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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


4 Discussion

There have been few studies investigating the effects of antifouling biocides to Australian macroalgal, invertebrate and vertebrate species. The results presented here for H. banksii suggest that this assay offers a relatively reproducible mechanism for assessing the toxicity of biocidal agents assayed.

Each of the biocides were significantly less toxic to H. banksii than TBTO. At 72h germination seanine 211 and zineb expressed the same toxicity to H. banksii and were the third most toxic compounds to H. banksii at this endpoint. Seanine 211 and ZPT expressed the same toxicity to H. banksii at 72h rhizoid elongation, being the second most toxic biocides to H. banksii following the toxicity of TBTO.

The ranges on sensitivity of H. banksii at both 48h and 72h rhizoid elongation endpoints observed during this study to TBTO were consistent with studies on marine species including P. comosa, Allorchesles compressa Dana, diatoms, fish, crustaceans and daphnia magna where sensitivity has been recorded in the 10-6 mgL-1 range.

The mean EC50 values for germination inhibition and rhizoid elongation of H. banksii using seanine 211 in this study are consistent with documented results for growth, germination and settlement inhibition for a range of other algal species and benthic organisms. Seanine 211 has been documented as having 'minimum inhibitory concentrations' for growth of freshwater algae (Chlorella, Chorococcum, Scenedesmus and Ulothrix) and blue green algae (Anabaena, Synechoccus, Nostoc, Scytonema, Microcystis and Oscillatoria) at concentrations below 1.3 mgL-1, and an LC50 of 0.34 mgL-1 was documented for the barnacle Balanus amphrite (Rohm and Haas 1992). The inhibition of spore germination has also been reported for two common marine algae, Enteromorpha intestinalis and Ectocarpus siliculosis, at concentrations of 0.1mgL-1 and 0.2 mgL-1 respectively (Rohm and Haas 1992). Jacobson and Willingham (2000) reported an LD50 for the diatom Amphora coffeaeformis at 0.003 mgL-1 and Fernandez Alba et al (2002) reported EC50 values for the crustacean D. magna, microalgae S. capricornotum and the bacteria V. ficheri at concentrations of 0.0004 mgL-1, 0.0003 mgL-1 and 0.0015mgL-1 respectively, which were slightly lower than those observed for H. banksii in the present study.

There is very little information on the toxicity of ZPT and zineb. Van Leeuwen et al. (1985) reported the effects of dithiocarbamates (zineb) to a range of aquatic species including the guppie, P. reticulata, D. magna, the green alga, C. pyrenoidosa, and three bacteria, P. phosphoreum, Nitrosomonas/Nitrobacter, in acute bioassays ranging from 15mins for the bacteria to 3 hours for the two nitrifying bacteria. The reported EC50 value for the algae to zineb was 1.8 mgL-1 and LC50 for D. magna 0.97 (0.56-1.80) mgL-1 which are similar to the results found for H. banksii in the present study following exposure to zineb. Results for the guppie, and three bacteria in Van Leeuwen et al (1985) study ranged 4.8-8.0 mgL-1 for P. phosphoreum to 18 mgL-1 for the nitrifying bacteria. These later values were higher than the mean EC50 values observed for germination and rhizoid elongation for H. banksii in the present study.

There were no results found for the effects of ZPT to other algal species, however Goka (1999) reported significant teratogenic effects on larvae of the zebra fish and Japanese meduka at 0.3 mgL-1 and 0.1mgL-1, and 0.03 mgL-1 and 0.01mgL-1 for each species respectively. Ninety-six hour LC50 values for the Bluegill sunfish and Rainbow trout have also been reported at 0.01mgL-1 and 0.003 mgL-1 (Arch chemicals 1996), which are slightly lower than the results observed for ZPT in the present study.

Diuron required a higher concentration to achieve the EC50 for germination and rhizoid elongation compared to the four other biocides. The toxicity of diuron has been reported by a number of authors to various aquatic organisms. In an investigation of the effects of three antifoulant paint biocides TBT, seanine 211 and diuron, Fernandez Alba et al. (2002) found that diuron was the least toxic of the three compounds to a crustacean, zooplankton, bacterium and algae. LOEC values were reported as 23 mgL-1 for the bacterium V. fischeri and 3.5 mgL-1 for the crustacean D. magna (Fernandez Alba et al. 2002). These results were relatively higher for the bacterium and lower for the crustacean than those observed in this study for diuron. Haynes et al. (2000b) reported concentrations of 0.002 mgL-1, 0.01-6.17 mgL-1 and 0.01mgL-1 to cause reduced photosynthesis in marine periphyton, reduction in growth of marine phytoplankton and inhibition of photosynthesis of seagrass respectively. The 48h EC50 for Undaria pinnatifida germination was reported as 23.5 mgL-1 by Gorski and Burridge (in prep.) which was also relatively higher than the sensitivity of H. banksii germination observed in the present study (mean EC50 values for 48h and 72h germination were 6.29 (5.59, 6.99) mgL-1 and 6.86 (5.33, 8.39) mgL-1 respectively).

The mechanisms for toxicity of each of the biocides towards H. banksii were not investigated in this study and are uncertain. However, mechanisms of toxicity towards other plant and algal species have been documented for some of the biocides and it is hypothesized that similar mechanisms of toxicity could be occurring in H. banksii.

Diuron's toxic action to plant photosynthesis is well understood, being reported to affect the photosynthetic electron transport system (PET) of photosystem II (PSII) in the photosynthetic process of plants (Haynes et al. 2000a; Haynes et al. 2000b). The herbicide inhibits the photoreduction side of PSII by binding with high affinity at the QB-binding site of the PSII photosynthetic complex and preventing QB from binding at this location. Exclusion of the QB from its binding site blocks electron transport from QA to QB, therefore limiting electron flow in PSII (Dayan et al. 2000; Haynes et al. 2000a; Haynes et al. 2000b). Evidence has shown germination and elongation of the rhizoid is a function of the spore's ability to photosynthesize. If PET is inhibited then the plant cannot photosynthesize and therefore may not have the energy requirements to germinate show elongation of the rhizoid (Anderson and Hunt 1988).

Van Leeuwen et al. (1985) concluded that the actions of zineb to inhibit algal growth was by means of inhibiting carbon fixation and noted that over a broad range of concentrations photosynthesis and respiration inhibition occurred. Zineb is a member of the dithiocarbamates, which are pesticides that posses insecticidal properties and have been used since 1934 to control a number of species belonging to taxonomically different groups including bacteria, fungi, nematodes and molluscs (Van Leeuwen et al. 1985). The mechanisms of action observed by Van Leeuwen et al. (1985) of inhibition of photosynthesis by zineb may be caused by the same actions as diuron and this may have been how these two biocides caused inhibition of germination and rhizoid elongation observed in H. banksii during the present study. The differences in mean EC50 values between diuron and zineb suggest that zineb has a greater mechanism of toxic action to H. banksii, even more so at the germination endpoint than the rhizoid elongation endpoint. This may mean that zineb posses other mechanisms of toxicity, but these are unknown.

TBT has been implicated as a respiratory uncouplar through its ability to inhibit the production and metabolism of ATP. Polarization and initial rhizoid elongation of the zygote involves high respiratory demand and uncoupling of oxidative phosporlyation may restrict germination through inhibition of the development of the apico-basal axis associated with normal development and the adhesion to substratum. In the absence of polarization and normal development, embryos may simply become nonviable (Burridge et al. 1995; Burridge et al. 1996). The inhibition of ATP production would also affect photosynthesis output. The inhibition of respiratory and photosynthetic capacity together may have a greater affect on settlement and germination. The mechanisms of seanine 211 have been described to be similar to that for TBT (Gorski and Burridge in prep.).

Mechanisms for toxicity of ZPT have primarily been investigated in mammals. It has been reported that this biocide inhibits the membrane transport system in fungi and has been found to inhibit cell growth in mammals (Evans et al. 2000). This biocide may effect the movement of cations across cell membranes by disrupting membrane integrity (Anderson and Hunt 1988; Anderson et al. 1990; Burridge et al. 1999).

The fact that zineb at the rhizoid elongation endpoint and diuron at all endpoints require relatively higher concentrations to achieve 50% effect to H. banksii gametes, suggests that these compounds would be the best substitutes, out of the four assayed, in comparison to TBT. The fact that ZPT and seanine 211 were much closer in toxicity to TBT, in comparison to diuron and zineb, suggests that their use may need to be more limited and further investigations of their toxicity towards a wider range of aquatic organisms should be undertaken.

The mechanisms of toxicity of the biocides studied are not well understood and literature is very limited on their toxicity and partitioning in the aquatic environment. If we release these new antifouling biocides studied into the environment, then their unknown effects may cause detrimental affects to aquatic organisms and cause marine pollution similar to that observed for TBT presently. The antifouling biocide Irgarol 1051 is a good example of the problems caused when compounds are released into the environment with very little information regarding their potential effects to the receiving environment being known. Irgarol 1051 is presently not registered for use in Australia and has been reported in Queensland coastal environments at potentially toxic levels. The presence of this compound in Queensland at high levels indicates that it could be present in other Australian coastal environments (Scarlett et al. 1999).

Based on short-term environmental exposure to C. insidiosum, Diuron or Zineb would appear to be the safest biocides. This study however, did not take the relative environmental persistence of the biocides into account. Both TBT and Diuron have been found in detectable concentrations (2.19 x10-3 mgL-1 for Diuron and 7.459 ngL-1 for TBT) in marinas and harbours throughout the world (Martinez et al. 2002 and; Thomas et al. 2000; Evans et al. 1995; Alzieu et al. 1986 and; Mensink et al.1997; Nias et al. 1993; Regolia et al. 2001; Tolsa et al. 1996 and; Waite et al. 1991).

Martinez et al. 2002, who detected diuron on the Spanish Mediterranean coast, also found concentrations of Seanine 211 (up to 3.7 x10-3 mgL-1). This compound was one of the more toxic of the biocides that were assessed in this investigation, fitting in the moderately toxic category. Willingham, 2000 has determined the half-life of Seanine 211 to be up to 24hr in natural seawater and less than 1hr in a seawater microcosm.

Due to its moderate acute toxicity, ZPT shows no clear advantage over TBT in this assessment with regard to toxicity to non-target organisms. What ZPT has in its favour is relatively non-toxic metabolites and rapid degradation rates, in the order of hours (half-life = 2-22hrs) (Turley et al., 2000). It has been suggested by Turley et al., 2000 that this occurs through photodegradation and/or biodegradation. One aspect of concern also demonstrated by Turley et al., 2000, is ZPTs slower degradation rate in the absence of light. This could increase toxic effects due to environmental persistence in buried sediments or turbid waters (Thomas et al. 2002).

Zineb had a relatively low toxicity to C.insidiosum compared to the other biocides. This is consistent with other observations that have been reported. It is rapidly excreted from rainbow trout Salmo gairdneri once exposure has occurred (Van Leuween et al. 1986). Zineb's relatively low toxicity has been shown in the freshwater Cladoceran, D. magna, 48hr EC50 0.97 mgL-1 (Van Leuween et al. 1985).

Aside from the investigations by Van Leeuwen et al. (1985-1986), there is little available information on the toxicity of Zineb, in particular, to marine organisms. Characteristics that may interfere with the bioavailability of the biocides to organisms include; adsorption to substrate, this includes amphipod carapace (Jacobson et al. 1993) and rapid degradation (Turley et al. 2000).

The expected outcomes from the chronic sublethal exposures of the biocides to M. edulis, A. butcherii and P. bassensis would include those found by previous studies.

Zineb

Past studies have been conducted by Van Leeuwen et al. 1986, using rainbow trout Salmo gairdneri to investigate the uptake, distribution and retention of Zineb. Using 14C-labled Zineb they found that the compound was excreted to the point of non-detection by the end of 4 days. Other studies conducted by Van Leeuwen et al. 1985-6 investigated teratogenicity, histopathology and embryo-larval studies with S. gairdneri, of which no significant effects were detected. There was no published data obtained on the effect of Zineb on molluscs.

Diuron

Studies on fish using Diuron have included uptake and elimination of the compound in S. gairdneri and flathead minnows (Call et al. 1987). This investigation studied aspects of lethal concentration in flathead minnow eggs and fry. The elimination of Diuron was undertaken on S. gairdneri and adult flathead minnows. 90% elimination occurred after 24hrs. Okamura et al. 2002, conducted a 28 day EC50 using Diuron on juvenile S. gairdneri. The EC50 value obtained was the least sensitive response at 0.23mgL-1.

ZPT

ZPT has been studied relatively well. It is an active ingredient in anti-dandruff shampoos and has been widely used for over 20 years (Shuster, 1984). An assessment by Goka, 1996 used the eggs and larval stages of the zebra fish, Brachydanio rerio, and Japanese Medaka, Oryzias latipes. Significant Teratogenic results were observed on the larvae of both the zebra fish and the Japanese Medaka at concentrations as low as 0.01mgL-1.

Another assessment, was conducted by Okamura et al. 2002, and used a variety of antifouling compounds including ZPT on juvenile S. gairdneri and suspension cultured fish cell from chinook salmon, Oncorhynchus tshawytshca. This produced a 28 day LC50 of 0.0046 mgL-1 and an EC50 of 0.18 mgL-1 on S. gairdneri and O. tshawytshca respectively.

Seanine 211

The toxicity of Seanine 211 has been assessed using teleost fish. A 28 day LC50 of 0.014 mgL-1 has been determined by Okamura et al. (2000). This study was conducted using juvenile S. gairdnerii and verified that Seanine 211 was less toxic than ZPT by a factor of 10 (0.0046 mgL-1). Another study conducted by Willingham and Jacobson, (1993) reported results of a 49 day bioconcentration exposure on the bluegill sunfish, which demonstrated that bioaccumulation of Seanine 211 was essentially nil.

TBT

The effects of TBT are well documented throughout the world (Evans et al. 1995). Most effects have been documented using molluscs as they seem to be the most sensitive to any TBT presence. Some of the side-effects have been observed to be; imposex in gastropods (Evans et al. 1995), shell thinning in oysters (Alzieu et al. 1981-82), inhibition of growth (Chargot et al. 1990) and bioaccumulation (Goulan and Yong 1995).

Many studies have been conducted using molluscs but few on teleost fish. The gill ATPase activities were tested, after exposure to TBT, on the estuarine fish, striped bass, Morone saxatilis, and mummichog, Fundulus heteroclitus (Punkney et al. 1989). This study demonstrated that TBT places stress on the ionic and osmotic regulatory systems of estuarine species.