MTI Analyses of Interactions

To classify the types of all interactions in the combinations examined, MTI values were calculated from the EC50 values of single chemicals and their combinations

3 300

100 0

3 300

100 0

5 10 15 20 25 Incubation time (min)

Figure 5 Inhibition of BLI of V. fischeri in the presence of the mixture, CuSO4 and Ziram. BLI changes in V. fischeri were followed in the presence of the mixture composed from CuSO4 and Ziram as a function of the incubation time. Different concentrations of CuSO4 and Ziram were added by 0.03 and 0.04 (closed circles), 0.05 and 0.06 (open triangles), 0.07 and 0.09 (closed triangles), 0.10 and 0.14 (open squares), and 0.15 and 0.21 mgl_1 (closed squares), respectively. Control samples containing ASW with 1 % (v/v) DMSO are shown as open circles. All the data were obtained from triplicate experiments, averages of which were described.

5 10 15 20 25 Incubation time (min)

Figure 5 Inhibition of BLI of V. fischeri in the presence of the mixture, CuSO4 and Ziram. BLI changes in V. fischeri were followed in the presence of the mixture composed from CuSO4 and Ziram as a function of the incubation time. Different concentrations of CuSO4 and Ziram were added by 0.03 and 0.04 (closed circles), 0.05 and 0.06 (open triangles), 0.07 and 0.09 (closed triangles), 0.10 and 0.14 (open squares), and 0.15 and 0.21 mgl_1 (closed squares), respectively. Control samples containing ASW with 1 % (v/v) DMSO are shown as open circles. All the data were obtained from triplicate experiments, averages of which were described.

Figure 6a, INH(%) values of both Ziram and CuSO4 were quite low when they were present as single forms. By mixing them, however, they increased to as high as 57% and 76% at the incubation times of 15 and 30min, respectively. Similar toxicity enhancement with mixing was observed for the combination of Zn-pt and CuSO4.

CuSO4 Ziram CuSO4 + Ziram Zn-pt CuSO4 + Zn-pt

50 40

CJT 30

CuSO4 Ziram CuSO4 + Ziram Zn-pt CuSO4 + Zn-pt

SEA-NINE 211 TPBP SEA-NINE 211 + TPBP

Diuron

Cu-pt Diuron + Cu-pt

Figure 6 Typical patterns of interactions in some combinations of antifouling chemicals. The INH (%) values were calculated based on the reductions of BLI for single chemicals as well as their combinations at 15 and 30 min of incubations, which are shown by open and shaded bars, respectively. As typical patterns, the combinations of CuSO4 with Zn-pt or with Ziram, SEA-NINE 211 with TPBP, and Diuron with Cu-pt are shown in (a), (b), and (c), respectively. For both single and mixed forms, the concentrations of CuSO4, Zn-pt, Ziram, SEA-NINE 211, TPBP, Diuron, and Cu-pt were fixed to be 0.06, 0.04, 0.08, 0.19, 0.45, 5.00, and 0.10mgl_1, respectively. All the data were obtained from triplicate experiments, averages of which were described with standard deviations.

Diuron

Cu-pt Diuron + Cu-pt

Figure 6 Typical patterns of interactions in some combinations of antifouling chemicals. The INH (%) values were calculated based on the reductions of BLI for single chemicals as well as their combinations at 15 and 30 min of incubations, which are shown by open and shaded bars, respectively. As typical patterns, the combinations of CuSO4 with Zn-pt or with Ziram, SEA-NINE 211 with TPBP, and Diuron with Cu-pt are shown in (a), (b), and (c), respectively. For both single and mixed forms, the concentrations of CuSO4, Zn-pt, Ziram, SEA-NINE 211, TPBP, Diuron, and Cu-pt were fixed to be 0.06, 0.04, 0.08, 0.19, 0.45, 5.00, and 0.10mgl_1, respectively. All the data were obtained from triplicate experiments, averages of which were described with standard deviations.

at 30 min of incubation. As shown in Figure 7, MTI values of the mixed chemicals examined were positive except for the combination of Cu-pt and Diuron, —0.006. As realized from the data shown in Figure 6c, the toxicity of Cu-pt and Diuron in combination was just the same as that of single Cu-pt, suggesting that the addition of Diuron did not bring about significant contribution. This MTI value, however, was almost zero, and thus we can conclude that the interaction of this combination has little additive effect. Other combinations were positive and thus regarded as having no antagonistic effects.

MTI values of the mixtures consisting of CuSO4 with Irgarol 1051, Zn-pt, Ziram, and Cu-pt resulted in >1.73, 2.20, 2.21, and 2.33, respectively (Figure 7). Their MTI values were in good agreement with their synergistic interactions, as indicated by the toxicity comparisons of single and combined chemicals described above. MTI values of 1.2-1.3 were obtained for the following mixtures: DCF with Irgarol 1051, CuSO4 with Diuron, Zn-pt with Ziram, and CuSO4 with SEA-NINE 211. Since MTI values of these combinations were larger than 1, they were regarded as synergistic. The levels of toxicity enhancement, however, were lower than those in the cases of CuSO4-containing combinations as mentioned above. On the other hand, the interactions of IPBC-con-taining mixtures with Diuron, TPBP, or SEA-NINE 211 were classified as concentration additive, since their MTI values were nearly 1. MTI values of other mixtures were 0-1, partially additive, which form the majority of the data shown in Figure 7. A typical example of partially additive is shown in Figure 6b, where the toxicity of combinations became larger than that of either SEA-NINE 211 or TPBP alone, but it was smaller than the sum of their theoretical toxicities of single chemicals.

Among the four chemicals showing marked synergistic effects in the mixture with CuSO4, they commonly contain metal, sulfur, and nitrogen atoms in their molecular structures, except for Irgarol 1051 (see Figure 1). In addition, the metals in three of the chemicals are loosely bounded with sulfide linkage through electron donation. These molecular characteristics should be taken into consideration to clarify the mechanism of these interactions in future investigations.

According to the MTI analyses, there was no antagonistic effect among the present combinations examined. Additive effects were observed in the mixtures containing IPBC with Diuron, TPBP, or SEA-NINE 211. Certain synergistic effects were found in the combinations such as DCF with Irgarol 1051, CuSO4 with Diuron, Zn-pt with Ziram, and CuSO4 with SEA-NINE 211. Significant stimulation of toxicity occurred when Irgarol 1051, Ziram, Zn-pt, or Cu-pt were combined with CuSO4, which is classified as synergistic effects. The other combinations were regarded as partially additive.

0

Irgarol 1051

Diuron

Ziram

Cu-pt

Zn-pt

TPBP

SEA-NINE 211

DCF

CuSO4

IPBC

UDa

>0.333b

>0.557b

>0.785b

UDa

>0.291b

>1.183b

>1.729b

UDa

Irgarol 1051

0.223

-0.006

0.399

0.125

0.591

0.424

1.267

1.000

Diuron

0.858

1.295

0.381

0.562

0.556

2.210

0.758

Ziram

0.568

0.366

0.435

0.602

2.326

0.370

Cu-pt

0.399

0.577

0.570

2.198

0.544

Zn-pt

0.751

0.737

0.839

0.985

TPBP

0.510

1.201

1.078

SEA-NINE 211

0.871

0.308

DCF

0.360

CuSO4

IPBC

Figure 7 MTI values for the mixed chemicals examined. The MTI values are calculated from EC50 values of single chemicals and their mixture at 30min incubations. aUD: undetectable due to the low toxicity of the mixture containing Irgarol 1051. bSincethe EC50 value of Irgarol 1051 was >40 mg l_1, the MTI values were larger than those shown here.

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