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Evaluation of the safety of new anti-fouling agents for use in Australian temperate waters

Final Report for the Department of the Environment and Heritage
S Duda, JH Myers and S Hoffman
Victorian Department of Natural Resources and Environment, 2003


3 Results

3.1 H. banksii

Contents

3.1.1 Physicochemical parameters

Water temperatures varied slightly over the collection period for H. banksii to conduct biocide bioassays and ranged from 11-14.5oC. The pH over the collection period ranged from 8.1 to 8.8. For the majority of the collection period pH variations were small. Salinity was stable over the H. banksii collection period ranging from 35.5-36.0 .

3.1.2 Control Success

The response of H. banksii to each biocide showed a high level of consistency in germination and rhizoid elongation success of controls. Seawater and solvent control survival in each of the five tests undertaken for each biocide met the test acceptability criteria of 85%. Solvent and seawater control germination ranged between 90-100% in all cases. Control rhizoid elongation ranged from 0.30 to 0.60mm. There was no difference in the percent germination and rhizoid elongation of seawater controls and solvent controls therefore both DMSO and methanol were at minimal levels and effects observed at higher concentrations of toxicant were not caused by the presence of these solvents.

3.1.3 Test Precision and Variability

Precision is used to assess the accuracy of EC50 estimates and can be measured through investigating the 95% confidence intervals surrounding the EC50 estimates. Tighter 95% confidence intervals surrounding the test endpoint are an indication of greater test reliability.

3.1.4 TBT

The EC50 estimates for TBTO 48h rhizoid elongation ranged from 0.0002mgL-1 to 0.0005 mgL-1 with a standard deviation of 0.0001mgL-1. The mean EC50 was 0.00034 (0.0001, 0.0006) mgL-1. At 72h rhizoid elongation the mean EC50 was 0.00032 (0.0002, 0.0006) mgL-1. The range of the five test EC50 estimates was from 0.0001mgL-1 to 0.0005mgL-1 with a standard deviation of 0.0002mgL-1 (Table 1). Germination mean EC50 at 48h was 0.00018 (0.00007, 0.0003) mgL-1. The range of EC50 estimates was from 0.0001mgL-1 to 0.0005 mgL-1 and the standard deviation was 0.0002 mgL-1. At 72h germination TBTO EC50 estimates for replicate tests ranged from 0.0001mgL-1 to 0.00025 mgL-1 with a standard deviation of 0.00007 mgL-1. The mean EC50 at 72h germination was 0.00017 (0.00004, 0.0003) mgL-1 (Table 1).

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Table 1: Summary of EC50 estimates (95% CI) and threshold responses (NOEC and LOEC) for five replicate tests for TBTO.
Toxicant Test endpoint Test Number NOEC(mgL-1) LOEC(mgL-1) EC50(mgL-1) 95% confidence intervals
TBTO (mgL-1) 48h Germination 1 0.00001 0.0001 0.0002 0.0002 - 0.0002
    2 0.00001 0.0001 0.00023 0.0002-0.00027
    3 0.00001 0.0001 0.0001 0.0001-.0001
    4 0.00001 0.0001 0.0002 0.0002 - 0.0002
    5 0.00001 0.0001    
             
  72h Germination 1 0.00001 0.0001 0.0002 0.0002 - 0.0002
    2 0.00001 0.0001 0.00025 0.00021 - 0.00029
    3 0.00001 0.0001 0.0001 0.0001-0.0002
    4 0.00001 0.0001 0.0002 0.0002 - 0.0003
    5 0.00001 0.0001 0.0001 0.0001 - 0.0001
             
  48h rhizoid elongation 1 0.000001 0.00001 0.0003 0.0002 - 0.0004
    2 0.0001 0.001 0.0005 0.0003 - 0.0006
    3 0.00000001 0.0000001 0.0004 0.0003 - 0.0004
    4 0.00001 0.00001 0.0003 0.0002 - 0.0004
    5 0.00001 0.0001 0.0002 0 - 0.0003
             
  72h rhizoid elongation 1 0.00001 0.0001 0.0003 0 - 0.0004
    2 0.00000001 0.000001 0.0005 0.0004 0 0.0006
    3 0.00001 0.0001 0.0003 0.0001 - 0.0003
    4 0.00001 0.0001 0.0004 0.0002 - 0.0004
    5 0.00001 0.0001 0.0001 0-0.0002
             
*Test concentrations: 0, 10-8, 10-7, 10-6, 10-5, 10-4, 10-3 mgL-1.

3.1.5 Zineb

The mean EC50 value at the 48h rhizoid elongation endpoint was 2.04 (-0.4, 4.49) mgL-1. EC50 estimates ranged from 0.79mgL-1 to 3.58 mgL-1 zineb with a standard deviation of 1.22 mgL-1. At 72h rhizoid elongation EC50 estimates ranged from 0.47 mgL-1 to 3.87 mgL-1 with a standard deviation of 1.5 mgL-1. The mean EC50 value at this endpoint was 2.12 (-0.88, 5.12) mgL-1 (Table 2). The range of EC50 estimates at the 48h germination endpoint was from 0.66 mgL-1 to 1.19 mgL-1 with a standard deviation of 0.2 mgL-1. The mean EC50 for this endpoint being 0.87 (0.46, 1.28) mgL-1. The mean EC50 at the 72h germination endpoint was 0.49 (0.02, 0.95) mgL-1. The range and standard deviation for EC50 estimates at 72h germination was from 0.3 mgL-1 to 0.87 mgL-1 and 0.23 mgL-1 respectively (Table 2).

Table 2: Summary of EC50 estimates (95% CI) and threshold responses (NOEC and LOEC) for five replicate tests for Zineb.
Toxicant Test endpoint Test Number NOEC(mgL-1) LOEC(mgL-1) EC50(mgL-1) 95% confidence intervals
Zineb (mgL-1) 48h Germination 1 0.1 0.5 0.93 0.86 - 1.02
    2 0.01 0.1 0.82 0.61-1.11
    3 0.1 0.5 1.19 0.88 - 1.75
    4 0.01 0.1 0.75 0.46 - 1.23
    5 0.01 0.1 0.66 0.61 - 2.70
             
  72h Germination 1 0.01 0.1 0.30 0.27 - 0.33
    2 0.1 0.5 0.47 0.43 - 0.52
    3 0.01 0.1 0.49 0.44 - 0.53
    4 0.1 0.5 0.87 0.80 - 0.95
    5 0.1 0.5 0.30 0.27 - 0.33
             
  48h rhizoid elongation 1 0.01 0.1 0.86 0.69 - 2.70
    2 < 0.001 0.001 2.84 2.00 - 3.93
    3 0.01 0.1 2.15 0.28 - 4.49
    4 0.01 0.1 3.58 3.16 - 4.15
    5 0.01 0.1 0.79 0.68 - 1.00
             
  72h rhizoid elongation 1 0.001 0.01 0.47 0.45 - 0.91
    2 0.1 0.5 3.87 2.76 - 5.38
    3 0.5 1.0 2.70 0.62 - 3.92
    4 0.01 0.5 2.93 1.86 - 4.49
    5 0.001 0.01 0.63 0.63 - 1.47
             
*Test concentrations: 0, 0.001, 0.01, 0.1, 0.5, 1.0, 10.0 mgL-1.

3.1.6 Diuron

The mean EC50 for diuron at 48h rhizoid elongation was 6.75 (5.53, 7.96) mgL-1. The range in EC50 estimates at this endpoint was from 5.93 mgL-1 to 7.59 mgL-1 with a standard deviation of 0.61mgL-1. At 72h rhizoid elongation the EC50 estimates ranged from 6.35 mgL-1 to 8.59 mgL-1 with a standard deviation of 0.9 mgL-1. The mean EC50 was 7.27 (5.47, 9.08) mgL-1 (Table 3). Diuron EC50EC50 estimates at the 48h germination endpoint ranged 5.93 mgL-1 to 6.83 mgL-1 with a standard deviation of 0.35 mgL-1. The mean EC50 at this endpoint was 6.29 (5.59, 6.99) mgL-1. Germination at 72h had a mean EC50 of 6.86 (5.33, 8.39) mgL-1. The range of EC50 estimates was from 5.98 mgL-1 to 7.96 mgL-1 with a standard deviation of 0.77 mgL-1 (Table 3).

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Table 3: Summary of EC50 estimates (95% CI) and threshold responses (NOEC and LOEC) for five replicate tests for Diuron.
Toxicant Test endpoint Test Number NOEC(mgL-1) LOEC(mgL-1) EC50(mgL-1) 95% confidence intervals
Diuron (mgL-1) 48h Germination 1 2.5 5.0 6.11 5.64 - 6.55
    2 2.5 5.0 6.16 6.02 - 6.30
    3 2.5 5.0 6.83 6.19 - 7.35
    4 2.5 5.0 5.93 5.77 - 6.09
    5 2.5 5.0 6.43 6.28 - 6.58
             
  72h Germination 1 5 7.5 6.42 5.75 - 6.91
    2 2.5 5.0 6.71 6.54 - 6.86
    3 < 0.5 0.5 7.96 7.58 - 8.34
    4 2.5 5.0 5.98 5.85 - 6.12
    5 2.5 5.0 7.24 7.07 - 7.40
             
  48h rhizoid elongation 1 5.0 7.5 6.63 5.89 - 7.14
    2 1.0 2.5 6.98 6.63 - 7.78
    3 2.5 5.0 7.59 7.19 - 8.25
    4 2.5 5.0 5.93 5.67 - 6.17
    5 1.0 2.5 6.60 6.44 - 7.06
             
  72h rhizoid elongation 1 2.5 5.0 6.51 6.01 - 6.93
    2 5.0 7.5 7.40 7.16 - 7.66
    3 5.0 7.5 8.59 8.25 - 8.88
    4 5.0 7.5 6.35 6.06 - 6.97
    5 5.0 7.5 7.52 7.16 - 8.24
             
*Test concentrations: 0, 0.5, 1.0, 2.5, 5.0, 7.5, 10.0 mgL-1.

3.1.7 ZPT

The mean EC50 for ZPT at 48h rhizoid elongation was 0.31 (0.17, 0.45) mgL-1. The EC50 estimates ranged from 0.21mgL-1 to 0.38 mgL-1 with a standard deviation of 0.07 mgL-1. At 72h rhizoid elongation EC50 estimates for the 5 tests ranged from 0.15 mgL-1 to 0.36 mgL-1 with a standard deviation of 0.08 mgL-1. The mean EC50 was 0.24 (0.09, 0.40) mgL-1 (Table 4). Forty-eight hour germination EC50 estimates had a range of 0.16 mgL-1 to 0.24 mgL-1 and a standard deviation of 0.04mgL-1. The mean EC50 at this endpoint was 0.21 (0.13, 0.29) mgL-1. At 72h germination the mean EC50 was 0.19 (0.07, 0.3) mgL-1. The range of EC50 estimates over the 5 tests was from 0.12 mgL-1 to 0.28 mgL-1 and the standard deviation 0.06 mgL-1 (Table 4).

Table 4: Summary of EC50 estimates (95% CI) and threshold responses (NOEC and LOEC) for five replicate tests for ZPT.
Toxicant Test endpoint Test Number NOEC(mgL-1) LOEC(mgL-1) EC50(mgL-1) 95% confidence intervals
ZPT (mgL-1) 48h Germination 1 0.05 0.15 0.18 0.17 - 0.20
    2 0.05 0.15 0.22 0.21 - 0.24
    3 0.05 0.15 0.24 0.20 - 0.28
    4 0.05 0.15 0.25 0.23 - 0.26
    5 0.008 0.05 0.16 0.15 - 0.17
             
  72h Germination 1 0.05 0.15 0.18 0.17 - 0.19
    2 0.05 0.15 0.18 0.17 - 0.19
    3 0.001 0.01 0.28 0.24 - 0.32
    4 0.05 0.15 0.18 0.17 - 0.19
    5 0.05 0.15 0.12 0.11 - 0.12
             
  48h rhizoid elongation 1 0.001 0.01 0.26 0.15 - 0.40
    2 0.05 0.15 0.34 0.23 - 0.44
    3 0.05 0.15 0.36 0.25 - 0.47
    4 0.05 0.15 0.38 0.35 - 0.45
    5 0.05 0.15 0.21 0.12 - 0.27
             
  72h rhizoid elongation 1 0.01 0.05 0.26 0.24 - 0.32
    2 0.001 0.01 0.21 0.16 - 0.31
    3 0.05 0.15 0.36 0.27 - 0.43
    4 < 0.0005 0.0005 0.24 0.29 - 0.38
    5 0.05 0.15 0.15 0.13 - 0.24
             
*Test concentrations: 0, 0.0005, 0.008, 0.05, 0.15, 0.5, 1.0 mgL-1.

3.1.8 Seanine 211

Seanine 211 mean EC50 at 48h rhizoid elongation was 0.43 (0.17, 0.7) mgL-1. The range in EC50 estimates was from 0.29 mgL-1 to 0.65 mgL-1 with a standard deviation of 0.13 mgL-1. At 72h rhizoid elongation the mean EC50 was 0.46 (0.27, 0.69) mgL-1. Ranges of EC50 estimates were from 0.34 mgL-1 to 0.58 mgL-1 and the standard deviation was 0.11 mgL-1 (Table 5). Mean EC50s at 48h germination and 72h germination were 0.34 (0.21, 0.47) mgL-1 and 0.42 (0.22, 0.62) mgL-1 respectively. Ranges of EC50 estimates at 48h germination was from 0.28 mgL-1 to 0.44 mgL-1 with a standard deviation of 0.06 mgL-1. At 72h germination EC50s ranged from 0.29 mgL-1 to 0.55 mgL-1 with a standard deviation of 0.10 mgL-1 (table 5)

Table 5: Summary of EC50 estimates (95% CI) and threshold responses (NOEC and LOEC) for five replicate tests for Seanine 211.
Toxicant Test endpoint Test Number NOEC(mgL-1) LOEC(mgL-1) EC50(mgL-1) 95% confidence intervals
Seanine 211 (mgL-1) 48h Germination 1 0.25 0.50 0.32 0.31 - 0.34
    2 0.005 0.04 0.30 0.27 - 0.31
    3 0.25 0.50 0.36 0.35 - 0.38
    4 0.50 1.00 0.44 0.42 - 0.46
    5 0.04 0.25 0.28 0.02 - 0.50
             
  72h Germination 1 0.25 0.50 0.45 0.41 - 0.49
    2 0.04 0.25 0.29 0.27 - 0.31
    3 0.04 0.25 0.35 0.33 - 0.36
    4 0.25 0.50 0.44 0.42 - 0.46
    5 0.25 0.50 0.55 0.53 - 0.57
             
  48h rhizoid elongation 1 0.25 0.50 0.41 0.35 - 0.53
    2 0.04 0.25 0.40 0.34 - 0.48
    3 0.25 0.50 0.41 0.34 - 0.49
    4 0.25 0.50 0.65 0.56 - 0.74
    5 0.04 0.25 0.29 0.09 - 0.68
             
  72h rhizoid elongation 1 0.5 1.0 0.58 0.35 - 0.75
    2 0.001 0.005 0.34 0.30 - 0.39
    3 0.25 0.50 0.39 0.36 - 0.45
    4 0.04 0.25 0.55 0.48 - 0.59
    5 0.001 0.01 0.54 0.42 - 0.65
             
*Test concentrations: 0, 0.001, 0.005, 0.04, 0.25, 0.5, 1.0 mgL-1.

3.1.9 Evaluation of H. banksii Sensitivity to the biocides

The sensitivity of H. banksii to each of the biocides at both 48h germination and 48h rhizoid elongation was as follows:
TBTO > ZPT > seanine 211 > zineb > diuron

3.2 C. insidiosum

The EC50 values for TBT ranged from 0.035 mgL-1 (0.028 - 0.044), NOEC and LOEC values 0.005 and 0.01 mgl-1 respectively, to 0.083 mgL-1 (0.057-0.149), NOEC and LOEC values 0.005 and 0.01 mgl-1 respectively, indicating that it was highly toxic to the amphipods (Table 6).

The two compounds ZPT and Seanine 211 showed moderate toxicity to C. insidiosum. Exposures to ZPT resulted in EC50 values ranging from 0.22 mgL-1 (0.15-0.35), NOEC and LOEC values 0.01 and 0.05 mgl-1 respectively, to 0.36 mgL-1 (0.26-0.52), NOEC and LOEC values 0.01 and 0.05 mgl-1 respectively. The EC50 values for Seanine 211, ranged from 0.27mgL-1 (-), NOEC and LOEC values 0.05 and 0.1 mgl-1 respectively, to 0.37 mgL-1 (-), NOEC and LOEC values 0.05 and 0.1 mgl-1 respectively (Table 6).

Zineb demonstrated lower toxicity to C. insidiosum with EC50 values ranging from 0.59 mgL-1 (0.43-0.81), NOEC and LOEC values 0.01 and 0.1 mgl-1 respectively, to 1.10 mgL-1 (0.70-1.69) NOEC and LOEC values 0.1 and 1.0 mgl-1 respectively (Table 6).

Diuron was the least toxic out of all biocides examined with EC50 values ranging from 2.02 mgL-1 (-) NOEC and LOEC values 0.5 and 2.0 mgl-1 respectively to 3.0 mgL-1 (-), NOEC and LOEC values 0.5 and 2.0 mgl-1 respectively (Table 6).

3.2.1 Evaluation of C. insidiosum Sensitivity to the biocides

The sensitivity of C. insidiosum to each of the biocides was as follows:
TBTO > ZPT > seanine 211 > zineb > diuron

Table 6: Summary of EC50 estimates (95% CI) and threshold responses (NOEC and LOEC) for three replicate tests for effects of antifouling biocides on C. insidiosum.
Toxicant (mgL-1) Test Number NOEC(mgL-1) LOEC(mgL-1) EC50(mgL-1) 95% confidence intervals
TBTO 1 0.025 0.05 0.047 0.040-0.054
  2 0.005 0.01 0.083 0.057-0.149
  3 0.005 0.01 0.035 0.028-0.044
           
Seanine 211 1 0.05 0.1 0.27 -
  2 0.05 0.1 0.37 -
  3 0.05 0.1 0.35 -
           
ZPT 1 0.01 0.05 0.22 0.15-0.35
  2 0.1 0.18 0.28 0.22-0.34
  3 0.01 0.05 0.36 0.26-0.52
           
Zineb 1 0.01 0.1 0.59 0.43-0.81
  2 0.1 1.0 1.10 0.70-1.69
  3 0.01 0.1 1.07 -
           
Diuron 1 0.5 2.0 2.02 -
  2 0.5 2.0 3.0 -
  3 0.5 2.0 2.99 2.72-3.23
           

3.3 Mussels and Fish

The chronic 6 week sublethal exposure for Sand flathead, Platycephalus bassensis was terminated due to problems encountered with the maintenance of Sand flathead in tanks at the MAFRI. The condition of wild caught animals was not optimal. Fish would not feed and mortalities were occurring in flow through tanks. There were no mortalities encountered due to the biocides in either the A. butcherii or M. edulis exposures. This indicates that at the concentrations used for exposures, the biocides were not toxic to either A. butcherii or M.edulis. It was anticipated that sublethal parameters would be undertaken on these two species due to their tolerance of the biocides, however, due to unforseen circumstances and mechanical failure of the refrigeration unit containing samples, this was not possible.