal Renewal periods: 72 hours and then 48 hours Duration of test: 7 days Test Species: Ceriodaphnia dubia Age at test start: <24 hours Source: EECS Lab culture Location Test Room No. F27 Chamber No. F29 Test Vessel Type: 100 ml Glass beaker Test Volume: 50 mL Test Temperature: 25 oC Test Photoperiod: 16 : 8 L:D Light intensity at surface of Test Vessels: <800 Lux Dilution Water Source:Filtered Sydney mains+ Na2S2O3 + 5% mineral water+ Sea Water Conductivity: 505 μS/cm pH: 8.0 Hardness: 100 mg/L as CaCO3 Alkalinity: 50 mg /L as CaCO3 Test Design Concentrations tested: Control (Diluent water). Samples 12102/12106 and 12104/12108:10, 30 and 100% Sample 12103/12107: 30 and 100% Test Concentrations: Nominal Number of replicate test vessels per concentration and control/s: 10 Number of animals per replicate: 1 Statistical Methods:

Data transformation for LOEC: Untransformed or log (x+1) Statistical Analysis Method: Dunnett’s Multiple Comparison Test or Steel Many-One Rank Test for LOEC

Linear Interpolation (bootstrap) for point estimates (IC20 and IC50)


Test Results of the 7-day Cladoceran Reproduction Impairment Test.


% Concentration Sample

No Test Endpoints 0 10 30 100

PMSD

#

Mean No. of Young / surviving adult (SE)

25.2 (6.7)

Control



(1.8) 20.3 97.7)

3.1* (3.9)

12102/ 12106



(2.7) 23.5 (3.1)

12103/ 12107



(4.7) 26.6 (2.1)

23.9 (5.3)

12104/ 12108


*Significantly Different to Control (p<0.05) The test validity criteria were met: (a) the control group produced >15 young per adult on average after three broods; (b) parental mortalities was <20% in control treatment. # PMSD (Percent Minimum Significant Difference) is an estimation of the smallest % decrease in reproduction from the control that could be determined as statistically significant for this test.

Table 2. Estimated NOEC, LOEC, IC20 and IC50 Values for Reproductive Impairment of C.dubia

Inhibition Concentrations (IC) of sample calculated to cause a 20% or 50% reduction in

reproduction in C.dubia (95% confidence intervals)

Sample Number

No Observed Effect

Concentration NOEC

(% Sample)


Concentration LOEC

(% Sample) IC20 IC50 12102/ 12106 30 100 24

(16-35) 48

(35-58) 12103/ 12107 100 NC NC NC

12104/ 12108 100 NC NC NC

NC or NA: Not calculable; LTC= Lowest Tested concentration for the sample

Table 3. Physico-Chemical Variables in Test Solutions pH Temperature

Conductivity

(μS/cm) Dissolved Oxygen

(%Saturation) Nominal

Test Conc. (% dilution) Fresh

(0 hrs) 72 h old

Fresh (0 hrs)

48 h old

Fresh (0 hrs)

48 h old

Fresh (0 hrs)

48 h old

Control 8.0 8.3 24.0 24.9 505 643 99 1147.4 8.1 24.9 25.1 506 550 113 96Diluent 8.0 8.1 25.0 24.3 519 565 97 109

12102/121068.4 8.6 24.1 24.0 643 754 100 1147.9 8.1 24.2 25.4 507 538 114 9510 8.4 8.4 24.7 24.7 638 654 97 1078.7 8.9 24.3 24.0 973 1190 100 1148.6 8.8 24.2 25.4 932 973 115 9430 8.7 8.8 25.1 24.7 887 895 97 1058.8 9.3 24.4 24.4 1915 2030 98 1138.9 9.2 24.4 25.3 1952 2000 114 93100 8.8 9.1 25.3 24.9 1801 1826 99 106

12103/12107 8.1 8.1 24.5 24.5 395 589 99 1147.9 8.0 24.5 25.4 409 426 115 9330 8.0 8.0 25.4 24.9 420 455 97 1068.1 7.6 24.7 24.3 167 308 99 1147.6 7.6 24.3 25.7 169 190 113 94100 7.6 7.7 25.5 24.5 251 264 102 107

12104/121088.2 8.4 24.2 24.1 552 596 596 988.1 8.3 24.3 25.9 533 552 552 11410

8.4 8.3 25.9 24.5 584 591 591 968.5 8.7 24.2 24.6 666 892 98 1148.6 8.8 24.7 25.8 782 805 120 9330 8.6 8.6 25.7 24.7 740 780 96 1078.7 9.0 24.3 24.3 995 1063 97 1148.7 8.8 24.0 25.7 765 802 118 92100 8.8 9.0 25.6 24.7 1278 1303 99 104

. This Report should only be reproduced in full and with the written approval of the Manager Ecotoxicology and Environmental Contaminants Section, OEH.


M Julli


7 August 2012



Report on the Acute Toxicity of Samples from West Cliff to Larvae of the Rainbowfish,

Melanotaenia duboulayi (Castelnau, 1878) Office of Environment and Heritage

Ecotoxicology and Environmental Contaminants Section 480 Weeroona Road, Lidcombe NSW 2141

Date of Issue of Report: 12 July 2012

Test Outline

The test was conducted to assess the potentially harmful effects of the samples to larvae of the native freshwater fish species Melanotaenia duboulayi. In this test the loss of balance (imbalance) is used as the endpoint as opposed to mortality, i.e. where possible, fish are removed from the test solution once they lose the ability to remain normally positioned.

Following exposure for 72 hours to various concentrations of the sample/s, the number of M. duboulayi affected was counted. This data is statistically analysed to determine sample concentrations causing a significant adverse effect to M. duboulayi relative to a control group

If more than 50% of exposed animals are imbalanced in any of the tested sample concentrations, a 72-hour EC50 (imbalance) value is calculated, which is the Effective Concentration of the sample which causes immobilisation in 50% of exposed M. duboulayi.


A conductivity adjusted control (CAC) was also tested, having similar conductivity to the test samples.


Samples 12106 and 12108 had no observable acute effect on M. duboulayi, in that there was

no immobilisation in exposed M. duboulayi during the 72 hour exposure period.

Samples 12107 and 12109 had minimal observable effect on M. duboulayi, in that imbalance in exposed M. duboulayi was not statistically different to that occurring in the Control group.




12106 201202109 LDP 10 Discharge from Brennans Creek 11:20

12107 201202104 LDP 11 Upstream Georges River 10:40

12108 201202099 LDP 12 downstream Georges River 10:52

12109 201202114 Inside Brennans Creek Dam 11:36


Test Methods and Conditions Test Commencement Date: 2 July 2012 Test Conducted by: R. Patra and R. Sunderam Test Method Protocol No ECOTOX 3. The test method is based on procedures published by the USEPA (2002), Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, 5th Edition. EPA-821-R-02-012. It differs from this guideline in that Australian fish species are used, and animals are cultured and bred in the OEH laboratory. The current animal research authority is (OEH AEC approval number 110620/02) Deviations from Protocol: None Test Type: Fish, Acute, Static Duration of test: 72 Hours Test Species: Melanotaenia duboulayi Age: 4 days Source: EECS Culture Tank B2 Test Location: Room No. F.27 Test Room No.: F.29 Test Vessel Type: 100 mL beaker Test Volume: 50 mL Test Temperature: 25 oC Test Photoperiod: 16 h L: 8 h D Light intensity at surface of Test Vessels: <800 Lux Dilution Water Source: Filtered Sydney mains water with conductivity adjusted to 500 μS/cm with filtered seawater. Conductivity: 512 μS/cm pH: 7.8 Hardness: 78 mg/L as CaCO3 Alkalinity: 42 mg/L as CaCO3 Conductivity Adjusted Control (CAC): Diluent above with additional filtered seawater to increase conductivity to 2000µS/cm


Test Design Concentrations tested: Test Concentrations: Nominal Number of replicate test vessels per concentration and control/s: 4 Number of animals per replicate: 5 Statistical Methods Data Transformation for Lowest Observed Effect Concentration (LOEC): Angular(uncorrected) Statistical Analysis Method: Steel Many-One Rank Test for LOEC Results Table 1. Imbalance of Melanotaenia duboulayi in test solutions

EECS Sample Number

Nominal Test Concentration (% sample)


imbalanced after 24 hours

exposure



exposure



exposure

Percent Minimum Significant

Difference # (PMSD)

Control Diluent 0 0 0

CAC Diluent +Seawater 0 0 0 -

10 0 0 0 30 0 0 0 12106 100 0 0 0

10 0 0 5 30 0 0 5 12107 100 0 0 0

6.9

10 0 0 30 0 0 0 12108 100 0 0 0

-

10 0 5 10 30 0 5 5 12109 100 0 0 0

11

*Significantly different from the control (p ≤ 0.05). 72-h data only analysed. # PMSD is an estimation of the smallest percentage increase in imbalance (relative to the control), that could be determined as statistically significant for this test. PMSD is based on parametric test calculations and PMSD for non-parametric analyses (as used here) may differ slightly from indicated values. A test validity criterion of Control group survival (greater than 90%) was met. Samples 12106-12109 The No Observed Effect Concentration (NOEC) of samples 12106-12109 was 100 %.


Table 2. Physico-chemical Variables in Test Solutions

Test Conc.

Temperature (0C)



(%) 0 h 72 h 0 h 72 h 0 h 72 h 0 h 72 h Control

Diluent 24.9 24.1 7.8 8.0 512 550 98 100 Conductivity adjusted control

Dil+SW 24.7 24.2 7.8 8.1 2030 2170 98 100 Sample 12106

10 24.4 24.1 8.4 8.5 635 677 99 100 30 24.4 24.2 8.6 8.8 888 942 100 100

100 24.1 24.2 8.8 9.2 1783 1810 100 99 Sample 12107

10 24.8 24.1 8.0 8.1 483 510 98 100 30 25.0 24.1 8.0 8.0 431 482 100 100

100 25.0 24.2 8.0 7.8 249 268 107 99 Sample 12108

10 24.7 24.0 8.3 8.4 583 612 98 99 30 24.7 24.1 8.5 8.7 734 796 100 99

100 24.7 24.2 8.7 9.1 1263 1314 112 99 Sample 12109

10 24.8 23.9 8.4 8.5 617 767 97 98 30 24.9 24.0 8.6 8.8 834 907 98 98




12 July 2012





Date of Issue of Report: 5 July 2012

Test Outline


Following exposure for 96 hours to various concentrations of the samples, the number of M. duboulayi affected was counted. This data is statistically analysed to determine sample concentrations causing a significant adverse effect to M. duboulayi relative to a control group




Samples 12102-12104 had minimal observable effect on M. duboulayi, in that imbalance in exposed M. duboulayi was not statistically different to that occurring in the Control group.




12102 201201943 LDP 10 Discharges from Brennans Creek

12103 201201948 LDP 11 U/S Georges River

12104 201201953 LDP 12 D/S Georges River


Test Methods and Conditions Test Commencement Date: 19 June 2012 Test Conducted by: R. Patra, R. Sunderam and M. Julli Test Method Protocol No ECOTOX 3. The test method is based on procedures published by the USEPA (2002), Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, 5th Edition. EPA-821-R-02-012. It differs from this guideline in that Australian fish species are used, and animals are cultured and bred in the OEH laboratory. The current animal research authority is (OEH AEC approval number 110620/02) Deviations from Protocol: None Test Type: Fish, Acute, Static Duration of test: 96 Hours Test Species: Melanotaenia duboulayi Age: 10 day Source: EECS Culture Tank B2 Test Location: Room No F.27 Test Room No.: F.29 Test Vessel Type : 100 mL beaker Test Volume: 50 mL Test Temperature: 25 oC Test Photoperiod: 16 h L: 8 h D Light intensity at surface of Test Vessels: <800 Lux Dilution Water Source: Filtered Sydney mains water with conductivity adjusted to 500 μS/cm (if necessary, depending on the source) with filtered seawater. Conductivity: 509 μS/cm pH: 8.0 Hardness: 86 mg/L as CaCO3 Alkalinity: 38 mg/L as CaCO3


Test Design Concentrations tested: Samples 12102 and 12104: 10, 30 and 100% Sample 12103: 100% Test Concentrations: Nominal Number of replicate test vessels per concentration and control: 4 Number of animals per replicate: 5 Statistical Methods Data Transformation for Lowest Observed Effect Concentration (LOEC): Angular(uncorrected) Statistical Analysis Method: Fisher Exact/Dunnett’s Multiple Comparison Test for LOEC Results Table 1. Imbalance of Melanotaenia duboulayi in test solutions EECS

Sample Number




exposure



exposure



exposure



exposure


Difference # (PMSD)

Control Diluent 0 0 0 0

10 0 0 0 0 30 0 0 0 10 12102 100 5 10 15 15

11

12103 100 10 5 5 5 - 10 5 5 5 5 30 15 15 15 15

12104

100 0 0 0 0 11

*Significantly different from the control (p ≤ 0.05). 96-hour data only analysed. # PMSD is an estimation of the smallest percentage increase in imbalance (relative to the control), that could be determined as statistically significant for this test. PMSD is based on parametric test calculations and PMSD for non-parametric analyses (as used here) may differ slightly from indicated values. A test validity criterion of Control group survival (greater than 90%) was met.



Test Conc.

Temperature (0C)



(%) 0 h 72 h 96 h 0 h 72 h 96 h 0 h 48 h 96 h 0 h 72 h 96 h Control

Dil 24.7 24.9 24.8 8.0 8.2 8.1 509 539 540 97 100 99 Sample 12102

10 24.6 24.4 24.6 8.4 8.5 8.5 649 670 672 98 98 97 30 24.4 24.4 24.5 8.6 8.9 8.8 933 970 974 98 96 96

100 24.4 24.3 24.6 8.8 9.3 9.3 1909 1971 1978 98 96 96 Sample 12103

100 24.3 24.3 24.4 7.9 8.2 8.2 168 176 176 97 96 96 Sample 12104

10 24.2 24.0 24.4 8.2 8.2 8.3 555 585 590 97 97 97 30 24.3 24.2 24.3 8.5 8.6 8.6 653 672 670 96 97 97

100 24.4 24.2 24.3 8.7 9.0 9.1 988 1023 1025 97 97 97 This Report should only be reproduced in full and with the written approval of the Manager Ecotoxicology and Environmental Contaminants Section, OEH.



5 July 2012

of 3 Ecotox 3 Test Report No. 12096-A




Replacement for Report Ecotox 3 Test Report No.12096 Date of Issue of Report: 7 August 2012 Test Outline

The test was conducted to assess the potentially harmful effects of the sample/s to larvae of the native freshwater fish species Melanotaenia duboulayi. In this test the loss of balance (imbalance) is used as the endpoint as opposed to mortality, i.e. where possible, fish are removed from the test solution once they lose the ability to remain normally positioned.

Following exposure for 48 hours to various concentrations of the sample/s, the number of M. duboulayi affected was counted. This data is statistically analysed to determine sample concentrations causing a significant adverse effect to M. duboulayi relative to a control group

If more than 50% of exposed animals are imbalanced in any of the tested sample/s concentrations, a 48-hour EC50 (imbalance) value is calculated, which is the Effective Concentration of the sample which causes immobilisation in 50% of exposed M. duboulayi.



Samples 12096 and 12100 had minimal observable effect on M. duboulayi, in that

imbalance in exposed M. duboulayi was not statistically different to that occurring in the Control group.

Samples 12099 and 12101 had no observable acute effect on M. duboulayi, in that there

were no exposed M. duboulayi larvae imbalanced in undiluted samples.


Sample Information EECS Sample

Number EFAS Sample

Number Additional Sample Identification


12099 201201760 U/S LDP 11 Georges River

12100 201201765 D/S LDP 12 Georges River

12101 201201770 Inside Brennans Creek Dam Sample Details Collection Date: 4 June 2012 Sample/s Collected By: J. Byrne Date of delivery to EECS: 4 June 2012 Storage Conditions: 4oC Comments on Sample Appearance: Clear Laboratory Accreditation does not extend to sample collection

Test Methods and Conditions Test Commencement Date: 5 June 2012 Test Conducted by: R. Patra, R. Sunderam and M. Julli Test Method Protocol No ECOTOX 3. The test method is based on procedures published by the USEPA (2002), Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, 5th Edition. EPA-821-R-02-012. It differs from this guideline in that Australian fish species are used, and animals are cultured and bred in the OEH laboratory. The current animal research authority is (OEH AEC approval number 110620/02) Deviations from Protocol: Two replicates per sample treatment Test Type: Fish, Acute, Static Duration of test: 48 Hours Test Species: Melanotaenia duboulayi Age: 14 days Source: EECS Culture Tank B2 Test Location: Room No F.27 Test Room No.: F.29 Test Vessel Type: 100 mL beaker Test Volume: 50 mL Test Temperature: 25 oC Test Photoperiod: 16 h L: 8 h D Light intensity at surface of Test Vessels: <800 Lux Dilution Water Source: Filtered Sydney mains water with conductivity adjusted to 500 μS/cm (if necessary, depending on the source) with filtered seawater. Conductivity: 517 μS/cm pH: 8.0 Hardness: 86 mg/L as CaCO3 Alkalinity: 38 mg/L as CaCO3 Test Design Concentrations tested: 100% Test Concentrations: Nominal Number of replicate test vessels per concentration and control: Two per sample treatment Four per Control Number of animals per replicate: 5


Results Table 1. Imbalance of Melanotaenia duboulayi in test solutions EECS Sample

Number Nominal Test Concentration (% sample)





exposure

Percent Minimum Significant Difference # (PMSD)

Control Diluent 5 5

12096 100 10 20 38 12099 100 0 0 - 12100 100 10 10 28 12101 100 0 0 -

# PMSD is an estimation of the smallest percentage increase in imbalance (relative to the control), that could be determined as statistically significant for this test. A test validity criterion of Control group survival (greater than 90%) was met. Table 2. Physico-chemical Variables in Test Solutions

Test Conc.

Temperature (0C)




Diluent 24.9 24.7 8.0 8.0 517 534 100 96 Sample 12096

100 24.4 24.8 8.7 9.1 1960 1985 94 96 Sample 12099

100 24.4 24.8 8.0 7.9 184 187 100 96 Sample 12100

100 24.4 24.8 8.4 8.7 673 689 100 96 Sample 12101




7 August 2012





Replacement for Report Ecotox 3 Test Report No.12082 Date of Issue of Report: 7 August 2012 Test Outline






Sample 12082 caused 30% imbalance in exposed M. duboulayi at a concentration of 100%.

This effect was not statistically significant. Sample 12083 caused significant imbalance in exposed M. duboulayi at a concentration of

30%. The sample would need to be diluted approximately 10 times to avoid these acute toxic effects on M. duboulayi (based on the tested concentrations).

The 96-hour EC50 (imbalance) concentration was calculated to be 25% sample. Sample 12084 had no observable acute effect on M. duboulayi, in that there was no imbalance in exposed M. duboulayi during the 96-hour exposure period. Sample 12085 caused significant imbalance in exposed M. duboulayi at a concentration of 100%. The sample would need to be diluted approximately 3 times to avoid these acute toxic effects on M. duboulayi (based on the tested concentrations). The 96-hour EC50 (imbalance) concentration was calculated to be 55% sample.


Sample Information EECS Sample Number EFAS Sample Number Additional Sample Identification 12082 201201633 Inside Brennans Creek Dam


12084 201201643 U/S LDP 11

12085 201201648 D/S LDP 12

Sample Details Collection Date: 28 May 2012 Sample/s Collected By: J. Byrne Date of delivery to EECS: 28 May 2012 Storage Conditions: 4oC Comments on Sample Appearance: Clear Laboratory Accreditation does not extend to sample collection Test Methods and Conditions Test Commencement Date: 29 May 2012 Test Conducted by: R. Patra and R. Sunderam Test Method Protocol No ECOTOX 3. The test method is based on procedures published by the USEPA (2002), Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, 5th Edition. EPA-821-R-02-012. It differs from this guideline in that Australian fish species are used, and animals are cultured and bred in the OEH laboratory. [The current animal research authority is (OEH AEC approval number 110620/02) Deviations from Protocol: Two replicates per sample treatment Test Type: Fish, Acute, Static Duration of test: 96 Hours Test Species: Melanotaenia duboulayi Age: 5 days Source: EECS Culture Tank B3 Test Location:Room No F.27 Test Room No.: F. 29 Test Vessel Type : 100 mL beaker Test Volume: 50 mL Test Temperature: 25 oC Test Photoperiod: 16 h L: 8 h D Light intensity at surface of Test Vessels: <800 Lux Dilution Water Source: Filtered Sydney mains water with conductivity adjusted to 500 μS/cm (if necessary, depending on the source) with filtered seawater. Conductivity: 507 μS/cm pH: 7.9 Hardness: 86 mg/L as CaCO3 Alkalinity: 38 mg/L as CaCO3 Test Design Concentrations tested: Samples 12082-12085: 10, 30 and 100% Test Concentrations: Nominal Number of replicate test vessels per concentration and control/s: Two per sample Four per control Number of animals per replicate: 5 Statistical Methods Data Transformation for Lowest Observed Effect Concentration (LOEC): Angular(uncorrected) Statistical Analysis Method: Trimmed Spearman-Kärber/Binomial graphical Estimates for EC50 Fisher exact / Bonferroni Adj t Test for LOEC


Results Table 1. Imbalance of Melanotaenia duboulayi in test solutions

EECS Sample Number




exposure



exposure



exposure


Difference # (PMSD)


10 0 0 10 30 0 0 0 12082 100 0 0 30

-

10 0 0 0 30 0 0 60* 12083 100 0 0 60*

53.0

10 0 0 0 30 0 0 0 12084 100 0 0 0

-

10 0 0 0 30 0 0 0 12085 100 0 0 100*

-

*Significantly different from the control (p ≤ 0.05). 96-h data only analysed. # PMSD is an estimation of the smallest percentage increase in imbalance (relative to the control), that could be determined as statistically significant for this test. PMSD is based on parametric test calculations and PMSD for non-parametric analyses (as used here) will differ slightly from indicated values. A test validity criterion of Control group survival (greater than 90%) was met. Sample 12082 The Lowest Observed effect concentration (LOEC) of sample 12082 was 100 % solution. The No Observed Effect Concentration (NOEC) was 30%. The 96-Hour EC50 (Imbalance) for sample 12082 was not calculable as there was less than 50% imbalance at the highest tested concentration of 100%. Sample 12083 The Lowest Observed effect concentration (LOEC) of sample 12083 was 30 % solution. The No Observed Effect Concentration (NOEC) was 10%. The 96-Hour EC50 (Imbalance) for sample 12083 was 25% (95%CL = 16 - 40%) Sample 12084 There was no imbalance in the undiluted sample. Sample 12085 The Lowest Observed effect concentration (LOEC) of sample 12085 was 100 % solution. The No Observed Effect Concentration (NOEC) was 30% The 96-Hour EC50 (Imbalance) for sample 12082 was 55% (95% CL = 37 - 80%)



Test Conc.

Temperature (0C)



(%) 0 h 48 h 96 h 0 h 48 h 96 h 0 h 48 h 96h 0 h 48 h 96 h Control

Diluent 24.6 24.6 24.4 7.9 7.9 7.9 507 515 505 99 99 96 Sample 12082

10 24.6 24.2 24.3 8.4 8.4 8.3 633 648 668 100 97 96 30 24.5 24.3 24.3 8.6 8.7 8.6 882 895 915 100 98 96

100 24.5 24.3 24.4 8.8 9.1 9.1 1743 1790 1822 100 97 96 Sample 12083

10 24.5 24.1 24.2 8.3 8.4 8.5 631 652 673 99 98 95 30 24.5 24.3 24.1 8.5 8.7 8.7 880 906 920 100 97 96

100 24.6 24.1 24.2 8.7 9.1 9.1 1733 1872 1871 100 98 97 Sample 12084

10 24.6 24.1 24.3 7.9 7.7 8.3 475 480 504 99 98 97 30 24.6 24.1 24.4 7.8 7.8 8.2 406 414 431 100 97 96

100 24.7 24.3 24.3 7.6 7.7 8.0 156 163 180 100 97 97 Sample 12085

10 24.5 24.1 24.3 8.2 8.0 8.0 579 593 599 100 97 96 30 24.5 24.2 24.2 8.4 8.5 8.5 723 738 759 100 98 96

100 24.5 24.2 24.3 8.6 9.0 9.0 1222 1260 1307 100 98 97 This Report should only be reproduced in full and with the written approval of the Manager Ecotoxicology and Environmental Contaminants Section, OEH.



7 August 2012

Report on the Acute Toxicity of Samples from West Cliff to Larvae of the Rainbowfish, Melanotaenia duboulayi (Castelnau, 1878)


480 Weeroona Road, Lidcombe NSW 2141 Replacement for Report Ecotox 3 Test Report No.12071 Date of Issue of Report: 7 August 2012

Test Outline

The test was conducted to assess the potentially harmful effects of the samples to larvae of the native freshwater fish species Melanotaenia duboulayi. In this test the loss of balance (imbalance) is used as the endpoint as opposed to mortality, i.e. were possible, fish are removed from the test solution once they lose the ability to remain normally positioned.





Sample 12071 had minimal observable effect on M. duboulayi, in that imbalance in exposed

M. duboulayi was not statistically different to that occurring in the Control group.

Sample 12072 caused significant imbalance in exposed M. duboulayi at a concentration of 30% (lowest tested concentration). As imbalance was above 50% in the lowest tested sample concentration of 30%, an EC50 (imbalance) value was not calculable

. Sample 12073 caused significant imbalance in exposed M. duboulayi at a concentration of 30%. The sample would need to be diluted approximately 10 times to avoid these acute toxic effects on M. duboulayi (based on the tested concentrations). The 96-hour EC50 (imbalance) concentration was calculated to be 25% sample. Sample 12074 caused significant imbalance in exposed M. duboulayi at a concentration of 30%. The sample would need to be diluted approximately 10 times to avoid these acute toxic effects on M. duboulayi (based on the tested concentrations). The 96-hour EC50 (imbalance) concentration was calculated to be 24% sample.

Page 1 of 5 Ecotox 3 Test Report No. 12071-A

Sample Information EECS Sample Number EFAS Sample Number Additional Sample Identification 12071 201201474 U/S LDP 11

12072 201201469 D/S LDP 12, George River

12073 201201479 Discharge LDP 10

12074 201201484 Inside BCD

Sample Details Collection Date: 21 May 2012 Sample/s Collected By: J. Byrne Date of delivery to EECS: 21 May 2012 Storage Conditions: 4oC Comments on Sample Appearance: Clear Laboratory Accreditation does not extend to sample collection

Test Methods and Conditions Test Commencement Date: 22 May 2012 Test Conducted by: R. Sunderam, R Patra and A. Rose Test Method Protocol No ECOTOX 3. The test method is based on procedures published by the USEPA (2002), Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, 5th Edition. EPA-821-R-02-012. It differs from this guideline in that Australian fish species are used, and animals are cultured and bred in the OEH laboratory. [The current animal research authority is (OEH AEC approval number 110620/02) Deviations from Protocol: None Test Type: Fish, Acute, Static Duration of test: 96 Hours Test Species: Melanotaenia duboulayi Age: 1 day Source: EECS Culture B3 Test Location: Room No F.27 Constant Temperature Room No.: F.29 Test Vessel Type: 100 mL beaker Test Volume: 50 mL Test Temperature: 25 oC Test Photoperiod: 16 h L: 8 h D Light intensity at surface of Test Vessels: <800 Lux Dilution Water Source: Filtered thiosulphate treated Sydney mains water with conductivity adjusted to 500 μS/cm with filtered seawater. Conductivity: 521μS/cm pH: 8.0 Hardness: 86 mg/L as CaCO3 Alkalinity: 38 mg/L as CaCO3


Test Design Concentrations tested: Sample 12071 and 12072: 30 and 100% Sample 12073 and 12074: 10, 30 and 100% Test Concentrations: Nominal Number of replicate test vessels per concentration and control/s: 2 and 4 Number of animals per replicate: 5 Statistical Methods Data Transformation for Lowest Observed Effect Concentration (LOEC): Angular(uncorrected) Statistical Analysis Method: Trimmed Spearman-Kärber for EC50 Bonferroni Adj t Test / STP 2x2 Contingency Tables for LOEC Results Table 1. Imbalance of Melanotaenia duboulayi in test solutions

EECS Sample Number


(% sample)


immobilised after 48 hours

exposure



exposure



exposure


Difference # (PMSD)


30 0 0 20 12071

100 0 0 20 57

30 0 20 70* 12072

100 10 50 100* 68

10 0 0 10 30 0 10 70* 12073 100 0 50 100*

73

10 0 0 20 30 0 10 70* 12074 100 0 10 90*

47

*Significantly different from the control (p ≤ 0.05). 96-h data only analysed. # PMSD is an estimation of the smallest percentage increase in imbalance (relative to the control), that could be determined as statistically significant for this test. PMSD is based on parametric test calculations and PMSD for non-parametric analyses (as used here for 12072-12074) will differ slightly from indicated values. A test validity criterion of Control group survival (90%) was met.


Sample 12071 had minimal observable effect on M. duboulayi, in that imbalance in exposed M.duboulayi was not statistically different to that occurring in the Control group. Sample 12072 The Lowest Observed effect concentration (LOEC) of sample 12072 was 30 % solution (the lowest tested concentration). As imbalance was above 50% in the lowest tested sample concentration of 30%, the 96-Hour EC50 (Imbalance) for sample 12072 was not calculable, that is the EC50 is less than 30% sample. Sample 12073

The Lowest Observed effect concentration (LOEC) of sample 12073 was 30 % solution. The No Observed Effect Concentration (NOEC) was 10 % sample.

The 96- Hour EC50 (Imbalance) for sample 12073 was 25% (95% CL=18 - 36%) Sample 12074

The Lowest Observed effect concentration (LOEC) of sample 12074 was 30 % solution. The No Observed Effect Concentration (NOEC) was 10 %

The 96Hour EC50 (Imbalance) for sample 12074 was 24% (95% CL=15 - 40%) Table 2. Physico-chemical Variables in Test Solutions

Test Conc.

Temperature (0C)



(%) 0 h 96 h 0 h 96 h 0 h 96 h 0 h 96h Control

Diluent 24.0 24.4 8.0 7.9 521 537 98 97 sample 12071

30 24.0 24.3 8.1 8.0 479 496 99 96 100 24.1 24.4 7.8 8.0 220 246 116 95

Sample 12072 30 24.0 24.5 8.5 8.7 756 769 100 95

100 24.2 24.4 8.7 9.1 1301 1303 116 94 Sample 12073

10 24.0 24.4 8.4 8.5 647 660 98 95 30 24.0 24.5 8.6 8.8 907 929 99 95

100 24.0 24.5 8.8 9.2 1751 1733 116 95 Sample 12074

10 24.0 24.2 8.5 8.5 626 647 98 95 30 24.0 24.3 8.6 8.8 844 871 100 95

100 24.1 24.7 8.8 9.2 1552 1533 113 95


This Report should only be reproduced in full and with the written approval of the Manager Ecotoxicology and Environmental Contaminants Section, OEH.



7 August 2012


Report on the Acute Toxicity of Samples from West Cliff to Larvae

of the Rainbowfish, Melanotaenia duboulayi (Castelnau, 1878) Office of Environment and Heritage


Replacement for Report Ecotox 3 Test Report No.12050 Date of Issue of Report: 7 August 2012

Test Outline

The test was conducted to assess the potentially harmful effects of the samples to larvae of the native freshwater fish species Melanotaenia duboulayi. In this test the loss of balance (imbalance) is used as the endpoint as opposed to mortality, i.e. were possible, fish are removed from the test solution once they lose the ability to remain normally positioned.

Following exposure for 96 hours to various concentrations of the sample/s, the number of M.duboulayi affected was counted. This data is statistically analysed to determine sample concentrations causing a significant adverse effect to M. duboulayi relative to a control group

If more than 50% of exposed animals are imbalanced in any of the tested sample/s concentrations, a 96-hour EC50 (imbalance) value is calculated, which is the Effective Concentration of the sample which causes immobilisation in 50% of exposed M. duboulayi.



Sample 12050 had minimal observable effect on M. duboulayi, in that imbalance in exposed M. duboulayi was not statistically different to that occurring in the Control group.

Sample 12052 caused significant imbalance in exposed M. duboulayi at a concentration of

30%. As imbalance was > 50% in the lowest tested sample concentration of 30%, an EC50 (imbalance) value was not calculable.


30%. The sample would need to be diluted approximately 10 times to avoid these acute toxic effects on M. duboulayi (based on the tested concentrations). The 96-hour EC50 (imbalance) concentration was calculated to be 17% sample.


100%. The sample would need to be diluted approximately 3 times to avoid these acute toxic effects on M. duboulayi (based on the tested concentrations). The 96-hour EC50 (imbalance) concentration was calculated to be 34% sample.


Sample Information EECS Sample Number EFAS Sample Number Additional Sample Identification 12050 201201383 U/S LDP 11

12052 201201389 D/S LDP 12

12054 201201395 Discharge Brennans Creek LDP 10

12056 201201401 Brennans Creek Dam

Sample Details Collection Date: 14 May 2012 Sample/s Collected By: M. Julli Date of delivery to EECS: 14 May 2012 Storage Conditions: 4oC Comments on Sample Appearance: Solutions Clear. 12056 had high numbers of rotiferan

fauna which were removed by filtration through a 100µm nylon mesh prior to testing.

Laboratory Accreditation does not extend to sample collection

Test Methods and Conditions Test Commencement Date: 15 June 2012 Test Conducted by: R. Sunderam, R. Patra, M. Julli Test Method Protocol No ECOTOX 3. The test method is based on procedures published by the USEPA (2002), Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, 5th Edition. EPA-821-R-02-012. It differs from this guideline in that Australian fish species are used, and animals are cultured and bred in the OEH laboratory.The current animal research authority is (OEH AEC approval number 110620/02) Deviations from Protocol: None Test Type: Fish, Acute, Static Duration of test: 96 Hours Test Species: Melanotaenia duboulayi Age: 6 days Source: EECS Culture;Tank B 2 Test Location:Room No F.27 Test Room No.: F.29 Test Vessel Type : 100 mL beaker Test Volume: 50 mL Test Temperature: 25 oC Test Photoperiod: 16 h L: 8 h D Light intensity at surface of Test Vessels: <800 Lux Dilution Water Source: Filtered Sydney mains water with conductivity adjusted to 500 μS/cm (if necessary, depending on the source) with filtered seawater. Conductivity: 505 μS/cm pH: 7.6 Hardness: 86 mg/L as CaCO3 Alkalinity: 38 mg/L as CaCO3


Test Design Concentrations tested: Samples 12050 and 12052: 30 and 100% Samples 12054 and 12056: 10, 30 and 100% Test Concentrations: Nominal Number of replicate test vessels per concentration and control: 2 samples / 4 controls Number of animals per replicate: 5 Statistical Methods Data Transformation for Lowest Observed Effect Concentration (LOEC): Angular uncorrected Statistical Analysis Method: Trimmed Spearman-Kärber for EC50 Wilcoxon/ Bonferroni Adj Test for LOEC Results Table 1. Imbalance of Melanotaenia duboulayi in test solutions

EECS Sample Number


(% sample)

Percentage of animals imbalanced

after 24 hours

exposure



exposure



exposure



exposure

Percent Minimum SignificantDifference # (PMSD)

Control Diluent 0 0 0 0

30 0 0 0 10 12050

100 0 0 0 10

30 20 20 20 100* 12052

100 0 0 0 100* -

10 0 0 10 10 30 0 0 0 90* 12054 100 0 0 40 100*

19

10 10 10 20 30 30 0 0 10 40 12056 100 0 0 0 90*

46

*Significantly different from the control (p ≤ 0.05). 96-h data only analysed. # PMSD is an estimation of the smallest percentage increase in imbalance (relative to the control), that could be determined as statistically significant for this test. PMSD is based on parametric test calculations and PMSD for non-parametric analyses (as used here for 12054 and 12056) may differ slightly from indicated values. A test validity criterion of Control group survival (greater than 90%) was met. Sample 12050 No significant effects (imbalance) were observed in M. duboulayi larvae following 96 hours exposure to sample 12050. Page 3 of 5 Ecotox 3 Test Report No. 12050-A

Sample 12052 The 96-hour No Observed Effect Concentration (NOEC) for sample 12052 was less than 30 % sample, the lowest concentration tested. The 96- hour EC50 (Imbalance) for sample 12052 was not calculable as 100% imbalance of M. duboulayi occurred in the lowest tested concentration of 30% (i.e. the 96-h EC50 is less than 30% sample). Sample 12054 The Lowest Observed effect concentration (LOEC) of sample 12054 was 30% solution. The No Observed Effect Concentration (NOEC) was10 %. The 96Hour EC50 (Imbalance) for sample 12054 was 17% (95% CL = 14 - 21%). Sample 12056 The Lowest Observed effect concentration (LOEC) of sample 12056 was 100% solution. The No Observed Effect Concentration (NOEC) was 30 %. The 96Hour EC50 (Imbalance) for sample 12056 was 34% (95% CL= 14 - 86%). Table 2. Physico-chemical Variables in Test Solutions

Test Conc.

Temperature (0C)



(%) 0 h 48 h 96h 0 h 48 h 96h 0 h 48 h 96 h 0 h 48 h 96 h Control

Diluent 24.9 24.6 24.7 7.6 7.9 7.9 505 520 505 95 99 98 Sample 12050

30 25.2 24.2 24.5 7.6 8.2 7.7 384 392 432 99 95 99 100 24.8 24.3 24.4 7.3 8.2 7.8 175 180 198 100 94 98

Sample 12052 30 24.9 24.4 24.5 8.4 8.4 7.2 695 738 736 97 99 94

100 24.6 24.3 24.3 8.6 9.0 8.9 1142 1167 1189 100 94 93 Sample 12054

10 25.0 24.2 24.4 8.3 8.5 8.3 617 642 654 96 95 99 30 25.1 24.3 24.2 8.5 8.7 8.6 845 852 885 94 94 98

100 25.0 24.2 24.4 8.6 9.1 9.0 1632 1653 1662 91 94 95 Sample 12056

10 25.0 24.4 24.3 8.4 8.5 8.3 607 674 647 94 94 96 30 24.9 24.2 24.3 8.6 8.7 8.5 825 845 880 95 93 95

100 24.7 24.3 24.3 8.8 9.1 9.0 1582 1653 1643 96 92 98


This Report should only be reproduced in full and with the written approval of the Manager Ecotoxicology and Environmental Contaminants Section, OEH.



7 August 2012




Report on the Acute Toxicity of Samples from West Cliff to the Cladoceran, Ceriodaphnia dubia Richard 1894


480 Weeroona Road, Lidcombe NSW 2141 Date of Issue of Report: 18 June 2012

Test Outline

The test was conducted to assess the potentially harmful effects of the sample/s to juveniles of the freshwater crustacean zooplankton species Ceriodaphnia dubia.

Following exposure for 48 hours to various concentrations of the sample/s, the number of C. dubia immobilised was counted. In this test immobilisation is considered similar to lethality.


If more than 50% of exposed animals are immobilised in any of the tested sample/s concentrations, a 48-hour EC50 (immobilisation) value is calculated, which is the Effective Concentration of the sample which causes immobilisation in 50% of exposed C. dubia.



Sample 12050, 12052, 12054 and 12056 had no observable acute effect on C. dubia, in that

there was no immobilisation in exposed C. dubia in any tested sample.

Page 1 of 4 Ecotox 1 Test Report No. 12050



12050 201201383 U/S LDP 11

12052 201201389 D/S LDP 12

12054 201201395 Discharge Brennans Creek LDP 10

12056 201201401 Brennans Creek Dam

Sample Details Collection Date: 14 May 2012 Samples Collected By: M. Julli Date of delivery to EECS: 14 May 2012 Storage Conditions: 4oC Comments on Appearance: Solutions Clear. 12056 had high numbers of rotiferan fauna

which were removed by filtration through a 100µm nylon mesh prior to testing.

Laboratory Accreditation does not extend to sample collection

Test Methods and Conditions Test Commencement Date: 15 May 2012 Test Conducted by: R. Patra, R. Sunderam, M. Julli Test Method Protocol No.: Test Method Protocol No.: ECOTOX 1. The test method is based on procedures published by the USEPA (2002), Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, 5th Edition. EPA-821-R-02-012. It differs from this guideline in that an Australian cladoceran species is used. Deviations from Protocol: None. Test Type: Cladoceran, Acute, Static Duration of test: 48 Hours Test Species: Ceriodaphnia dubia Age:<24 hours Source: EECS Culture Location: Room No F.27 Constant Temperature Room No.: F. 29 Test Vessel Type : 100 mL beaker Test Volume: 50 mL Test Temperature: 25 oC Test Photoperiod: 16 h L: 8 h D Light intensity at surface of Test Vessels: <800 Lux Dilution Water Source: Filtered thiosulphate-treated Sydney mains water with 5% mineral water and conductivity adjusted to 500μS/cm with filtered seawater Conductivity: 484 μS/cm pH: 8.0 Hardness: 100mg/L as CaCO3 Alkalinity: 50 mg/L as CaCO3


Test Design Concentrations tested: Samples 12050 and 12052:30 and 100% Samples 12054 and 12056: 10, 30 and 100% Test Concentrations: Nominal Number of replicate test vessels per concentration and control/s: 4 Number of animals per replicate: 5 Statistical Methods Statistical Analysis Method: Probit for reference toxicant EC50


EECS Sample

Number


(% sample)

Percentage of

animals

immobilised after

24 hours exposure

Percentage of

animals

immobilised after

48 hours exposure


12050 100 0 0 30 0 0

12052 100 0 0 10 0 0 30 0 0 12054 100 0 0 10 0 0 30 0 0 12056 100 0 0

A test validity criterion of Control group survival (greater than 90%) was met. Samples 12050, 12052, 12054 and 12056 There was no immobilisation of C. dubia at any tested concentrations of the samples.



Test Conc.

Temperature (0C)




Diluent 24.7 24.4 8.0 8.0 484 495 97 95 Sample 12050

30 24.6 24.3 7.9 8.0 387 406 97 96 100 24.8 24.2 7.5 7.8 161 178 100 96

Sample 12052 30 24.5 24.2 8.4 8.3 680 699 97 96 100 24.8 24.2 8.6 8.9 1141 1181 100 94

Sample 12054 10 24.0 24.2 8.2 8.4 595 618 94 96 30 24.3 24.2 8.4 8.7 826 881 94 95 100 24.9 24.3 8.6 9.1 1637 1659 92 94

Sample 12056 10 24.7 24.1 8.3 8.5 590 602 94 95 30 25.0 24.2 8.5 8.7 811 836 96 95 100 25.5 24.3 8.7 9.1 1584 1610 98 95



Head, Ecotoxicology Testing Services 18 June 2012




Report on the Acute Toxicity of Samples from Brennans Creek to the Cladoceran, Ceriodaphnia dubia Richard 1894


480 Weeroona Road, Lidcombe NSW 2141 Date of Issue of Report: 20 June 2012

Test Outline

The test was conducted to assess the potentially harmful effects of the sample/s to juveniles of the freshwater crustacean zooplankton species Ceriodaphnia dubia.

Following exposure for 48 hours to various concentrations of the samples, the number of C. dubia immobilised was counted. In this test immobilisation is considered similar to lethality.


If more than 50% of exposed animals are immobilised in any of the tested samples concentrations, a 48-hour EC50 (immobilisation) value is calculated, which is the Effective Concentration of the sample which causes immobilisation in 50% of exposed C. dubia.



Samples 12071-12074 had no observable acute effect on C. dubia, in that no immobilisation

occurred in C. dubia exposed to any of the undiluted samples.




12071 201201474 U/S LDP 11

12072 201201469 D/S LDP 12, George River

12073 201201479 Discharge LDP 10

12074 201201484 Inside BCD

Sample Details Collection Date: 21 May 2012 Sample/s Collected By: J. Byrne Date of delivery to EECS: 21 May 2012 Storage Conditions: 4oC Comments on Appearance: Clear Laboratory Accreditation does not extend to sample collection

Test Methods and Conditions Test Commencement Date: 22 May 2012 Test Conducted by: R. Patra, R. Sunderam and A. Rose Test Method Protocol No.: Test Method Protocol No.: ECOTOX 1. The test method is based on procedures published by the USEPA (2002), Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, 5th Edition. EPA-821-R-02-012. It differs from this guideline in that an Australian cladoceran species is used. Deviations from Protocol: Two replicates per sample treatments Test Type: Cladoceran, Acute, Static Duration of test: 48 Hours Test Species: Ceriodaphnia dubia Age:<24 hours Source: EECS Culture Location: Room No F.27 Constant Temperature Room No.: F.29 Test Vessel Type : 100 mL beaker Test Volume: 50 mL Test Temperature: 25 oC Test Photoperiod: 16 h L: 8 h D Light intensity at surface of Test Vessels: <800 Lux Dilution Water Source: Filtered thiosulphate-treated Sydney mains water with 5% mineral water and conductivity adjusted to 500μS/cm with filtered seawater Conductivity: 500 μS/cm pH: 8.0 Hardness: 100 mg/L as CaCO3 Alkalinity: 50 mg/L as CaCO3

Test Design Concentrations tested: Sample 12071 and 12072: 30 and 100% Sample 12073 and 12074: 10, 30 and 100% Test Concentrations: Nominal Number of replicate test vessels per concentration and control/s: Two for sample treatments Four for control/Reference test Number of animals per replicate: 5 Statistical Methods Statistical Analysis Method: Probit for reference toxicant EC50



EECS Sample

Number


(% sample)






12071 100 0 0 30 0 0

12072 100 0 0 10 0 0 30 0 0 12073 100 0 0 10 0 0 30 0 0 12074 100 0 0

. A test validity criterion of Control group survival (greater than 90% ) was met. Samples 12071-12074: There was no immobilisation of any exposed C. dubia at any tested concentrations of the samples. Table 2. Physico-chemical Variables in Test Solutions

Test Conc.

Temperature (0C)




Diluent 24.6 24.6 8.0 7.9 487 503 97 97 Sample 12071

30 24.0 24.5 8.0 8.0 413 427 99 96 100 24.1 24.4 7.8 8.0 220 246 116 95

Sample 12072 30 24.2 24.4 8.5 8.7 734 753 100 95 100 24.2 24.4 8.7 9.1 1301 1303 116 94

Sample 12073 10 24.0 24.4 8.4 8.5 596 614 99 96 30 24.0 24.4 8.6 8.8 880 899 100 96 100 24.0 24.5 8.8 9.2 1751 1733 116 95

Sample12074 10 24.3 24.2 8.4 8.6 598 610 99 95 30 24.0 24.4 8.6 8.8 814 831 100 95 100 24.1 24.7 8.9 9.2 1552 1533 113 95




Head, Ecotoxicology Testing Services 20 June 2012


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