fungicide in leather

8/13/2019 Fungicide in Leather

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The toxicity of leather tannery effluent to apopulation of the blue musselMytilus edulis(L.)

A N D R E W R . W A L S H 3 and JOHN O HALLORAN

Depa rtme nt of Zool ogy and Ani mal Eco logy, Lee Malt ings , Prosp ect Row, Univ ers ity Col lege Cork , Irela nd

Received 24 July 1995; accepted 17 January 1996

The toxicity of leather tannery effluent affecting a population ofMytilus edulis in the Colligan estuary,

Ireland was investigated. At the whole animal level, the growth, condition and chromium

concentrations were measured in transplanted, local and control mussels. The fitness of the mussels

was assessed by their tolerance to aerial exposure. At the cellular level, the degree of lipid

peroxidation was measured in the tissues of field sampled mussels. In addition, mussels were exposed

in the laboratory to components of the effluent thought likely to enduce peroxidation, i.e. Cr(VI),

Cr(III)protein and the fungicide, Busan 30WB containing 2-(thiocyanomethylthio) benzothiazole

(TCMTB). The growth, condition and chromium concentrations of transplanted mussels were

significantly higher than controls after 1 year of exposure. The fitness of mussels at most sites in the

Colligan estuary was comparable to the controls except those closest to the tannery outfall. At the

cellular level, lipid peroxidation was demonstrated in the digestive cells of field sampled mussels,

while in laboratory exposures Busan 30WB was found to enduce lipid peroxidation in the digestive

gland and amoebocyte proliferation in the gill. Cr(VI) and Cr(III)protein exposure, by comparison,

gave a negative peroxidative response. The results indicate that the enhanced growth and condition

seen may be due to the high nutritive content of the effluent while the lipid peroxidation observed was

reasoned to be a result of fungicide exposure. Chromium toxicity, however, could not be detected,

although such an effect could not be ruled out.

Keywords: chromium; Busan 30WB

; benzothiazole; TCMTB; tannery effluent; Mytilus edulis; lipid

peroxidation.

Introduction

The present study set out to assess the toxicity of leather tannery discharges to apopulation of mussels in a well-mixed estuary by paying particular attention topotentially toxic and persistent components identified previously in the effluent of thetannery under study (Walsh and OHalloran, in press). In that study, the speciation andtransformations of Cr(III), in particular, were followed as this effluent component has

been identified as being of particular environmental concern due to the possibility ofoxidation and/or direct toxicity (e.g. USEPA, 1979; WHO, 1988; UNEP/IEO, 1991). Inwaters receiving leather tannery waste, aquatic animals have been found to accumulatechromium to very high concentrations in their tissues (e.g. Aislabie and Loutit, 1986;

Neill, 1991;Walsh et al., 1994a). These accumulations have been linkedto the enhancedbioavailability of Cr(III)protein complexes in the effluent(Walsh and OHalloran, 1996)

Ecotoxicology6, 137152 (1997)

09639292 1997 Chapman & Hall

3 To whom correspondence should be addressed.


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and as a result it is questioned whether this increased bioavailability leads to a parallelincrease in toxicity?

Alternatively, the fungicide Busan 30WB , containing the active ingredient 2-(thiocyanomethylthio)-benzothiazole (TCMTB), which is widely used by the industry(Reemtsma et al., 1995), was also considered to be potentially toxic to mussels as itwas previously found to be lethal to zebra mussels Dreissena polymorpha at aconcentration of 500 g l (Martin et al., 1993) and to cause gill damage in salmonidsat concentrations as low as 6 g l (Nikl and Farrell, 1993).

On initial inspection of the ecology of the receiving estuary, a direct lethal effect ofthe effluent to Mytilus edulis appeared to be unlikely as large mussel beds could befound within 200 m of the tannery outfall, in direct contact with the effluent plumeduring discharge. As a result it was decided to concentrate on the chronic effects of thedischarges to the health of mussels in the area. Therefore, the accumulation ofchromium was measured in local mussels and in mussels transplanted into the estuaryfrom a clean site. TCMTB concentrations were not measured as the development ofanalytical methods for this substance was outside the scope of the study. Biologicalindices of the growth and condition were also measured in the transplanted mussels andthe physiological fitness of local and transplanted mussels was assessed. At thesubcellular level, lipid peroxidation was measured in the gill and digestive gland offield sampled mussels and in mussels subjected to conditions of prolonged thermal

stress. This approach was chosen as lipid peroxidation is a common product of organicand inorganic xenobiotic exposure in marine molluscs (Viarengo, 1985; Livingstone etal., 1990; Moore, 1991). In addition, laboratory exposure to Cr(VI), Cr(III)protein andBusan 30WB

was conducted for comparative purposes.

Materials and methods

Study site

The river Colligan enters the sea through the town of Dungarvan on the south coast ofIreland. The tannery is situated near the mouth of the estuary and a composite effluentfrom the various stages of leather production is discharged on each ebb tide (336 m perday and 685 kg Cr per day) (Neill, 1989, 1991). As judged by the sediment totalchromium concentrations corrected for grain size, particulate effluent components aredeposited both upstream and downstream of the emission point (Neill, 1991).The estuaryof the Cork Blackwater was chosen as the control site as previous work found it tocontain background concentrations of chromium (Walsh et al., 1994a).

Transplant experiment

Mussels growing on an uncontaminated exposed rocky shore with a mean shell length of24.2 mm were collected in February 1993 and divided into five subsamples consisting of100 individuals in each group. They were subsequently transplanted in oyster spat bags tothe control Blackwater estuary site K1 and Colligan estuary sites C1, C2, C3 and C4 at a

position 1.1 m above chart datum (CD), to ensure that the degree of aerial exposureduring the tidal cycle would be similar. Sites C1 and C2 were upstream relative to thedischarge, while sites C3 and C4 were downstream. The experiment was conducted over

a period of 12 months. The shell length was monitored quarterly and the total chromiumand condition indices were analysed at the end of the exposure period. The condition

138 Walsh and OHalloran


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index ((dry tissue weight/shell weight) 2 1000) was used as described by Lawerence andScott (1982).

Chromium analysis

Chromium analyses were conducted as described byWalsh and OHalloran (1996) and allreagents used in the analysis were AnalaR grade obtained from BDH chemicals. Standardreference oyster tissue (SRM 1566a) was also prepared and analysed as above to validatethe method.

Survival in air

A modification of the survival in air technique described byEertmann et al. (1993) wasemployed in this study to assess the physiological fitness of mussels in the Colliganestuary. This involved the use of intertidal mussels as opposed to the sublittoral musselsdescribed. In November 1993 therefore, mussels with a shell length range of between 57and 64 mm were sampled intertidally from the Blackwater estuary. A control group of 35mussels was placed in an oyster bag at a position 1.1 m CD at the control site. Fourfurther samples of 35 mussels each were transplanted to sites C1, C2, C3 and C4 in theColligan estuary, again on trestles at a position 1.1 m CD. Local mussels of a similarshell length distribution were also collected and placed on trestles at these sites. Themussels were then left in position for a period of 3 months. In March 1994 the mussels

were collected and transported to the laboratory where they were kept at 138C inrecirculating 100% seawater to depurate for 72 h. A subsample of five mussels from eachgroup was removed for chromium analysis of the gill, digestive gland, kidney, mantle andadductor mucle as described above. The remainder were placed in a constant temperaturechamber under air at 188C and mortalities were recorded twice a day until all themussels had died. Death was determined by shell gape and a lack of response tomechanical stimulus.

Lipid peroxidation

Mussels in the shell length range 5764 mm were collected from the control site andfrom site C4 in the Colligan estuary in April 1994. On return to the laboratory, themussels were depurated in recirculating 100% seawater for 72 h at 138C. Twenty musselsfrom each group were initially subsampled and their digestive glands and gills wereremoved and immediately frozen in liquid nitrogen. The remaining mussels were placedin an incubator under air at 28 8C. After 12 and 24 h, respectively, mussels from eachgroup were subsampled as before. The remaining mussels were then returned torecirculating seawater at 138C. Further samples were taken after 12 and 24 h recovery.The sampled tissues were stored at 708C prior to analysis.

The degree of lipid peroxidation was assessed by measuring the concentration of thesoluble peroxidative product malondialdhyde (MDA) in the cytosol of the sampledtissues. The method involved the homogenization of 1.52 g of tissue pooled from fourto six individuals in 10 ml of homogenization media using an ultraturax homogenizer.The media contained 0.4 M KCl, 1.00 mM EDTA and 1.00 mM dl-dithiothreitol (DTT) in

phosphate buffer at pH 7.2. The concentration of MDA was subsequently determinedspectrophotometrically in the supernatant after centrifugation at 30 000 g for 1 h

according to the method of Wills (1987). The coloured product was extracted into n-butanol prior to analysis. Concentrations were expressed as nM MDA g dry weight of

Toxicity of tannery effluent to mussels 139


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tissue. The dry weight of the tissues was calculated from a wet weight/dry weightconversion factor determined for a subsample of tissues taken at each sampling period.Expression in terms of dry weight was preferred as mussels from different environmentswere found to have considerable variability in the wet weight of tissues and cytosolic

protein, the two most commonly employed parameters used to express concentrations ofsoluble cellular components (P.J. Fitzpatrick, A.R. Walsh, D. Sheehan and J. OHalloranin preparation).

To assess the long-term effects of tannery effluent exposure on lipid peroxidation, the

degree of lipofuscin accumulation in the gill and digestive gland of the mussels at thevarious sites in the Colligan estuary was determined histochemically and compared tothe controls. Therefore, the digestive gland and gill tissue from the mussels from thecontaminated and control sites were excised, fixed in Bakers formal saline and paraffinwax embedded. Sections (10 m) of the tissues were stained using the Schmorlsreaction as described by Pearse (1985).

Laboratory exposure

Mussels in the length range 4050 mm were collected from the control site and dividedinto seven groups of ten individuals and placed in 40 l aquaria with 100% seawater.Cr(III)albumin was added at a concentration of 0.1 and 1.0 mg Cr l in the first twotanks. This was prepared by reacting bovine serum albumin with CrCl

.6H

O at a 10:1ratio at pH 4.5 for 6 h. The pH was then adjusted to 8.1 followed by filtration to removeany non-complexed chromium. Busan 30WB was obtained from a commercial batch atthe tannery and was added at a concentration of 0.05 and 0.5 mg TCMTB l in tanks 3and 4 (the commercial preparation comes as a 30% TCMTB solution). Due to the

photosensitivity of the compound (Brownlee et al., 1992), the commercial sample waskept in the dark prior to use and the pH of the sample was adjusted to 10 before theexperiment to imitate tannery effluent conditions. In addition, mussels in tanks 5 and 6were exposed to Cr(VI) as K

Cr

O

at 0.1 and 1 mg l concentrations. The seventh tankacted as a control. The exposures were conducted over a 14 day period and the water andchemicals were changed every 3 days. During exposure, the filtering activity of themussels was observed and the mortalities measured. At the end of the period, the mussels

were sampled and the gills and digestive gland were dissected, fixed and stained forlipofuscin as described above.

Statistical analysis

Differences in the condition and chromium concentrations in mussels were analysedusing the Students t-test. Differences in the growth curves and temporal variation in theMDA concentrations were determined using a two-way analysis of variance (ANOVA). Inthe survival in air experiment, LT

values were determined for each group by PROBITanalysis. The 95% confidence intervals for the LT

values were calculated according toDixon andNewman (1991). Differences in the survivorship curves were determined byapplying the general linear model (GLM) using minitab. Percentage mortalities were arc-

sin transformed and the exposure time was log transformed for the analysis. In thismodel, the differences were accepted at the 99% confidence level.



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Results

Transplant experiment

The growth rate of mussels was measured at 4 month intervals over the exposure periodand the growth curves generated are illustrated in Fig. 1. The growth rate of the musselsat sites C1 and C2 was superior to that in the Blackwater estuary (C1, F

, 7.6,p, 0.001 and C2, F

, 9.1, p, 0.001). In a comparison between the two Colligansites, there was no significant difference between the curves. Due to vandalism, mussels

from sites C3 were lost after 5 months exposure and therefore only one sample wasavailable for comparison. At this time the mean shell length of the C3 mussels wassignificantly higher than the controls (C3, t 1.98, p, 0.05). The C4 transplants werelost at an earlier stage and were therefore unavailable for comparison.

After 1 year of exposure, the chromium concentrations in the whole soft parts of themussels transplanted into the Colligan estuary (C1, 20.9 g g and C2, 16.2 g g )were significantly elevated compared with mussels from the Blackwater estuary (K1,1.5 g g , p, 0.001). However, there was no difference between the two Colligansites. Chromium analysis of standard reference oyster tissue gave a result within the95% confidence interval of the quoted mean. The condition indices of both Colligangroups (C1, 81.8 and C2, 92.1) were also significantly higher compared to the controls(K1, 72.2, p, 0.01).

Survival in air

Due to a combination of storm damage at site C4 and excessive siltation at site C1, thetransplanted mussels at these sites were unavailable for the survival in air experiment.The calculated LT

values and statistics for the remaining sites are detailed inTable 1.There was no significant difference in survivorship between the control mussels, C4

38

36

34

32

30

28

26

24

22Feb. June Oct. Jan.

a Significantly different from controls p < 0.001b Significantly different from C2 p < 0.05

a

a

a

ab

a

a

c2

c1

Control

Shell

length(mm)

Fig. 1. Mean shell lengths6 SD and growth curves for mussels transplanted to theBlackwater (K1) and Colligan (C1 and C2) estuaries over a 12 month period.



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locals and C2 transplants while the C1 locals, C3 transplants and C3 locals hadsignificantly reduced survival times (p, 0.001). The C2 locals were found to have ahigher LT

compared to the controls (p, 0.001).The total chromium concentrations in the tissues of mussels from the various sites

are given in Table 2. The highest concentrations occur in the gills of mussels from allsites with high concentrations also occurring in the digestive gland and kidney. Lowerconcentrations were found in the mantle and muscle tissues. Between sites, C3 localshad the highest concentrations of chromium followed by C2, C4 and C1 locals,respectively.

Lipid peroxidation

The changes in the concentrations of MDA in the digestive gland and gill of the C4locals and control mussels during a combination of heat and aerial stress followed byrecovery are summarized in Fig.2. Comparing the two groups, the MDA concentrationswere higher after 12 h aerial exposure in the C4 mussels compared to the controls in boththe gill (F

,

6.16, p, 0.001) and digestive gland (F ,

3.66, p 0.015) tissues.Lipofuscin granules could be detected in the gill of both the control and

contaminated groups of mussels although there was no apparent increase in the

incidence of the granules in the Colligan mussels at any of the sites. There was also nodiscernable alteration in the structure of the gills in the Colligan mussels compared tothe controls. Lipofuscin granules were more apparent in the digestive cells of both the

Table 1. Calculated LT

values for survivorship curves in survival in air experiment

LT

6 95%

Site (days) Cl Equation r Best fit f p

Control 7.22 0.27 Y 1.164X 1.778 0.986

C1(loc) 6.78 0.28 Y 1.674X 2.680 0.970 Non-parallel 26.15 , 0.001

C2(loc) 8.05 0.26 Y 1.194X 1.968 0.955 Parallel 30.7 , 0.001

C2(tr) 7.16 0.27 Y 1.396X 2.22 0.951 NS

C3(loc) 6.39 0.28 Y

1.279X

1.850 0.943 Parallel 37.2 , 0.001C3(tr) 6.50 0.28 Y 1.289X 1.889 0.939 Parallel 28.13 , 0.001

C4(loc) 7.09 0.27 Y 1.171X 1.782 0.962 NS

Equations of transformed regression lines are given along with relevant statistics.loc, local; tr, transplanted.

Table 2. Mean6 SD total chromium concentrations ( g per g dry weight) in the tissues of local (loc)and transplanted (tr) mussels used in survival in air experiment after 3 months exposure

Tissue/site Digestive gland Gill Kidney Mantle Muscle

Control 3.8 6 1.5 4.9 6 2.1 3.6 6 0.6 1.6 6 0.3 1.2 6 0.4

C1(loc) 14.1 6 5.8 57.6 6 19 18.8 6 4.8 12.3 6 3.6 1.5 6 0.2

C2(loc) 64.4 6 23 163 6 46 69.3 6 24 19.6 6 4.9 15 6 4.9

C2(tr) 5.6 6 1.9 7.7 6 3.8 4.7 6 2.1 1.9 6 0.5 15 6 4.9

C3(loc) 74.8 6 27.8 588 6 154 161 6 45 60.7 6 18 2.2 6 0.8

C3(tr) 16.1 6 4.3 24.2 6 10 10.4 6 2.6 8.6 6 2.1 23.4 6 10

C4(loc) 11.0 6 3.4 134 6 52 12.2 6 3.2 12.3 6 4.4 6.2 6 1.1



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Colligan and control animals. However, the intensity, number and size of the granuleswas greater in the mussels sampled from sites C2 and C4 and in particular in the

mussels from site C3 compared to the controls (Figs 36). Lipofuscin staining in thedigestive gland of the C1 mussels appeared similar to the controls.

Fig. 2. Mean6 SD of cytosolic malonyldialdehyde (MDA) concentrations in (a) the gill and

(b) the digestive gland of Colligan and Blackwater control mussels subjected to 24 h aerial

exposure at 288C followed by 24 h recovery in seawater at 13 8C.

1,200

1,000

800

600

400

200

00 12 24 36 48

(a)

a

Time (h)

[MDA]nM/g

(D.Wt)

c4

Control

700

600

500

400

300

200

100

00 12 24 36 48

b

(b)

[MDA]nM/g(D.Wt)

c4

Control

Time (h)

Significantly different from controls ap < 0.039b < 0.015



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Fig. 3. Section (10 m) of digestive tubules from control mussels. Lysosomal lipofuscinaccumulation can be seen in the digestive cells ( 2 400 magnification; l, lipofuscin granules).

Fig. 4. Section (10 m) of digestive tubules from site C2 mussels. Lysosomal lipofuscinaccumulation can be seen in the digestive cells (

2 400 magnification; l, lipofuscin granules).



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Fig. 5. Section (10 m) of digestive tubules from site C3 mussels. Lysosomal accumulationcan be seen in the digestive cells ( 2 400 magnification; l, lipofuscin granules).

Fig. 6. Section (10 m) of digestive tubules from site C4 mussels. Lysosomal accumulationcan be seen in the digestive cells (

2 400 magnification, l, lipofuscin granules).



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Laboratory exposure

No mortalities were recorded over the exposure period. However, the filtering activity ofthe mussels exposed to 0.5 mg TCMTB l was seen to be inhibited, as the amount of

pseudofaeces produced was much lower than the other groups. In addition, the valves ofthe mussels tended to remain closed for extended periods. At the 1.0 mg Cr(VI) l

exposure concentration, byssus production was severely reduced compared to thecontrols. In contrast, this effect was not seen in the mussels exposed to the lower Cr(VI)

concentration and no outward physiological signs of toxicity were evident in Cr(III)albumin-exposed mussels.

On microscopic examination, a much higher incidence of lipofuscin was evident inthe digestive gland of mussels exposed to both 0.05 and 0.5 mg TCMTB l comparedto the controls (Figs7 and 8). In comparison, there was no evidence of peroxidation inthe digestive gland of Cr(III)- or Cr(VI)-exposed mussels at either concentration. In thegill, neither TCMTB or chromium was found to induce peroxidation in excess of thatseen in the controls. However, in the case of mussels exposed to the fungicide, therewas a marked relative proliferation of amoebocytes throughout the gill lumen (Figs 9and 10).

Discussion

The ecotoxicological consequences of leather tannery discharges and of chromium inparticular have been questioned on many occasions (e.g.Guruprasadaand Nanda Kumar,1981; Aislabie and Loutit, 1986; Bartlett and James, 1988; Aboul Dahab et al., 1990;Fuller et al., 1990), but few field studies have attempted to assess the threat of tannery

Fig. 7. Section (10 m) of digestive tubules from control mussels. Lysosomal lipofuscinaccumulation can be seen in the digestive cells (

2 400 magnification; l, lipofuscin granules).



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Fig. 8. Section (10 m) of digestive tubules from mussels exposed to 500 g TCMTB l .Lysosomal lipofuscin accumulation can be seen in the digestive cells ( 2 400 magnification; l,

lipofuscin granules).

Fig. 9. Section (10 m) of gill filaments from control mussels ( 2 400 magnification; a,amoebocytes; la, lamellae; ij, intralamellar junction).



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discharges in the context of specific effluent and environmental conditions. As is evidentin the present study, the physiological response of mussels to tannery effluent exposure iscomplex and the results suggest that there may be synergistic and/or antagonistic effects

between components in the effluent.For example, the growth and condition of the mussels transplanted to sites C1 and

C2 were not adversely affected by exposure to the tannery effluent for 1 year. Althoughthe growth rates of mussels have been found to vary considerably between sites aroundIreland (Aldrich, 1990), a relative depression in the growth of toxic stressed mussels

would have been expected compared to mussels sampled from a clean estuary. Theactual growth pattern measured certainly contradicts this hypothesis. The increases seenare possibly a result of growth promoting factors in the effluent, i.e. a high proteindischarge (5041140 kg protein per day) (Neill, 1991; Walsh and OHalloran, in press)and/or a thermal effect (22238C throughout the year; Neill 1989). In addition, asexceptionally good condition in the grey mullet, Chelon labrosus, sampled from theestuary has also been linked to the high protein content of the effluent (Walsh et al.,1994b), growth enhancement in detrital feeding animals appears to be a commonfeature in the estuary. As a result this effect should be considered when assessing theimpact of tannery discharges in similar environments.

In contrast, it is evident that mussels in the Colligan estuary are subject toconsiderable oxidative stress, the severity of such being highly correlated with the

tannery effluent exposure. In terms of cause and effect, the aquarium experimentstrongly suggests that exposure to the fungicide Busan 30WB

is responsible, with

Fig. 10. Section (10 m) of gill filaments from mussels exposed to 500 g TCMTB l illustrating amoebocyte proliferation ( 2 400 magnification; a, amoebocytes; la lamellae; ij,

intralamellar junction).



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induction occurring specifically in the digestive cells of both field sampled mussels andmussels exposed to the fungicide in the laboratory. In addition, environmental factorsalso appear to modify the peroxidative response. This is evidenced in the thermal stressexperiment. Here it is notable that the increase in the MDA concentrations occurredspecifically during the initial aerial exposure period, indicating that the limitedatmospheric oxygen utilization which occurs in M. edulis during aerial exposure(Zandee et al., 1986), is sufficient to initiate the peroxidation seen. As a result, it is

possible that aerial exposure during prolonged periods of hot weather may induce

increased peroxidation in the Colligan mussels. In addition, the reduction of the MDAconcentrations back to control values after a further 12 h aerial exposure indicates thatnatural antioxidant defence mechanisms may be brought into play. Although none weremonitored in the present study, Viarengo et al. (1989) found that in parallel withchanges in the MDA concentrations in the mussel tissues, the cytosolic glutathione(GSH) and vitamin E concentrations were also altered which may explain the reductionin MDA seen after prolonged aerial exposure.

In addition to the peroxidation seen, it is evident that the fungicide is also capable ofcausing a number of other toxicological responses, e.g. a reduction in the filtration rateand significant gill damage at high concentrations. Moreover, the proliferation ofamoebocytes seen in the gill at both concentrations is indicative of a generalinflammatory response (Lowe and Moore, 1979). While given that no gill damage was

evident in the field sampled mussels, it is apparent that such an effect is probable athigher exposure concentrations which may occur in less well-mixed receivingwaterbodies.

In terms of the environmental chemistry of benzothiozoles, Brownlee et al. (1992)provided evidence that TCMTB does not tend to persist or accumulate in aquaticenvironments as it is rapidly decomposed. However, a recent study of the fungicidespecifically in tannery effluent by Reemtsma et al. (1995) indicated that degradation

products such as benzothiazole (BT), 2-mercaptobenzothiazole (MBT) and 2-(methylthio) benzothiazole (MTBT) are also potentially toxic and persistent. In factthe latter study demonstrated that the alkaline conditions of tannery effluent will resultin rapid hydrolysis of the parent compound with MBT being the predominant product.Therefore, it is difficult to determine whether the parent compound or (more likely) a

breakdown derivative is responsible for the lipid peroxidation seen in both aquariumand field exposed mussels. Such information is certainly required given the widespreaduse of this family of compounds by the leather(Parbery and Taylor, 1989;Reemtsma etal., 1995), rubber (Jungclaus et al., 1976) and lumber (Brownlee et al., 1992)industries. In addition, the bioaccumulation of benzothiazole derivatives also requiresquantification from both a public health and ecotoxicological point of view. Certainlythe potential for accumulation exists as benzothiazoles have been detected in flounderliver in San Francisco bay (Spies et al., 1987) and high rates of BT uptake wererecorded in leeches by Metcalf et al. (1989). In the latter study, although the half lifefor BT was found to be relatively short, i.e. 7 days, considering the ability of molluscsto accumulate lipophilic xenobiotics in general (e.g. Viarengo, 1985; Moore, 1991), theaccumulation of benzothiazoles by this group of animals requires quantification.

In terms of chromium, the negative effect of Cr(VI) on lipid peroxidation is contrary

to the results for mammalian exposures (Lefebvre and Pezerat, 1994; Shi and Dalal,1994). A possible explanation is that seawater conditions, i.e. high salinity (Olsen and



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