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Disappearance of malachite green residues in fry of rainbow trout (Oncorhynchus mykiss) after treatment of eggs at the hatching stage
Aquaculture 297 (2009) 25–30
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Disappearance of malachite green residues in fry of rainbow trout (Oncorhynchus mykiss) after treatment of eggs at the hatching stage Kirsi Niska a, Tiina Korkea-aho b,1, Erja Lindfors a,⁎, Tapio Kiuru c, Markku Tuomainen d, Jouni Taskinen e, Kimmo Peltonen a a
Finnish Food Safety Authority (Evira), Chemistry and Toxicology Unit, Mustialankatu 3, FI-00790 Helsinki, Finland University of Kuopio, Department of Biosciences, PO Box 1627, FI-70211 Kuopio, Finland Finnish Game and Fisheries Research Institute Aquaculture Unit, P.O. Box 46, FI-41341 Laukaa, Finland d Fish Innovation Centre, Tervontie 4, FI-72210 Tervo, Finland e University of Jyväskylä, Department of Biological and Environmental Sciences, P.O. Box 35, FI-40014 University of Jyväskylä, Finland b c
a r t i c l e
i n f o
Article history: Received 23 January 2009 Received in revised form 27 August 2009 Accepted 28 August 2009 Keywords: Malachite green Leucomalachite green Fish fry Saprolegnia Residue analysis
a b s t r a c t The disappearance of malachite green (MG) residues was determined in fry of rainbow trout (Oncorhynchus mykiss) after six repeated treatments of the eggs at the hatching stage with MG oxalate at exposure levels of 1, 3 and 6 mg l− 1 for 30 min. Fry samples were taken from newly hatched fry (0 days post-hatch, d.p.h.) and at regular time intervals at 16, 31, 43, 57 and 96 d.p.h. The residues of MG and its major metabolite, leucomalachite green (LMG), were found to accumulate in the fry after MG treatments of eggs, with the highest residue levels being determined in the newly hatched fry. After exposures of 3 mg l− 1 MG, mean concentrations of 1170 ± 106 µg kg− 1 and 276 ± 38.6 µg kg− 1 (n = 3) were found in fry for LMG and MG, respectively. However, the disappearance of residues occurred rapidly in the fry, such that by 43 d.p.h. only low levels of LMG could be determined. To confirm the elimination of residues, determinations were made also in fry muscle at 96 d.p.h. but no residues were detected. The residues of MG in fry were determined by liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis with a limit of detection (LOD) of 0.5 µg kg− 1 and a limit of quantification (LOQ) of 1.0 µg kg− 1. The accumulation as well as the elimination of residues correlated well with the level of exposure. During the study, the fry increased their weight, such that at the end of the study, their mean body weight was about 150 times greater than the mean body weight of the newly hatched fry. As the disappearance of residues occurred in conjunction with the growth of fry, the present results indicate that no residues of MG will remain in the fish intended for human consumption, if MG treatment takes place at the hatching stage under controlled conditions. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The triphenylmethane dye, malachite green (MG), has been extensively used in the freshwater fish farming industry for the prevention and treatment of fungal infections and ectoparasites in the farmed fish. MG had been in use as a fungicide since the 1930s, for example against Oomycetes Saprolegnia which initially grows on dead fish eggs but can also spread readily to uninfected eggs. As the incubation period takes several weeks, fungal attack may evoke severe egg mortality unless this is prevented and controlled by regular fungicide treatments (Alderman, 1985). MG has also several toxic effects on mammalian cells (Culp and Beland, 1996; Culp, 2004; Srivastava et al., 2004; US Department of Health and Human Services, 2005) which has led to its prohibition in fish farming in the European Union. The European
⁎ Corresponding author. Tel.: +358 20 772 4420; fax: +358 20 772 4359. E-mail address: erja.lindfors@evira.fi (E. Lindfors). 1 Current address: University of Stirling, Stirling FK9 4 LA, United Kingdom. 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.08.037
Commission has designated that any analytical method used to determine these residues in meat of aquaculture products has to reach a minimum required performance limit (MRPL) of 2 µg kg− 1 for the sum of MG and its metabolite leucomalachite green (LMG). Alternatives to MG have been sought to control fungal infections of fish eggs, and several compounds, e.g. hydrogen peroxide, formalin and bronopol have been proposed (Waterstrat and Marking, 1995; Schreier et al., 1996; Barnes et al., 1998; Pottinger and Day, 1999; Rach et al., 2004). However, these alternative fungicides have not been as effective as MG in all situations, with their efficacies depending on culture conditions, hatchery systems and laborious treatment methods. Despite the fact that it has been prohibited for several years, MG/LMG residues are still detected in fish in the residue monitoring programs of EU member states (European Commission, 2008) emphasizing of the advantages of MG when compared to the alternative methods. Frequent reports of MG findings can also be seen in the Rapid Alert System of Food and Feed (RASFF, European Commission). The potential to permit a restricted use of MG in the treatment of fish eggs and small fry was debated at a meeting of fish experts from different EU countries (Rahkonen and Koski, 2002).
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In order to avoid any potential risks to the consumer attributable to the use of MG in the production of fish eggs and fry, it is essential that the possible residues of MG in food fish are known. However, only a few studies have examined the pharmacokinetics of MG in pre-exposed adult fish, fish eggs and fry. MG is easily absorbed by fish during waterborne exposure and it is distributed into certain fish tissues (Alderman and Clifton-Hadley, 1993; Plakas et al., 1996). After absorption, MG is rapidly metabolized to its reduced form LMG, which in fact is the actual target analyte in programs monitoring MG abuse. (Bauer et al., 1988; Plakas et al., 1996). After a single exposure of MG (0.8 mg l− 1, 1 h), the elimination half-life of LMG from catfish muscle was reported to be 10 days, but quantifiable traces of LMG have been determined (LOQ 5 µg kg− 1) for up to 42 days (Plakas et al., 1996). In another study, the persistence of gentian violet (GV) was determined in catfish muscle after water-borne exposure (0.1 mg ml− 1, 1 h). GV is a triphenylmethane dye related to MG, which is also metabolized in fish tissues to a leuco metabolite. In that study, leucogentian violet (LGV) was still detectable at a level of 3.1 µg kg− 1 in fish muscle at the 79 day measurement point (Thompson et al., 1999). The low excretion rate of the leuco metabolite seems to be related to the fat content of fish tissues (Bauer et al., 1988; Plakas et al., 1996). Environmental factors, like water temperature and pH have also been reported to influence the pharmacokinetics of MG residues in fish (Alderman and Clifton-Hadley, 1993; Plakas et al., 1996). Residues of MG have also been found in salmon eggs and newly hatched fry, when mature parent fish were treated with MG (0.1 mg l− 1, 24 h) before spawning. However, the elimination of residues in fry was not determined (Allen and Hunn, 1986). Only one study has been conducted to determine the disposition of residues in rainbow trout eggs and fry exposed directly to MG such that the elimination of residues in the fry was also examined. The eggs were found to accumulate MG residues during the exposure period, with LMG being the predominant residue found both in eggs and fry. The decline of MG residues in the fry was determined over a time period of 28 days, with residues still being detected at the end of the experiment (Meinertz et al., 1995). In another study, juvenile eels were treated with MG and the persistence of residues was determined over a 100 day sampling period. LMG was still present in the eels at the end of this time at an average concentration of 15 µg kg− 1 (LOD 1 µg kg− 1) (Bergwerff et al., 2004). These previous studies indicate that the residues of LMG may be present in fish muscle and fry for a long time after a single MG treatment. Accumulation of residues may take place after repeated exposures and this can cause a more prolonged elimination of residues. However, the eggshell of fish eggs constitutes a major barrier between the embryo and water-borne chemicals (von Westernhagen, 1988) and to some degree it may prevent the penetration of MG into the embryo. Since one focus of interest is in the restricted use of MG in the treatment of fish eggs, more information is needed about the fate of MG in fish exposed only at the egg stage. In the present study, we determined the residues of MG and LMG in rainbow trout fry hatched from eggs treated with three different exposure levels of MG, and followed the disappearance of residues for as long as possible. The final determinations were made in both whole fry and fry muscle samples to confirm the complete elimination of residues. 2. Materials and methods 2.1. Exposure of eggs Rainbow trout (Oncorhynchus mykiss) eggs were obtained in summer 2004 from a commercial fish farm in Northern Finland. Eggs from one female were fertilized with sperm obtained from three males and these were transported to the Fisheries Research Unit of the University of Kuopio. The eggs were divided into 12 egg-hatching jars (7 l of volume, 23 cm in bottom diameter and 40 cm in upper diameter), 70 ml of eggs in each jar. MG oxalate (dye content ≥90%, Merck) was diluted in distilled water to make a stock solution of 60 mg l− 1. The eggs
were exposed at 7 days post-fertilization (d.p.f.) to 0,1, 3 and 6 mg l− 1 of MG oxalate for 30 min. Each exposure level studied included three replicate hatching jars. During the exposure, the water flow was stopped, the water volume was set to 5 l and the required quantity of stock solution was added with a pipette to obtain the desired concentration of MG in the jars. Subsequently, the exposure was continued twice a week, on 10, 14, 17, 21 and 25 d.p.f. Hatching of eggs started on 27 d.p.f., and all the eggs had hatched on 29 d.p.f. 2.2. Fish maintenance During the one month incubation, a constant water flow was obtained from Lake Kallavesi, through a sand filter. The source water was not checked for MG or LMG prior to the study because Lake Kallavesi has an area of 500 km2 and no fish farms or other potential sources of MG or LMG are located in the drainage area of the Lake Kallavesi. Each hatching jar was supplied with its own incoming water flow of 1.5–1.8 l/ min. Temperature and oxygen were measured at least three times a week by an OxyGuard® oxygen electrode. The temperature ranged from 10.5 to 13.9 °C in the hatching jars (12.3 ± 1.2 °C, n = 11 measurements). The dissolved oxygen ranged from 7.15 to 10.2 mg DO l− 1 (8.7 ± 1 mg DO l− 1, n = 11 measurements) in the jars. Dead eggs and eggs infected by fungus were removed during the experiment. The eggs from three replicate control jars (exposed to 0 mg l− 1 of MG) were completely infested by fungus on 15 d.p.f., and removed. One of the three replicate jars treated with 6 mg l− 1 of MG was lost on 22 d.p.f., when the incoming water tube became blocked during the night and all the eggs died due to lack of oxygen. Fish were fed with an excess of a commercial dry food (Ecostart 17, Biomar) starting on 5 days post-hatch (d.p.h.). On 7 d.p.h., fry were removed to larger fibreglass tanks (70 l in volume, 60 cm in diameter). The tanks received a constant water flow from Lake Kallavesi through a drumfilter (40 µm mesh size). Water flow was set to 2.5–3 l/min per tank and the volume of water was set to 50 l. Initially, temperature and oxygen contents were measured similarly as in the hatching jars. After 26 d.p.h. temperature and oxygen contents were measured once a week. The pH was measured with a laboratory pH meter and BlueLine electrode (SCHOTT) on 16, 21, 35 and 70 d.p.h. from each tank. From 7 d.p.h. till the end of the experiment at 96 d.p.h., the temperature decreased from 15.5 to 10.1 °C in the tanks as the water temperature cooled during the autumn in Lake Kallavesi (13.3±1.9 °C, n =16 measurements). The dissolved oxygen content ranged from 8.1 to 10.2 mg DO l− 1 (9 ±0.5 mg DO l− 1, n =16 measurements). The pH in the tanks was 6.2 ±0.06 (n= 4 measurements). During the experiment, infestation of costia (Ichthyobodo necator) was found in some of the tanks. Therefore, two tanks exposed to 3 mg l− 1 of MG as eggs and one tank exposed to 6 mg l− 1 of MG as eggs were treated with formaldehyde solution on 36 d.p.h., and one tank exposed to 6 mg l− 1 of MG as eggs was treated on 40 d.p.h. In the treatment, 6.5 ml of 35–38% formaldehyde (Riedel-de Haën) was applied to 50 l of water in the tank (1:8000) for 30 min. 2.3. Eggs and fry sampling Samples from pooled eggs were taken before the eggs were divided into hatching jars. On 9 d.p.f., two days after the first MG treatment, the eggs were sampled, collecting about 100 eggs (5 g) from each of the 12 hatching jars. Sampling of fry was performed for all of the remaining 9 tanks on 0,16, 31, 43, 57 and 96 d.p.h. The fry sampled from 0 to 57 d.p.h. were sacrificed with a sharp blow on the head and frozen immediately, after which they were sent for further analyses to the National Veterinary and Food Research Institute EELA (nowadays Finnish Food Safety Authority Evira), Helsinki. The fry sampled on 96 d.p.h. were sacrificed in the same manner but sent fresh to EELA, where separate samples of whole fry and fry muscle were made and frozen before analysis.
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2.4. Analysis of residues in eggs and fry The method described for the determination of MG residues in fish muscle (Halme et al., 2004) was applied for fish eggs and fry with minor modifications. Egg samples were first homogenized with an Ultra-Turrax blender. Egg homogenates of 2.0 g were extracted with an aqueous ammonium acetate buffer (2 ml, 0.1 M, pH 4), hydroxylamine hydrochloride (600 µl, 0.25 g ml− 1), p-toluenesulfonic acid (1000 µl, 1.0 M) and acetonitrile (4 ml) on a platform shaker (300 min− 1, 5 min). Fry samples (including whole fry and fry muscle samples) were cut into small pieces and samples of 2.0 g were homogenized with an Ultra-Turrax blender in a buffer solution containing hydroxylamine hydrochloride and p-toluenesulfonic acid (as above). Acetonitrile (4 ml) was added and the samples were extracted on a platform shaker. After extraction, both egg and fry samples were handled in the same manner. The samples were centrifuged (3400 ×g, 10 min, 10 °C) and the supernatants were collected. Extraction with acetonitrile (4 ml) was repeated, samples were centrifuged and the supernatants were combined. Water (4 ml, Milli-Q quality, Millipore, Bedford, MA, USA) and methylene chloride (4 ml) were added to the supernatants and the samples were extracted and centrifuged as before. The lower layer was then recovered and the extraction with methylene chloride (4 ml) was repeated for the rest of the sample. The lower layers were combined and evaporated at 50 °C to about a volume of about 0.5 ml. The concentrated extracts were then purified on an automated solid-phase extraction system (ASPEC, Gilson, Villiers Le Bel, France) using neutral alumina (6 ml, 1 g; J. T. Baker, Deventer, Holland) and propylsulfonic acid (3 ml, 500 mg; Varian, Harbor City, CA, USA) SPE columns as described earlier (Halme et al., 2004). The residues were eluted from the PRS column with 3 ml of a mixture of ammonium acetate (0.1 M, pH 4) and acetonitrile (40:60, v/v). Ascorbic acid solution (30 µl, 1 mg ml− 1) was added, the volume of samples was adjusted to 3 ml and samples were filtered before analysis. A method based on liquid chromatography (LC-VIS) was used in the analysis of fish eggs and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used in the analysis of fry. The chromatographic separation and detection in the LC-VIS method were carried out as described earlier (Halme et al., 2004). In the LC-MS/MS system, a Zorbax Eclipse XDB-C18 analytical column (3.5 µm, 150× 2.1 mm i.d.; Agilent Technologies, Palo Alto, CA, USA) and a mobile phase of a mixture of ammonium acetate (25 mM, pH 4) and acetonitrile (25:75, v/v) were used. The flow rate of the mobile phase was 200 µl min− 1 and the column oven was operated at 40 °C. The injection volume was 10 µl. A post-column oxidation reactor with 100% lead(IV)oxide was used to convert LMG to MG before detection. Residues were determined using a Micromass Quattro Micro tandem mass spectrometer with an atmospheric pressure ionization (API) source operating in the positive-ion electrospray (ESI) mode (Micromass UK Ltd, Altrincham, Cheshire, UK). The following parameters were used: capillary voltage 3.5 kV; cone voltage 50 V; source temperature 150 °C; desolvation temperature 300 °C and a collision energy of 35 eV for both transitions. The ion transitions monitored with the Multiple Reaction Monitoring (MRM) mode were m/z 329 → 313 and 208. Quality control (QC) samples consisting of one blank sample and two fortified blank samples were analyzed along with each series of samples and used for the recovery correction in the quantification. Fortification levels in QC samples varied during the study and were in the range of 2–500 µg kg− 1 for MG and 2–1000 µg kg− 1 for LMG. The methods were validated according to ISO 17025. The limits of detection (LOD) for MG and LMG were determined on the basis of a 3:1 S/ N ratio to be 0.5 µg kg− 1 and the limit of quantification (LOQ) was calculated from LOD by multiplying LOD with a factor of two for MG and LMG respectively, resulting in a LOQ of 1.0 µg kg− 1. The recovery and repeatability of the LC-VIS method for fish eggs were tested in a validation procedure with six replicates of blank samples fortified at a concentration level of 2 µg kg− 1 and additionally with six replicates of samples fortified at levels of 1, 2, 5, 10 and 20 µg kg− 1 for both MG and LMG. The recovery
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(n=6) and repeatability (n=3) of the LC-MSMS method were tested with blank fish muscle samples fortified with 1, 2, 3 and 4 µg kg− 1. The mean recoveries over the tested concentration range of MG and LMG in eggs were 55.2% (range 50.9–63.7%, RSD 5.4–10.6%) and 54.3% (range 48.9–59.4%, RSD 6.4–6.8%) respectively. The mean recoveries over the tested concentration range of MG and LMG in muscle were 60.6% (range 58.3–67.1%, RSD 10.8–14.9%) and 66.5% (range 60.6–76.8%, RSD 8.8–12.8%) respectively. The validation of the methods is discussed in more detail elsewhere (Halme et al., 2004, 2006). 3. Results and discussion No background levels of MG residues were found in the rainbow trout eggs used for the study when they were analyzed before the MG exposures. The quality of eggs at the beginning of the study proved to be rather poor, since there were considerable numbers of dead eggs. Furthermore, attack by fungal pathogens was unavoidable since these organisms were in the water supply to the fish hatchery. Continual loss of eggs occurred in all hatching jars before the start of the MG exposure. All the control jars which were not exposed to MG became infected by fungus and all were lost. It is well known that without appropriate treatment, the fish eggs will succumb to fungal infection in greater quantities than eggs treated with an effective fungicide (e.g. Waterstrat and Marking, 1995). This was also observed in our study, where the proportion of infected eggs in the hatching jars treated with MG seemed to correlate with the concentration of MG, i.e. less infected eggs were found in those jars treated with higher concentrations of MG. As the amount of living eggs was unexpectedly low already at the beginning of the study, only one sampling of eggs was performed, on 9 d.p. f., two days after the first MG treatment. The eggs contained both MG and LMG residues. The mean concentrations (n=3) of MG in eggs were 55.8± 34.1 µg kg− 1 (exposure of 1 mg l− 1), 99.4±42.5 µg kg− 1 (exposure of 3 mg l− 1) and 220±19.5 µg kg− 1 (exposure of 6 mg l− 1). The mean concentrations of LMG in eggs were somewhat lower being 29.8±17.1 µg kg− 1 (exposure of 1 mg l− 1), 53.8±23.0 µg kg− 1 (exposure of 3 mg l− 1) and 110±15.8 µg kg− 1 (exposure of 6 mg l− 1). The presence of LMG residues in egg samples indicates that the metabolic reduction of MG can occur also in fish eggs. The reduction of MG to LMG has been previously reported to take place in fish, fish eggs and fry (e.g. Meinertz et al., 1995; Plakas et al., 1996; Bergwerff et al., 2004) whereas in water, MG exists as a dye ion or in the carbinol form (Alderman,1985) and no formation of LMG has been reported. Although the measured concentrations exhibited some variation between the three replicate hatching jars, the mean concentrations seem to correlate with the level of exposure. The eggs hatched during 27–29 d.p.f., after six repeated treatments of MG. In the newly hatched fry, high levels of both MG and LMG residues were determined. LMG was the major residue found in the fry at mean concentrations of 498 ± 59.6 µg kg− 1 (exposure of 1 mg l− 1), 1170 ± 106 µg kg− 1 (exposure of 3 mg l− 1) and 2400 ± 211 µg kg− 1 (exposure of 6 mg l− 1). The corresponding levels of MG in fry were 108 ± 17.6 µg kg− 1 (exposure of 1 mg l− 1), 276 ± 38.6 µg kg− 1 (exposure of 3 mg l− 1) and 569±117 µg kg− 1 (exposure of 6 mg l− 1). The high residue levels found in the newly hatched fry indicate that MG can penetrate through the eggshell into the embryo and further be metabolized to LMG when this fungicide is applied to eggs. The concentration of MG residues in fry was determined at regular time intervals until 57 d.p.h. Disappearance of MG residues in fry occurred rapidly as shown in Table 1. On 16 d.p.h., the mean concentrations of MG in fry were 1.4 ±0.1 µg kg− 1 (exposure of 1 mg l− 1), 1.8 ± 0.5 µg kg− 1 (exposure of 3 mg l− 1) and 4.6 ±0.4 µg kg− 1 (exposure of 6 mg l− 1). The residues of LMG had also substantially decreased being 29.2±7.3 µg kg− 1 (exposure of 1 mg l− 1), 70.9 ±6.2 µg kg− 1 (exposure of 3 mg l− 1) and 179± 33.2 µg kg− 1 (exposure of 6 mg l− 1). On 31 d.p.h., MG residues below 1 µg kg− 1 were determined in fry exposed to MG at a concentration of 6 mg l− 1. At the same time, LMG was determined in fry, the residue levels detected were below 1 µg kg− 1 (exposure of 1 mg l− 1),
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2.7 ± 0.3 µg kg− 1 (exposure of 3 mg l− 1) and 5.3 ± 1.4 µg kg− 1 (exposure of 6 mg l− 1). On 43 d.p.h., only LMG at levels below 1 µg kg− 1 were determined in fry exposed to treatments of 6 mg l− 1. On 57 d.p.h., no residues above LOD were found in any of the samples (Table 1). Analysis of samples taken from both whole fry and fry muscle on 96 d.p.h., when the fry were over three months of age, confirmed the complete elimination of residues. LC-MS/MS chromatograms of newly hatched fry and the same fry on 31 d.p.h also demonstrate the rapid disappearance of residues (Fig. 1). The obtained results are in agreement with the earlier study of Meinertz et al. (1995), in which rainbow trout eggs were repeatedly exposed to [methano-14C] labelled MG (1.0 mg l− 1, with immediate water flow). In that study, the eggs were also found to accumulate MG residues during the exposure period, with LMG being the predominant residue found both in eggs and fry. The total MG residues determined in the newly hatched fry after the exposure period were 271 ± 42 µg kg− 1 (Meinertz et al., 1995). The reported residue concentration was lower than in our study at the same exposure level (1 mg l− 1). However, a comparison of the data is difficult because of the differences in exposure and rearing conditions. The distribution of residues to MG and LMG was not reported by Meinertz et al. (1995), because the eggs they used contained measurable concentrations of MG and LMG as a background contamination. In that study, the total MG residues were found to decline during the relatively short 28-day period and were 55 ± 11 µg kg− 1. Growth dilution during this period was considered as a significant factor contributing to the decrease in the residues, accounting for 25% of the decrease in the total residue concentration (Meinertz et al., 1995). In another exposure study performed with juvenile eels by Bergwerff et al. (2004), the average tissue concentration of LMG at 15 µg kg− 1 was detectable at 100 days after exposure. The eels did not gain weight, and thus the change of body mass did not cause any dilution of the residues. The mean body weights of fry were also determined in our study at each sampling time. The growth of fry during the sampling period of 96 days is represented in Fig. 2. During this time, the body weights of fry increased linearly being followed by an exponential increase to the last measuring point. The mean body weights of fry at the end of the study had increased 150-fold compared to the mean body weights of the newly hatched fry. Had the disappearance of MG and LMG been a result exclusively of dilution caused by growth, then both the MG and LMG levels after 150-fold dilution at 96 d.p.h would still have been detectable (approximately 1–16 µg kg− 1). Some of the tanks were treated with formaldehyde solution for 30 min on 36 d.p.h and on 40 d.p.h because of an infestation with costia (Ichthyobodo necator). Formaldehyde is toxic to fish (Hohreiter and Rigg 2001) but no decrease concerning the growth of the body weight of fry could be detected after this formaldehyde treatment (Fig. 2). There is no data available on how formaldehyde could interact with MG and LMG in fish tissue. In this study the level of MG on 31 d.p.h had already decreased to below 1 µg kg− 1 and the concentrations of LMG continued to decline linearly after the treatment. The concentration of LMG in the formaldehyde treated tanks was at the same level as that determined in the untreated replicates. LMG is known to be metabolized in rats and mice through oxidative demethylation (Culp et al.,1999; Cho et al 2003). On the other hand, formaldehyde is a reducing agent but no reduction
Fig. 1. LC-MS/MS determination of MG (retention time about 2.7 min) and LMG (retention time about 11.4–11.5 min) in newly hatched fry (0 d.p.h.) (A.). The same fry on 31 d.p.h. after repeated exposure to 3 mg l− 1 MG at the hatching stage (see Section 2.1 in the text) (B.). In the newly hatched fry, residues of 255 µg kg− 1 MG and 1094 µg kg− 1 LMG were determined. During the period of 31 d.p.h., disappearance of residues was observed and the fry contained only 2.6 µg kg− 1 LMG. The ion transitions monitored were m/z 329 → 313 and 208 for both MG and LMG residues. The value of peak area is shown below the retention time.
pathways for LMG degradation have been reported. Therefore it seems most unlikely that this formaldehyde treatment would have had any effect on the disappearance of LMG residues in the fish under our test conditions. The quality of analyses was confirmed by analyzing QC samples along with authentic samples. For the analysis of egg samples, eggs sampled from the control hatching jars before exposure were used as
Table 1 Concentrations of MG and LMG in rainbow trout fry after repeated exposure of 1, 3 and 6 mg l− 1 of MG during the hatching stage. Exposure
0 d.p.h.
(mg/l)
MG
LMG
MG
LMG
MG
LMG
MG
LMG
MG
LMG
MG
LMG
108 ± 17.6 276 ± 38.6 569 ± 117
498 ± 59.6 1170 ± 106 2400 ± 211
1.4 ± 0.1 1.8 ± 0.5 4.6 ± 0.4
29.2 ± 7.3 70.9 ± 6.2 179 ± 33.2
nd nd <1
<1 2.7 ± 0.3 5.3 ± 1.4
nd nd nd
nd nd <1
nd nd nd
nd nd nd
nd nd nd
nd nd nd
a
1 3a 6b
16 d.p.h.
31 d.p.h.
The fry were sampled on 0, 16, 31, 43 and 96 d.p.h. Data is given as means ± S.D. a n = 3. b n = 2.
43 d.p.h.
57 d.p.h.
96 d.p.h.
K. Niska et al. / Aquaculture 297 (2009) 25–30
Fig. 2. The increase of mean body weight (mg) of the fry during the study period from 0 to 96 days.
the blank material for QC samples. The recovery in eggs was over 90% for both MG and LMG. However, determination of fry samples was problematic as all the control eggs were lost during incubation due to the extensive fungal growth, and thus no control fry samples for the study were available. Due to this, a blank fry sample obtained from another fish farm was used for the QC samples analyzed along with samples from 0 to 57 d.p.h. This blank fry sample was checked to be free of MG and LMG, divided into lots and frozen. It was not age matched with the study samples. This may lead to some inaccuracy in the results, however, it does not invalidate the major observation on disappearance of residues. The average recoveries were 75 ± 14% for LMG analyzed along with samples from 0 to 43 d.p.h., but only about 20% in the determination of samples at 57 d.p.h. The low recovery of the samples at this late time point was traced to problems in the automated solid-phase extraction instrument when it was treating the QC samples at the end of the set. The rest of the sample set as well as the other sets were treated without incident. The recoveries in other sample sets were satisfactory. There can be day to day variation in recoveries, suspected for example to be caused by changes in the relative humidity. However, the RSD values within sample sets were satisfactory and the results were corrected with the recovery of the QC samples of the set. LMG was not detected at 57 d.p.h. which is concordant with levels in samples taken before and after: LMG at 43 d. p.h. was already below 1 µg kg− 1 and at 96 d.p.h. not detectable (Table 1), so the not detected LMG level at 57 d.p.h. can be considered as reliable. The recovery of MG was generally low, 35 ± 7%. In the final determination (samples of 96 d.p.h), a whole fry sample (which was one of the authentic samples) and a blank fish muscle sample were used as QC samples. The recoveries in both determinations were about 80% for both MG and LMG. Despite the problems in recovery determination, the results are considered as reliable in demonstrating the time trend for residue disappearance after exposure during the hatching stage exposures. Additionally, the results of all three exposure concentrations with replicate tanks were in concordance with each other. In the earlier studies (Meinertz et al., 1995; Bergwerff et al., 2004), only a single exposure level has been studied with no replicates. Therefore, we believe that our data represents an important contribution to the study of the analysis of MG residues in the early stages of fish. 4. Conclusion The residues of MG accumulated into fry after repeated treatments of eggs, but the disappearance of residues in fry occurred rapidly and no residues of MG and LMG were determined in fry muscle at the end of the study, at 96 d.p.h. This indicates that the elimination of MG residues in
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combination with growth of the fry to market size fish resulting in a dilution factor of about 1000 (1 g → 1000 g of body weight), means that there are no remaining residues in the fish intended for human consumption. This is in accordance with the earlier findings (Meinertz et al.,1995; Bergwerff et al., 2004). Therefore, the present results indicate that recent findings of MG/LMG residues still being detected today in adult fish (RASFF, European Commission), cannot have originated from treatment of eggs with MG. In fact, we have demonstrated that MG can be used safely if it is applied restrictedly for the treatment of fish eggs to be used for the production of farmed fish. However, it is recognized that there could be problems with reintroducing MG use. The restricted permission for the use of MG and its control to avoid any possible abuse would represent a major challenge to the authorities. Another challenge would be the appropriate handling of MG in order to ensure zero exposure to the fish culturist or the environment. However, water volumes are very small during the incubation stage of fish rearing and appropriate water management would clearly be technically feasible. The outflow of water from hatcheries to fish pools would need to be blocked and the MG containing waste water handled in a special manner. The authorities would need to monitor any possible abuse by controlling for the detection of MG and LMG in fry and fish samples. At the present time, the use of MG is not permitted by EU legislation, since no maximum residue limit (MRL) has been established for this fungicide (Rahkonen and Koski, 2002). Acknowledgements The authors thank Riitta Linjakumpu, Finnish Food Safety Authority (Evira) for her skilful assistance in performing sample analysis and the personnel in Fisheries Research Unit of the University of Kuopio for the assistance in fish maintenance. The research was partly funded by a grant from Maj and Tor Nessling Foundation (JT). References Alderman, D.J., 1985. Malachite green: a review. J. Fish Dis. 8, 289–298. Alderman, D.J., Clifton-Hadley, R.S., 1993. Malachite green: a pharmacokinetic study in rainbow trout, Oncorhynchus mykiss (Walbaum). J. Fish Dis. 16, 297–311. Allen, J.L., Hunn, J.B., 1986. Fate and distribution studies of some drugs used in aquaculture. Vet. Hum. Toxicol. 28, 21–24. Barnes, M.E., Ewing, D.E., Cordes, R.J., Young, G.L., 1998. Observations on hydrogen peroxide control of Saprolegnia spp. during rainbow trout egg incubation. Prog. Fish-Cult. 60, 67–70. Bauer, K., Dangschat, H., Knöppler, H.-O., Neudegger, J., 1988. Aufnahme und Ausscheidung von Malachitgrün bei Regenbogenforellen. Arch. Lebensm.hyg. 39, 85–103. Bergwerff, A.A., Kuiper, R.V., Scherpenisse, P., 2004. Persistence of residues of malachite green in juvenile eels (Anguilla anguilla). Aquaculture 233, 55–63. Cho, B.P., Yang, T., Blankenship, L.R., Moody, J.D., Churchwell, M., Beland, F.A., Culp, J.C., 2003. Synthesis and characterization of N-demethylated metabolites of malachite green and leucomalachite green. Chem. Res. Toxicol. 16, 285–294. Culp, S.J., 2004. NTP technical report on the toxicity studies on malachite green chloride and leucomalachite green administered in feed to F344/N rats and B6C3F1 mice. US Department of Health and Human Services June 2004. National Toxicology Program, Toxicity report series. NIH Publication No. 04-4416. Available: http:// ntp.niehs.nih.gov/ntp/htdocs/ST_rpts/tox071.pdf. Culp, S.J., Beland, F.A., 1996. Malachite green: a toxicological review. J. Amer. Coll. Toxicol. 15, 219–238. Culp, J.C., Blankenship, L.R., Kusewitt, D.F., Doerge, D.R., Mulligan, L.T., Beland, F.A., 1999. Toxicity and metabolism of malachite green and leucomalachite green during short-term feeding to Fischer 344 rats and B6C3F1. Chem.-biol. Interact. 122, 153–170. European Commission, 2008. Commission staff working paper on the implementation of national residue monitoring plans in the member states in 2007, (Council Directive 96/23/EC). Brussels 23.12.2008. Available: http://ec.europa.eu/food/food/ chemicalsafety/residues/control_en.print.htm. European Commission. Rapid alert system for food and feed. RASFF. Week 2009/9 http://ec.europa.eu/food/food/rapidalert/archive_en.htm. Halme, K., Lindfors, E., Peltonen, K., 2004. Determination of malachite green residues in rainbow trout muscle with liquid chromatography and liquid chromatography coupled with tandem mass spectrometry. Food Addit. Contam. 21, 641–648. Halme, K., Lindfors, E., Peltonen, K., 2006. A confirmatory analysis of malachite green residues in rainbow trout with liquid chromatography-electrospray tandem mass spectrometry. J. Chromatogr., B, Biomed. Sci. Appl. 845, 74–79. Hohreiter, D.W., Rigg, D.K., 2001. Derivation of ambient water quality criteria for formaldehyde. Chemosphere 45, 471–486.
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Meinertz, J.R., Stehly, G.R., Gingerich, W.H., Allen, J.L., 1995. Residues of [14C]-malachite green in eggs and fry of rainbow trout, Oncorhynchus mykiss (Walbaum), after treatments of eggs. J. Fish Dis. 18, 239–247. Plakas, S.M., El Said, K.R., Stehly, G.R., Gingerich, W.H., Allen, J.L., 1996. Uptake, tissue distribution and metabolism of malachite green in the channel catfish (Ictalurus punctatus). Can. J. Fish. Aquat. Sci. 53, 1427–1433. Pottinger, T.G., Day, J.G., 1999. A Saprolegnia parasitica challenge system for rainbow trout: assessment of Pyceze as an anti-fungal agent for both fish and ova. Dis. Aquat. Org. 36, 129–141. Rach, J.J., Valentine, J.J., Schreier, T.M., Gaikowski, M.P., Crawford, T.G., 2004. Efficacy of hydrogen peroxide to control saprolegniasis on channel catfish (Ictalurus punctatus) eggs. Aquaculture 238, 135–142. Rahkonen, R., Koski, P., 2002. Roundtable 3. Post malachite green: alternative strategies for fungal infections and white spot disease. Bull. Eur. Assoc. Fish Pathol. 22, 152–157. Schreier, T.M., Rach, J.J., Howe, G.E., 1996. Efficacy of formalin, hydrogen peroxide, and sodium chloride on fungal-infected rainbow trout eggs. Aquaculture 140, 323–331.
Srivastava, S., Sinha, R., Roy, D., 2004. Toxicological effects of malachite green. Aquat. Toxicol. 66, 319–329. Thompson Jr., H.C., Rushing, L.G., Gehring, T., Lochmann, R., 1999. Persistence of gentian violet and leucogentian violet in channel catfish (Ictalurus punctatus) muscle after water-borne exposure. J. Chromatogr., B, Biomed. Sci. Appl. 723, 287–291. US Department of Health and Human Services, 2005. Technical report on the toxicity and carcinogenic studies on malachite green chloride and leucomalachite in F344/N rats and B6C3F1 mice (feed studies). National Toxicology Program NTP TR 527, NIH Publication No. 05-4463. Available: http://ntp.niehs.nih.gov/files/527-C_LowRes_Bookmark.pdf. Waterstrat, P.R., Marking, L.L., 1995. Clinical evaluation of formalin, hydrogen peroxide, and sodium chloride for the treatment of Saprolegnia parasitica on fall chinook salmon eggs. Prog. Fish-Cult. 57, 287–291. Von Westernhagen, H., 1988. Sublethal effects of pollutants on fish eggs and larvae. The physiology of developing fish. Part A. Eggs and Larvae. : In: Hoar, W.S., Randall, D.J. (Eds.), Fish physiology, vol. XI. Academic Press Inc, San Diego, pp. 253–346.
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Disappearance of malachite green residues in fry of rainbow trout (Oncorhynchus mykiss) after treatment of eggs at the hatching stage Kirsi Niska a, Tiina Korkea-aho b,1, Erja Lindfors a,⁎, Tapio Kiuru c, Markku Tuomainen d, Jouni Taskinen e, Kimmo Peltonen a a
Finnish Food Safety Authority (Evira), Chemistry and Toxicology Unit, Mustialankatu 3, FI-00790 Helsinki, Finland University of Kuopio, Department of Biosciences, PO Box 1627, FI-70211 Kuopio, Finland Finnish Game and Fisheries Research Institute Aquaculture Unit, P.O. Box 46, FI-41341 Laukaa, Finland d Fish Innovation Centre, Tervontie 4, FI-72210 Tervo, Finland e University of Jyväskylä, Department of Biological and Environmental Sciences, P.O. Box 35, FI-40014 University of Jyväskylä, Finland b c
a r t i c l e
i n f o
Article history: Received 23 January 2009 Received in revised form 27 August 2009 Accepted 28 August 2009 Keywords: Malachite green Leucomalachite green Fish fry Saprolegnia Residue analysis
a b s t r a c t The disappearance of malachite green (MG) residues was determined in fry of rainbow trout (Oncorhynchus mykiss) after six repeated treatments of the eggs at the hatching stage with MG oxalate at exposure levels of 1, 3 and 6 mg l− 1 for 30 min. Fry samples were taken from newly hatched fry (0 days post-hatch, d.p.h.) and at regular time intervals at 16, 31, 43, 57 and 96 d.p.h. The residues of MG and its major metabolite, leucomalachite green (LMG), were found to accumulate in the fry after MG treatments of eggs, with the highest residue levels being determined in the newly hatched fry. After exposures of 3 mg l− 1 MG, mean concentrations of 1170 ± 106 µg kg− 1 and 276 ± 38.6 µg kg− 1 (n = 3) were found in fry for LMG and MG, respectively. However, the disappearance of residues occurred rapidly in the fry, such that by 43 d.p.h. only low levels of LMG could be determined. To confirm the elimination of residues, determinations were made also in fry muscle at 96 d.p.h. but no residues were detected. The residues of MG in fry were determined by liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis with a limit of detection (LOD) of 0.5 µg kg− 1 and a limit of quantification (LOQ) of 1.0 µg kg− 1. The accumulation as well as the elimination of residues correlated well with the level of exposure. During the study, the fry increased their weight, such that at the end of the study, their mean body weight was about 150 times greater than the mean body weight of the newly hatched fry. As the disappearance of residues occurred in conjunction with the growth of fry, the present results indicate that no residues of MG will remain in the fish intended for human consumption, if MG treatment takes place at the hatching stage under controlled conditions. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The triphenylmethane dye, malachite green (MG), has been extensively used in the freshwater fish farming industry for the prevention and treatment of fungal infections and ectoparasites in the farmed fish. MG had been in use as a fungicide since the 1930s, for example against Oomycetes Saprolegnia which initially grows on dead fish eggs but can also spread readily to uninfected eggs. As the incubation period takes several weeks, fungal attack may evoke severe egg mortality unless this is prevented and controlled by regular fungicide treatments (Alderman, 1985). MG has also several toxic effects on mammalian cells (Culp and Beland, 1996; Culp, 2004; Srivastava et al., 2004; US Department of Health and Human Services, 2005) which has led to its prohibition in fish farming in the European Union. The European
⁎ Corresponding author. Tel.: +358 20 772 4420; fax: +358 20 772 4359. E-mail address: erja.lindfors@evira.fi (E. Lindfors). 1 Current address: University of Stirling, Stirling FK9 4 LA, United Kingdom. 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.08.037
Commission has designated that any analytical method used to determine these residues in meat of aquaculture products has to reach a minimum required performance limit (MRPL) of 2 µg kg− 1 for the sum of MG and its metabolite leucomalachite green (LMG). Alternatives to MG have been sought to control fungal infections of fish eggs, and several compounds, e.g. hydrogen peroxide, formalin and bronopol have been proposed (Waterstrat and Marking, 1995; Schreier et al., 1996; Barnes et al., 1998; Pottinger and Day, 1999; Rach et al., 2004). However, these alternative fungicides have not been as effective as MG in all situations, with their efficacies depending on culture conditions, hatchery systems and laborious treatment methods. Despite the fact that it has been prohibited for several years, MG/LMG residues are still detected in fish in the residue monitoring programs of EU member states (European Commission, 2008) emphasizing of the advantages of MG when compared to the alternative methods. Frequent reports of MG findings can also be seen in the Rapid Alert System of Food and Feed (RASFF, European Commission). The potential to permit a restricted use of MG in the treatment of fish eggs and small fry was debated at a meeting of fish experts from different EU countries (Rahkonen and Koski, 2002).
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In order to avoid any potential risks to the consumer attributable to the use of MG in the production of fish eggs and fry, it is essential that the possible residues of MG in food fish are known. However, only a few studies have examined the pharmacokinetics of MG in pre-exposed adult fish, fish eggs and fry. MG is easily absorbed by fish during waterborne exposure and it is distributed into certain fish tissues (Alderman and Clifton-Hadley, 1993; Plakas et al., 1996). After absorption, MG is rapidly metabolized to its reduced form LMG, which in fact is the actual target analyte in programs monitoring MG abuse. (Bauer et al., 1988; Plakas et al., 1996). After a single exposure of MG (0.8 mg l− 1, 1 h), the elimination half-life of LMG from catfish muscle was reported to be 10 days, but quantifiable traces of LMG have been determined (LOQ 5 µg kg− 1) for up to 42 days (Plakas et al., 1996). In another study, the persistence of gentian violet (GV) was determined in catfish muscle after water-borne exposure (0.1 mg ml− 1, 1 h). GV is a triphenylmethane dye related to MG, which is also metabolized in fish tissues to a leuco metabolite. In that study, leucogentian violet (LGV) was still detectable at a level of 3.1 µg kg− 1 in fish muscle at the 79 day measurement point (Thompson et al., 1999). The low excretion rate of the leuco metabolite seems to be related to the fat content of fish tissues (Bauer et al., 1988; Plakas et al., 1996). Environmental factors, like water temperature and pH have also been reported to influence the pharmacokinetics of MG residues in fish (Alderman and Clifton-Hadley, 1993; Plakas et al., 1996). Residues of MG have also been found in salmon eggs and newly hatched fry, when mature parent fish were treated with MG (0.1 mg l− 1, 24 h) before spawning. However, the elimination of residues in fry was not determined (Allen and Hunn, 1986). Only one study has been conducted to determine the disposition of residues in rainbow trout eggs and fry exposed directly to MG such that the elimination of residues in the fry was also examined. The eggs were found to accumulate MG residues during the exposure period, with LMG being the predominant residue found both in eggs and fry. The decline of MG residues in the fry was determined over a time period of 28 days, with residues still being detected at the end of the experiment (Meinertz et al., 1995). In another study, juvenile eels were treated with MG and the persistence of residues was determined over a 100 day sampling period. LMG was still present in the eels at the end of this time at an average concentration of 15 µg kg− 1 (LOD 1 µg kg− 1) (Bergwerff et al., 2004). These previous studies indicate that the residues of LMG may be present in fish muscle and fry for a long time after a single MG treatment. Accumulation of residues may take place after repeated exposures and this can cause a more prolonged elimination of residues. However, the eggshell of fish eggs constitutes a major barrier between the embryo and water-borne chemicals (von Westernhagen, 1988) and to some degree it may prevent the penetration of MG into the embryo. Since one focus of interest is in the restricted use of MG in the treatment of fish eggs, more information is needed about the fate of MG in fish exposed only at the egg stage. In the present study, we determined the residues of MG and LMG in rainbow trout fry hatched from eggs treated with three different exposure levels of MG, and followed the disappearance of residues for as long as possible. The final determinations were made in both whole fry and fry muscle samples to confirm the complete elimination of residues. 2. Materials and methods 2.1. Exposure of eggs Rainbow trout (Oncorhynchus mykiss) eggs were obtained in summer 2004 from a commercial fish farm in Northern Finland. Eggs from one female were fertilized with sperm obtained from three males and these were transported to the Fisheries Research Unit of the University of Kuopio. The eggs were divided into 12 egg-hatching jars (7 l of volume, 23 cm in bottom diameter and 40 cm in upper diameter), 70 ml of eggs in each jar. MG oxalate (dye content ≥90%, Merck) was diluted in distilled water to make a stock solution of 60 mg l− 1. The eggs
were exposed at 7 days post-fertilization (d.p.f.) to 0,1, 3 and 6 mg l− 1 of MG oxalate for 30 min. Each exposure level studied included three replicate hatching jars. During the exposure, the water flow was stopped, the water volume was set to 5 l and the required quantity of stock solution was added with a pipette to obtain the desired concentration of MG in the jars. Subsequently, the exposure was continued twice a week, on 10, 14, 17, 21 and 25 d.p.f. Hatching of eggs started on 27 d.p.f., and all the eggs had hatched on 29 d.p.f. 2.2. Fish maintenance During the one month incubation, a constant water flow was obtained from Lake Kallavesi, through a sand filter. The source water was not checked for MG or LMG prior to the study because Lake Kallavesi has an area of 500 km2 and no fish farms or other potential sources of MG or LMG are located in the drainage area of the Lake Kallavesi. Each hatching jar was supplied with its own incoming water flow of 1.5–1.8 l/ min. Temperature and oxygen were measured at least three times a week by an OxyGuard® oxygen electrode. The temperature ranged from 10.5 to 13.9 °C in the hatching jars (12.3 ± 1.2 °C, n = 11 measurements). The dissolved oxygen ranged from 7.15 to 10.2 mg DO l− 1 (8.7 ± 1 mg DO l− 1, n = 11 measurements) in the jars. Dead eggs and eggs infected by fungus were removed during the experiment. The eggs from three replicate control jars (exposed to 0 mg l− 1 of MG) were completely infested by fungus on 15 d.p.f., and removed. One of the three replicate jars treated with 6 mg l− 1 of MG was lost on 22 d.p.f., when the incoming water tube became blocked during the night and all the eggs died due to lack of oxygen. Fish were fed with an excess of a commercial dry food (Ecostart 17, Biomar) starting on 5 days post-hatch (d.p.h.). On 7 d.p.h., fry were removed to larger fibreglass tanks (70 l in volume, 60 cm in diameter). The tanks received a constant water flow from Lake Kallavesi through a drumfilter (40 µm mesh size). Water flow was set to 2.5–3 l/min per tank and the volume of water was set to 50 l. Initially, temperature and oxygen contents were measured similarly as in the hatching jars. After 26 d.p.h. temperature and oxygen contents were measured once a week. The pH was measured with a laboratory pH meter and BlueLine electrode (SCHOTT) on 16, 21, 35 and 70 d.p.h. from each tank. From 7 d.p.h. till the end of the experiment at 96 d.p.h., the temperature decreased from 15.5 to 10.1 °C in the tanks as the water temperature cooled during the autumn in Lake Kallavesi (13.3±1.9 °C, n =16 measurements). The dissolved oxygen content ranged from 8.1 to 10.2 mg DO l− 1 (9 ±0.5 mg DO l− 1, n =16 measurements). The pH in the tanks was 6.2 ±0.06 (n= 4 measurements). During the experiment, infestation of costia (Ichthyobodo necator) was found in some of the tanks. Therefore, two tanks exposed to 3 mg l− 1 of MG as eggs and one tank exposed to 6 mg l− 1 of MG as eggs were treated with formaldehyde solution on 36 d.p.h., and one tank exposed to 6 mg l− 1 of MG as eggs was treated on 40 d.p.h. In the treatment, 6.5 ml of 35–38% formaldehyde (Riedel-de Haën) was applied to 50 l of water in the tank (1:8000) for 30 min. 2.3. Eggs and fry sampling Samples from pooled eggs were taken before the eggs were divided into hatching jars. On 9 d.p.f., two days after the first MG treatment, the eggs were sampled, collecting about 100 eggs (5 g) from each of the 12 hatching jars. Sampling of fry was performed for all of the remaining 9 tanks on 0,16, 31, 43, 57 and 96 d.p.h. The fry sampled from 0 to 57 d.p.h. were sacrificed with a sharp blow on the head and frozen immediately, after which they were sent for further analyses to the National Veterinary and Food Research Institute EELA (nowadays Finnish Food Safety Authority Evira), Helsinki. The fry sampled on 96 d.p.h. were sacrificed in the same manner but sent fresh to EELA, where separate samples of whole fry and fry muscle were made and frozen before analysis.
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2.4. Analysis of residues in eggs and fry The method described for the determination of MG residues in fish muscle (Halme et al., 2004) was applied for fish eggs and fry with minor modifications. Egg samples were first homogenized with an Ultra-Turrax blender. Egg homogenates of 2.0 g were extracted with an aqueous ammonium acetate buffer (2 ml, 0.1 M, pH 4), hydroxylamine hydrochloride (600 µl, 0.25 g ml− 1), p-toluenesulfonic acid (1000 µl, 1.0 M) and acetonitrile (4 ml) on a platform shaker (300 min− 1, 5 min). Fry samples (including whole fry and fry muscle samples) were cut into small pieces and samples of 2.0 g were homogenized with an Ultra-Turrax blender in a buffer solution containing hydroxylamine hydrochloride and p-toluenesulfonic acid (as above). Acetonitrile (4 ml) was added and the samples were extracted on a platform shaker. After extraction, both egg and fry samples were handled in the same manner. The samples were centrifuged (3400 ×g, 10 min, 10 °C) and the supernatants were collected. Extraction with acetonitrile (4 ml) was repeated, samples were centrifuged and the supernatants were combined. Water (4 ml, Milli-Q quality, Millipore, Bedford, MA, USA) and methylene chloride (4 ml) were added to the supernatants and the samples were extracted and centrifuged as before. The lower layer was then recovered and the extraction with methylene chloride (4 ml) was repeated for the rest of the sample. The lower layers were combined and evaporated at 50 °C to about a volume of about 0.5 ml. The concentrated extracts were then purified on an automated solid-phase extraction system (ASPEC, Gilson, Villiers Le Bel, France) using neutral alumina (6 ml, 1 g; J. T. Baker, Deventer, Holland) and propylsulfonic acid (3 ml, 500 mg; Varian, Harbor City, CA, USA) SPE columns as described earlier (Halme et al., 2004). The residues were eluted from the PRS column with 3 ml of a mixture of ammonium acetate (0.1 M, pH 4) and acetonitrile (40:60, v/v). Ascorbic acid solution (30 µl, 1 mg ml− 1) was added, the volume of samples was adjusted to 3 ml and samples were filtered before analysis. A method based on liquid chromatography (LC-VIS) was used in the analysis of fish eggs and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used in the analysis of fry. The chromatographic separation and detection in the LC-VIS method were carried out as described earlier (Halme et al., 2004). In the LC-MS/MS system, a Zorbax Eclipse XDB-C18 analytical column (3.5 µm, 150× 2.1 mm i.d.; Agilent Technologies, Palo Alto, CA, USA) and a mobile phase of a mixture of ammonium acetate (25 mM, pH 4) and acetonitrile (25:75, v/v) were used. The flow rate of the mobile phase was 200 µl min− 1 and the column oven was operated at 40 °C. The injection volume was 10 µl. A post-column oxidation reactor with 100% lead(IV)oxide was used to convert LMG to MG before detection. Residues were determined using a Micromass Quattro Micro tandem mass spectrometer with an atmospheric pressure ionization (API) source operating in the positive-ion electrospray (ESI) mode (Micromass UK Ltd, Altrincham, Cheshire, UK). The following parameters were used: capillary voltage 3.5 kV; cone voltage 50 V; source temperature 150 °C; desolvation temperature 300 °C and a collision energy of 35 eV for both transitions. The ion transitions monitored with the Multiple Reaction Monitoring (MRM) mode were m/z 329 → 313 and 208. Quality control (QC) samples consisting of one blank sample and two fortified blank samples were analyzed along with each series of samples and used for the recovery correction in the quantification. Fortification levels in QC samples varied during the study and were in the range of 2–500 µg kg− 1 for MG and 2–1000 µg kg− 1 for LMG. The methods were validated according to ISO 17025. The limits of detection (LOD) for MG and LMG were determined on the basis of a 3:1 S/ N ratio to be 0.5 µg kg− 1 and the limit of quantification (LOQ) was calculated from LOD by multiplying LOD with a factor of two for MG and LMG respectively, resulting in a LOQ of 1.0 µg kg− 1. The recovery and repeatability of the LC-VIS method for fish eggs were tested in a validation procedure with six replicates of blank samples fortified at a concentration level of 2 µg kg− 1 and additionally with six replicates of samples fortified at levels of 1, 2, 5, 10 and 20 µg kg− 1 for both MG and LMG. The recovery
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(n=6) and repeatability (n=3) of the LC-MSMS method were tested with blank fish muscle samples fortified with 1, 2, 3 and 4 µg kg− 1. The mean recoveries over the tested concentration range of MG and LMG in eggs were 55.2% (range 50.9–63.7%, RSD 5.4–10.6%) and 54.3% (range 48.9–59.4%, RSD 6.4–6.8%) respectively. The mean recoveries over the tested concentration range of MG and LMG in muscle were 60.6% (range 58.3–67.1%, RSD 10.8–14.9%) and 66.5% (range 60.6–76.8%, RSD 8.8–12.8%) respectively. The validation of the methods is discussed in more detail elsewhere (Halme et al., 2004, 2006). 3. Results and discussion No background levels of MG residues were found in the rainbow trout eggs used for the study when they were analyzed before the MG exposures. The quality of eggs at the beginning of the study proved to be rather poor, since there were considerable numbers of dead eggs. Furthermore, attack by fungal pathogens was unavoidable since these organisms were in the water supply to the fish hatchery. Continual loss of eggs occurred in all hatching jars before the start of the MG exposure. All the control jars which were not exposed to MG became infected by fungus and all were lost. It is well known that without appropriate treatment, the fish eggs will succumb to fungal infection in greater quantities than eggs treated with an effective fungicide (e.g. Waterstrat and Marking, 1995). This was also observed in our study, where the proportion of infected eggs in the hatching jars treated with MG seemed to correlate with the concentration of MG, i.e. less infected eggs were found in those jars treated with higher concentrations of MG. As the amount of living eggs was unexpectedly low already at the beginning of the study, only one sampling of eggs was performed, on 9 d.p. f., two days after the first MG treatment. The eggs contained both MG and LMG residues. The mean concentrations (n=3) of MG in eggs were 55.8± 34.1 µg kg− 1 (exposure of 1 mg l− 1), 99.4±42.5 µg kg− 1 (exposure of 3 mg l− 1) and 220±19.5 µg kg− 1 (exposure of 6 mg l− 1). The mean concentrations of LMG in eggs were somewhat lower being 29.8±17.1 µg kg− 1 (exposure of 1 mg l− 1), 53.8±23.0 µg kg− 1 (exposure of 3 mg l− 1) and 110±15.8 µg kg− 1 (exposure of 6 mg l− 1). The presence of LMG residues in egg samples indicates that the metabolic reduction of MG can occur also in fish eggs. The reduction of MG to LMG has been previously reported to take place in fish, fish eggs and fry (e.g. Meinertz et al., 1995; Plakas et al., 1996; Bergwerff et al., 2004) whereas in water, MG exists as a dye ion or in the carbinol form (Alderman,1985) and no formation of LMG has been reported. Although the measured concentrations exhibited some variation between the three replicate hatching jars, the mean concentrations seem to correlate with the level of exposure. The eggs hatched during 27–29 d.p.f., after six repeated treatments of MG. In the newly hatched fry, high levels of both MG and LMG residues were determined. LMG was the major residue found in the fry at mean concentrations of 498 ± 59.6 µg kg− 1 (exposure of 1 mg l− 1), 1170 ± 106 µg kg− 1 (exposure of 3 mg l− 1) and 2400 ± 211 µg kg− 1 (exposure of 6 mg l− 1). The corresponding levels of MG in fry were 108 ± 17.6 µg kg− 1 (exposure of 1 mg l− 1), 276 ± 38.6 µg kg− 1 (exposure of 3 mg l− 1) and 569±117 µg kg− 1 (exposure of 6 mg l− 1). The high residue levels found in the newly hatched fry indicate that MG can penetrate through the eggshell into the embryo and further be metabolized to LMG when this fungicide is applied to eggs. The concentration of MG residues in fry was determined at regular time intervals until 57 d.p.h. Disappearance of MG residues in fry occurred rapidly as shown in Table 1. On 16 d.p.h., the mean concentrations of MG in fry were 1.4 ±0.1 µg kg− 1 (exposure of 1 mg l− 1), 1.8 ± 0.5 µg kg− 1 (exposure of 3 mg l− 1) and 4.6 ±0.4 µg kg− 1 (exposure of 6 mg l− 1). The residues of LMG had also substantially decreased being 29.2±7.3 µg kg− 1 (exposure of 1 mg l− 1), 70.9 ±6.2 µg kg− 1 (exposure of 3 mg l− 1) and 179± 33.2 µg kg− 1 (exposure of 6 mg l− 1). On 31 d.p.h., MG residues below 1 µg kg− 1 were determined in fry exposed to MG at a concentration of 6 mg l− 1. At the same time, LMG was determined in fry, the residue levels detected were below 1 µg kg− 1 (exposure of 1 mg l− 1),
28
K. Niska et al. / Aquaculture 297 (2009) 25–30
2.7 ± 0.3 µg kg− 1 (exposure of 3 mg l− 1) and 5.3 ± 1.4 µg kg− 1 (exposure of 6 mg l− 1). On 43 d.p.h., only LMG at levels below 1 µg kg− 1 were determined in fry exposed to treatments of 6 mg l− 1. On 57 d.p.h., no residues above LOD were found in any of the samples (Table 1). Analysis of samples taken from both whole fry and fry muscle on 96 d.p.h., when the fry were over three months of age, confirmed the complete elimination of residues. LC-MS/MS chromatograms of newly hatched fry and the same fry on 31 d.p.h also demonstrate the rapid disappearance of residues (Fig. 1). The obtained results are in agreement with the earlier study of Meinertz et al. (1995), in which rainbow trout eggs were repeatedly exposed to [methano-14C] labelled MG (1.0 mg l− 1, with immediate water flow). In that study, the eggs were also found to accumulate MG residues during the exposure period, with LMG being the predominant residue found both in eggs and fry. The total MG residues determined in the newly hatched fry after the exposure period were 271 ± 42 µg kg− 1 (Meinertz et al., 1995). The reported residue concentration was lower than in our study at the same exposure level (1 mg l− 1). However, a comparison of the data is difficult because of the differences in exposure and rearing conditions. The distribution of residues to MG and LMG was not reported by Meinertz et al. (1995), because the eggs they used contained measurable concentrations of MG and LMG as a background contamination. In that study, the total MG residues were found to decline during the relatively short 28-day period and were 55 ± 11 µg kg− 1. Growth dilution during this period was considered as a significant factor contributing to the decrease in the residues, accounting for 25% of the decrease in the total residue concentration (Meinertz et al., 1995). In another exposure study performed with juvenile eels by Bergwerff et al. (2004), the average tissue concentration of LMG at 15 µg kg− 1 was detectable at 100 days after exposure. The eels did not gain weight, and thus the change of body mass did not cause any dilution of the residues. The mean body weights of fry were also determined in our study at each sampling time. The growth of fry during the sampling period of 96 days is represented in Fig. 2. During this time, the body weights of fry increased linearly being followed by an exponential increase to the last measuring point. The mean body weights of fry at the end of the study had increased 150-fold compared to the mean body weights of the newly hatched fry. Had the disappearance of MG and LMG been a result exclusively of dilution caused by growth, then both the MG and LMG levels after 150-fold dilution at 96 d.p.h would still have been detectable (approximately 1–16 µg kg− 1). Some of the tanks were treated with formaldehyde solution for 30 min on 36 d.p.h and on 40 d.p.h because of an infestation with costia (Ichthyobodo necator). Formaldehyde is toxic to fish (Hohreiter and Rigg 2001) but no decrease concerning the growth of the body weight of fry could be detected after this formaldehyde treatment (Fig. 2). There is no data available on how formaldehyde could interact with MG and LMG in fish tissue. In this study the level of MG on 31 d.p.h had already decreased to below 1 µg kg− 1 and the concentrations of LMG continued to decline linearly after the treatment. The concentration of LMG in the formaldehyde treated tanks was at the same level as that determined in the untreated replicates. LMG is known to be metabolized in rats and mice through oxidative demethylation (Culp et al.,1999; Cho et al 2003). On the other hand, formaldehyde is a reducing agent but no reduction
Fig. 1. LC-MS/MS determination of MG (retention time about 2.7 min) and LMG (retention time about 11.4–11.5 min) in newly hatched fry (0 d.p.h.) (A.). The same fry on 31 d.p.h. after repeated exposure to 3 mg l− 1 MG at the hatching stage (see Section 2.1 in the text) (B.). In the newly hatched fry, residues of 255 µg kg− 1 MG and 1094 µg kg− 1 LMG were determined. During the period of 31 d.p.h., disappearance of residues was observed and the fry contained only 2.6 µg kg− 1 LMG. The ion transitions monitored were m/z 329 → 313 and 208 for both MG and LMG residues. The value of peak area is shown below the retention time.
pathways for LMG degradation have been reported. Therefore it seems most unlikely that this formaldehyde treatment would have had any effect on the disappearance of LMG residues in the fish under our test conditions. The quality of analyses was confirmed by analyzing QC samples along with authentic samples. For the analysis of egg samples, eggs sampled from the control hatching jars before exposure were used as
Table 1 Concentrations of MG and LMG in rainbow trout fry after repeated exposure of 1, 3 and 6 mg l− 1 of MG during the hatching stage. Exposure
0 d.p.h.
(mg/l)
MG
LMG
MG
LMG
MG
LMG
MG
LMG
MG
LMG
MG
LMG
108 ± 17.6 276 ± 38.6 569 ± 117
498 ± 59.6 1170 ± 106 2400 ± 211
1.4 ± 0.1 1.8 ± 0.5 4.6 ± 0.4
29.2 ± 7.3 70.9 ± 6.2 179 ± 33.2
nd nd <1
<1 2.7 ± 0.3 5.3 ± 1.4
nd nd nd
nd nd <1
nd nd nd
nd nd nd
nd nd nd
nd nd nd
a
1 3a 6b
16 d.p.h.
31 d.p.h.
The fry were sampled on 0, 16, 31, 43 and 96 d.p.h. Data is given as means ± S.D. a n = 3. b n = 2.
43 d.p.h.
57 d.p.h.
96 d.p.h.
K. Niska et al. / Aquaculture 297 (2009) 25–30
Fig. 2. The increase of mean body weight (mg) of the fry during the study period from 0 to 96 days.
the blank material for QC samples. The recovery in eggs was over 90% for both MG and LMG. However, determination of fry samples was problematic as all the control eggs were lost during incubation due to the extensive fungal growth, and thus no control fry samples for the study were available. Due to this, a blank fry sample obtained from another fish farm was used for the QC samples analyzed along with samples from 0 to 57 d.p.h. This blank fry sample was checked to be free of MG and LMG, divided into lots and frozen. It was not age matched with the study samples. This may lead to some inaccuracy in the results, however, it does not invalidate the major observation on disappearance of residues. The average recoveries were 75 ± 14% for LMG analyzed along with samples from 0 to 43 d.p.h., but only about 20% in the determination of samples at 57 d.p.h. The low recovery of the samples at this late time point was traced to problems in the automated solid-phase extraction instrument when it was treating the QC samples at the end of the set. The rest of the sample set as well as the other sets were treated without incident. The recoveries in other sample sets were satisfactory. There can be day to day variation in recoveries, suspected for example to be caused by changes in the relative humidity. However, the RSD values within sample sets were satisfactory and the results were corrected with the recovery of the QC samples of the set. LMG was not detected at 57 d.p.h. which is concordant with levels in samples taken before and after: LMG at 43 d. p.h. was already below 1 µg kg− 1 and at 96 d.p.h. not detectable (Table 1), so the not detected LMG level at 57 d.p.h. can be considered as reliable. The recovery of MG was generally low, 35 ± 7%. In the final determination (samples of 96 d.p.h), a whole fry sample (which was one of the authentic samples) and a blank fish muscle sample were used as QC samples. The recoveries in both determinations were about 80% for both MG and LMG. Despite the problems in recovery determination, the results are considered as reliable in demonstrating the time trend for residue disappearance after exposure during the hatching stage exposures. Additionally, the results of all three exposure concentrations with replicate tanks were in concordance with each other. In the earlier studies (Meinertz et al., 1995; Bergwerff et al., 2004), only a single exposure level has been studied with no replicates. Therefore, we believe that our data represents an important contribution to the study of the analysis of MG residues in the early stages of fish. 4. Conclusion The residues of MG accumulated into fry after repeated treatments of eggs, but the disappearance of residues in fry occurred rapidly and no residues of MG and LMG were determined in fry muscle at the end of the study, at 96 d.p.h. This indicates that the elimination of MG residues in
29
combination with growth of the fry to market size fish resulting in a dilution factor of about 1000 (1 g → 1000 g of body weight), means that there are no remaining residues in the fish intended for human consumption. This is in accordance with the earlier findings (Meinertz et al.,1995; Bergwerff et al., 2004). Therefore, the present results indicate that recent findings of MG/LMG residues still being detected today in adult fish (RASFF, European Commission), cannot have originated from treatment of eggs with MG. In fact, we have demonstrated that MG can be used safely if it is applied restrictedly for the treatment of fish eggs to be used for the production of farmed fish. However, it is recognized that there could be problems with reintroducing MG use. The restricted permission for the use of MG and its control to avoid any possible abuse would represent a major challenge to the authorities. Another challenge would be the appropriate handling of MG in order to ensure zero exposure to the fish culturist or the environment. However, water volumes are very small during the incubation stage of fish rearing and appropriate water management would clearly be technically feasible. The outflow of water from hatcheries to fish pools would need to be blocked and the MG containing waste water handled in a special manner. The authorities would need to monitor any possible abuse by controlling for the detection of MG and LMG in fry and fish samples. At the present time, the use of MG is not permitted by EU legislation, since no maximum residue limit (MRL) has been established for this fungicide (Rahkonen and Koski, 2002). Acknowledgements The authors thank Riitta Linjakumpu, Finnish Food Safety Authority (Evira) for her skilful assistance in performing sample analysis and the personnel in Fisheries Research Unit of the University of Kuopio for the assistance in fish maintenance. The research was partly funded by a grant from Maj and Tor Nessling Foundation (JT). References Alderman, D.J., 1985. Malachite green: a review. J. Fish Dis. 8, 289–298. Alderman, D.J., Clifton-Hadley, R.S., 1993. Malachite green: a pharmacokinetic study in rainbow trout, Oncorhynchus mykiss (Walbaum). J. Fish Dis. 16, 297–311. Allen, J.L., Hunn, J.B., 1986. Fate and distribution studies of some drugs used in aquaculture. Vet. Hum. Toxicol. 28, 21–24. Barnes, M.E., Ewing, D.E., Cordes, R.J., Young, G.L., 1998. Observations on hydrogen peroxide control of Saprolegnia spp. during rainbow trout egg incubation. Prog. Fish-Cult. 60, 67–70. Bauer, K., Dangschat, H., Knöppler, H.-O., Neudegger, J., 1988. Aufnahme und Ausscheidung von Malachitgrün bei Regenbogenforellen. Arch. Lebensm.hyg. 39, 85–103. Bergwerff, A.A., Kuiper, R.V., Scherpenisse, P., 2004. Persistence of residues of malachite green in juvenile eels (Anguilla anguilla). Aquaculture 233, 55–63. Cho, B.P., Yang, T., Blankenship, L.R., Moody, J.D., Churchwell, M., Beland, F.A., Culp, J.C., 2003. Synthesis and characterization of N-demethylated metabolites of malachite green and leucomalachite green. Chem. Res. Toxicol. 16, 285–294. Culp, S.J., 2004. NTP technical report on the toxicity studies on malachite green chloride and leucomalachite green administered in feed to F344/N rats and B6C3F1 mice. US Department of Health and Human Services June 2004. National Toxicology Program, Toxicity report series. NIH Publication No. 04-4416. Available: http:// ntp.niehs.nih.gov/ntp/htdocs/ST_rpts/tox071.pdf. Culp, S.J., Beland, F.A., 1996. Malachite green: a toxicological review. J. Amer. Coll. Toxicol. 15, 219–238. Culp, J.C., Blankenship, L.R., Kusewitt, D.F., Doerge, D.R., Mulligan, L.T., Beland, F.A., 1999. Toxicity and metabolism of malachite green and leucomalachite green during short-term feeding to Fischer 344 rats and B6C3F1. Chem.-biol. Interact. 122, 153–170. European Commission, 2008. Commission staff working paper on the implementation of national residue monitoring plans in the member states in 2007, (Council Directive 96/23/EC). Brussels 23.12.2008. Available: http://ec.europa.eu/food/food/ chemicalsafety/residues/control_en.print.htm. European Commission. Rapid alert system for food and feed. RASFF. Week 2009/9 http://ec.europa.eu/food/food/rapidalert/archive_en.htm. Halme, K., Lindfors, E., Peltonen, K., 2004. Determination of malachite green residues in rainbow trout muscle with liquid chromatography and liquid chromatography coupled with tandem mass spectrometry. Food Addit. Contam. 21, 641–648. Halme, K., Lindfors, E., Peltonen, K., 2006. A confirmatory analysis of malachite green residues in rainbow trout with liquid chromatography-electrospray tandem mass spectrometry. J. Chromatogr., B, Biomed. Sci. Appl. 845, 74–79. Hohreiter, D.W., Rigg, D.K., 2001. Derivation of ambient water quality criteria for formaldehyde. Chemosphere 45, 471–486.
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K. Niska et al. / Aquaculture 297 (2009) 25–30
Meinertz, J.R., Stehly, G.R., Gingerich, W.H., Allen, J.L., 1995. Residues of [14C]-malachite green in eggs and fry of rainbow trout, Oncorhynchus mykiss (Walbaum), after treatments of eggs. J. Fish Dis. 18, 239–247. Plakas, S.M., El Said, K.R., Stehly, G.R., Gingerich, W.H., Allen, J.L., 1996. Uptake, tissue distribution and metabolism of malachite green in the channel catfish (Ictalurus punctatus). Can. J. Fish. Aquat. Sci. 53, 1427–1433. Pottinger, T.G., Day, J.G., 1999. A Saprolegnia parasitica challenge system for rainbow trout: assessment of Pyceze as an anti-fungal agent for both fish and ova. Dis. Aquat. Org. 36, 129–141. Rach, J.J., Valentine, J.J., Schreier, T.M., Gaikowski, M.P., Crawford, T.G., 2004. Efficacy of hydrogen peroxide to control saprolegniasis on channel catfish (Ictalurus punctatus) eggs. Aquaculture 238, 135–142. Rahkonen, R., Koski, P., 2002. Roundtable 3. Post malachite green: alternative strategies for fungal infections and white spot disease. Bull. Eur. Assoc. Fish Pathol. 22, 152–157. Schreier, T.M., Rach, J.J., Howe, G.E., 1996. Efficacy of formalin, hydrogen peroxide, and sodium chloride on fungal-infected rainbow trout eggs. Aquaculture 140, 323–331.
Srivastava, S., Sinha, R., Roy, D., 2004. Toxicological effects of malachite green. Aquat. Toxicol. 66, 319–329. Thompson Jr., H.C., Rushing, L.G., Gehring, T., Lochmann, R., 1999. Persistence of gentian violet and leucogentian violet in channel catfish (Ictalurus punctatus) muscle after water-borne exposure. J. Chromatogr., B, Biomed. Sci. Appl. 723, 287–291. US Department of Health and Human Services, 2005. Technical report on the toxicity and carcinogenic studies on malachite green chloride and leucomalachite in F344/N rats and B6C3F1 mice (feed studies). National Toxicology Program NTP TR 527, NIH Publication No. 05-4463. Available: http://ntp.niehs.nih.gov/files/527-C_LowRes_Bookmark.pdf. Waterstrat, P.R., Marking, L.L., 1995. Clinical evaluation of formalin, hydrogen peroxide, and sodium chloride for the treatment of Saprolegnia parasitica on fall chinook salmon eggs. Prog. Fish-Cult. 57, 287–291. Von Westernhagen, H., 1988. Sublethal effects of pollutants on fish eggs and larvae. The physiology of developing fish. Part A. Eggs and Larvae. : In: Hoar, W.S., Randall, D.J. (Eds.), Fish physiology, vol. XI. Academic Press Inc, San Diego, pp. 253–346.