Impact of preparation method on gonad domoic acid levels in the scallop, Pecten maximus (L.)

Harmful Algae 2 (2003) 215–222

Impact of preparation method on gonad domoic acid levels in the scallop, Pecten maximus (L.) D.A. Campbell a , M.S. Kelly a,∗ , M. Busman b , E. Wiggins b , T.F. Fernandes c a c

Scottish Association for Marine Science, Oban, Argyll, Scotland PA37 1QA, UK b National Ocean Service, Charleston, SC 29412, USA School of Life Sciences, Napier University, Edinburgh, Scotland EH10 5DT, UK

Received 4 March 2003; received in revised form 25 March 2003; accepted 7 April 2003

Abstract The king scallop, Pecten maximus (L.), fishery is a valuable economic resource in the UK, and is reliant on supplying premium “roe-on” processed scallops to the continental market. A considerable degree of variability is observed in domoic acid (DA) levels among individual P. maximus and their body components, which complicates the management of the fishery during amnesic shellfish poisoning (ASP) events. This study examined the impact of professional processing and three differing laboratory preparation techniques on final gonadal DA levels. DA analysis was conducted using a LC–MS/MS procedure. The results demonstrate that different methods of preparation can significantly alter gonadal toxicities in scallops from the same site, and the extent to which DA within the digestive loop, which passes through the gonad, contributes to total gonadal DA. Mean gonadal toxicity attributed to the digestive loop contents was estimated at 4.7–24.7 ␮g DA g−1 . Despite large individual variations in toxin levels; in scallops with elevated gonadal toxicities resulting from higher digestive loop content toxicity, the effect of flushing out the contents of the digestive loop significantly reduced the DA content of the tissue and lowered the frequency of individuals harbouring levels above the 20 ␮g DA g−1 statutory safety limit. Removal of the digestive loop contents can potentially result in an 87% decrease in gonadal DA burden. Furthermore, the method applied by professional processors effectively removed the contents of the digestive loop and reduced gonadal DA to levels comparable with the laboratory techniques. Deliberate contamination with scallop mucus did not increase gonadal DA levels. The extent of toxin variation resulting from differing gonad preparations demonstrates the need to standardize scallop tissue preparation techniques during ASP events. Consequently, detailed protocols aimed at minimizing the contamination of edible components should be developed and adhered to by both processing facilities and monitoring bodies. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Amnesic shellfish poisoning; Domoic acid; Gonad; Professional processing; Pecten maximus; Scallop

1. Introduction The king scallop (Pecten maximus (L.)), fishery is a valuable economic resource in the UK, and is prin∗ Corresponding author. Tel.: +44-1631-559233; fax: +44-1631-559001. E-mail address: [email protected] (M.S. Kelly).

cipally exploited by scallop dredgers, which account for 97% of UK landings. An estimated 95% of the king scallops are processed as an adductor muscle and gonad “roe-on” product for the continental market, of which 60% is distributed as premium chilled product and 40% frozen (Denton, 1999). In July 1999, P. maximus harbouring the amnesic shellfish poisoning (ASP) toxin, domoic acid (DA),

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in gonadal tissue at levels above the statutory limit (20 ␮g DA g−1 ) were detected across areas of northern and western Scotland. This prompted a widespread closure of the king scallop fishery which persisted for more than 10 months. During the height of the incident the ban covered in excess of 19,000 square miles, to date the largest fisheries closures resulting from a harmful algal bloom (HAB) (Campbell and Kelly, 2001). The pennate diatom Pseudo-nitzschia australis was indicated as a potential causative agent of scallop toxicification, on the basis of its dominance within the phytoplankton and confirmation of its DA production capability in culture (Campbell et al., 2001). ASP events have occurred each year in Scotland since then, resulting in financial hardship for scallop dredging, diving and cultivation industries. A considerable degree of variation in DA level is encountered among individual P. maximus and their body components. Despite this, DA loading of the tissues follows a predictable rank order: all other tissue (digestive gland (hepatopancreas), mantle tissue and gills) > gonad > adductor (Arevalo et al., 1998; Campbell et al., 2001; Hess et al., 2001). Toxin levels within all other tissue account for 99% of the total DA burden and are consistently an order of magnitude over the statutory 20 ␮g DA g−1 limit. Thus during ASP events the consumption P. maximus digestive glands, mantles and gills would pose a high risk to public health. However, in Pectinids, a loop of intestine passes directly through the gonad (Purchon, 1977). The digestive loop in P. maximus can represent up to 6% of the gonadal volume (Mackie, 1986) and the contents therefore have the potential to contribute to gonadal toxicity. DA levels in gonad tissue are generally lower than the statutory 20 ␮g DA g−1 limit. However, the concentration of DA in gonad tissue can vary by an order of magnitude and DA levels above the statutory (20 ␮g DA g−1 ) safety level are regularly detected (Campbell et al., 2001). Consequently, strict regulatory regimes are now compulsory for the safe marketing of “roe-on” scallops. A total ban on harvesting is invoked when whole scallop toxicity exceeds 250 ␮g DA g−1 . Scallops from a fishing box with whole animal toxicities in the range from 20 to >250 ␮g DA g−1 can only be landed to a processor, having shown that the median gonadal toxicity is not greater than 4.6 ␮g DA g−1 , which ensures that

individuals with gonadal toxicities above 20 ␮g DA g−1 are not harvested at a 99% confidence level (Fryer et al., 2001). Adductor muscle toxicity contributes negligible amounts to the total body burden, and levels never exceed the statutory limit, even when toxin levels are extremely high in all other tissue (Campbell et al., 2001). The high individual variation in toxicities, and particularly the occurrence of DA in the gonad at levels above the regulatory limit, complicates the management of the king scallop fishery during ASP events. The variance of DA toxicity in scallop tissue may be attributed to one of, or a combination of three sources: (1) natural variation in tissue toxicity (Shumway and Cembella, 1993); (2) variable contamination of the tissue from toxic body components, during “shucking” and dissection; (3) analytical error. Characterizing variation in toxin levels is necessary both for ecological considerations and for the development of sound management protocols (Whyte et al., 1993). Given the extent to which digestive material can contaminate edible tissues, the potential to reduce the DA burden by appropriate preparation of gonad tissue should be established. The objectives of this study were to (a) examine the impact of professional processing and three different laboratory preparation techniques on final gonadal toxin levels, (b) evaluate the significance of the toxin content in the digestive loop running through the scallop gonad on final gonadal DA level and (c) assess scallop mucus as a potential source of DA contamination.

2. Materials and methods 2.1. Sampling In October 2000, and after obtaining a dispensation order from the Food Standards Agency (FSA) Scotland to fish, 80 specimens of adult P. maximus (shell height >90 mm, “market size”) were collected by SCUBA divers from each of fisheries boxes J9 (Jura), SM15 and SM16 (Sound of Mull) (sites 1, 2 and 3, respectively, Table 1). These locations are routinely used for monitoring ASP toxin levels by the FSA Biotoxin Monitoring Program and were chosen as a result of previous consistently high DA levels (above

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Table 1 Number of gonad samples per treatment and site (total number 240) Site

J9 SM15 SM16

Treatment

1 2 3

Gonad washed, digestive loop flushed

Gonad washed, digestive loop flushed and mucus contaminated

Gonad washed and digestive loop “unflushed”

Professionally processed

20 20 20

20 20 20

20 20 20

20 20 20

the 20 ␮g DA g−1 statutory level) within scallop gonad. Upon collection the 80 scallops from each site were randomly divided into four groups of 20 for each treatment (Table 1), individually sealed in zip-lock polythene bags, placed in cool boxes and transported to the Scottish Association for Marine Science Laboratory (SAMS) or the professional processing plant within 6 h, for immediate dissection and shucking, respectively. The gonad treatments were, Laboratory treatment 1: gonad dissected out, digestive tissue completely removed from the top of the gonad (digestive tissue and gonad interface), digestive loop flushed out with fresh distilled water (FDW) and the gonad washed and blotted dry to remove surface mucus. Laboratory treatment 2: same as treatment 1, but dipped into the mucus exudate of the same individual, to account for its contamination potential, either directly as a source of DA, or through indirectly transferring DA from more toxic tissues. Laboratory treatment 3: same as treatment 1, but the digestive loop content left intact and the gonad then washed with FDW and blotted dry to remove mucus. Professionally processed treatment: 1–2 professional staff shucked and trimmed the adductor muscle and gonad as a “roe on” product, which were then all rinsed with a fresh water pressure jet in one colander (approximately 30 s) under continual agitation; the gonad was subsequently removed. In the laboratory treatments, special care was taken to avoid artifactual contamination from adjacent tissues, by careful dissection, washing and drying of individual body components. All gonads were weighed to the nearest 0.001 g, sealed separately in zip-lock polythene bags and frozen at −20 ◦ C prior to DA extraction (Quilliam et al., 1989).

2.2. DA extraction and quantification in scallop tissue Gonad tissues were homogenized in a blender (3 min), which was cleaned and rinsed with methanol and then distilled water, between each sample. Four grams of tissue homogenate were re-homogenized (4 min) with 15 ml of 100% methanol, centrifuged (10 min at 5000 rpm), and a 5 ml subsample of the resulting supernatant was then filtered through a 25 mm inline filter holder with a disposable, 0.45 ␮m pore size, nylon filter membrane (Whatman). The extract was stored at −20 ◦ C prior to DA detection and quantification. The extracts were evaporated to dryness using vacuum centrifugation and resolubilized in 50/50 methanol and water prior to triplicate analysis. The samples were analyzed on a liquid chromatography– tandem mass spectrometry (LC–MS/MS) system consisting of an Agilent Model 1100 high performance liquid chromatography system, coupled to either a SCIEX API-III triple quadrupole or a Finnigan LCQ ion trap mass spectrometer. The chromatography was performed on a C18 reversed phase column with a 0.2 ml min−1 flow of a 1–95% gradient of methanol:water. All solvents had 0.1% trifluoroacetic acid added. A portion of the effluent from the HPLC system was directed into the electrospray ionization source of the mass spectrometer via a flow splitter. The mass spectrometer was operated in positive mode with [M+H]+ ions (−312 m/z) being isolated in a first stage of MS analysis. The isolated ions were subjected to ‘collision induced dissociation’ reaction conditions which are expected to stimulate the fragmentation of the [M+H]+ ions into characteristic product ions. All quantitations were based on the

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integrated chromatographic intensity areas of one of the fragment ions (at 267 m/z), and the appearance of other characteristic ions was used as confirmatory evidence for the DA ion’s identity (Scholin et al., 2000). 2.3. Statistical analysis Means and standard errors were calculated for all data sets. After ensuring the data were normally distributed (Anderson Darling Test) and achieved homogeneity of variance (Bartlet’s test), a two-factor ANOVA and Tukey’s multiple pairwise comparison tests were used to test for significant differences among treatments and sites. A non-parametric, Kolmogorov Smirnov Z-test was also used to assess significant differences among pair wise treatment data sets. The statistics packages Minitab® version 13.1 and SPSS® version 11 were used for all analyses.

3. Results 3.1. Gonad wet weight Normality and homoscedasticity were achieved by applying a log10 transformation to the raw data. Significant differences were found among the mean gonad wet weights (g) of scallops from each site (F = 140.4, P =< 0.001, d.f. = 2228). Individuals from site 3 had a significantly greater mean gonad wet weight than individuals from sites 1 and 2, and the mean gonad wet weight was greater in individuals from site 2 than 1. However, there was no significant difference in mean gonad size after the scallops had been randomly distributed among the treatments (F = 0.53, P = 0.659, d.f. = 3228). Therefore, the potential effect of allometric relationships between DA retention and scallop size with respect to the differing gonad preparation treatments were ignored in further analyses (Table 2). 3.2. Gonad DA toxicity Normality and homoscedasticity were achieved by applying a log10 transformation to the raw data. The mean coefficient of variation (CV%) for DA among triplicate analyses of any one sample accounted for by the detection method was ±11.8%, indicating that

Table 2 Mean and standard error (S.E.) for gonad wet weight (g) of P. maximus for each treatment and site (back transformed from the log10 scale) Variable

n

Mean gonad wet weight (g)

S.E.

Treatment 1 2 3 4

60 60 60 60

3.12 2.96 3.13 3.34

a a a a

1.130 1.101 1.102 1.086

Site 1 2 3

80 80 80

1.43 a 4.09 b 5.26 c

1.065 1.060 1.058

Means with different alphabets in the same column are significantly different (P < 0.05, two-factor ANOVA and Tukey’s test on log10 transformed data).

the variability observed among individual gonads was unlikely to be a result of analytical error. In each treatment, scallops with gonadal toxicities (␮g DA g−1 wet weight) exceeding the 20 ␮g DA g−1 statutory limit were encountered. The frequency of these individuals ranged from 6 out of 60 individuals (treatment 4—professionally processed) to 25 out of 60 (treatment 3—digestive loop unflushed), of which 22 of these individuals occurred in scallops from sites 1 and 3, where overall mean DA levels where observed to be highest (Table 3). Significant differences in mean gonadal DA toxicity were observed among sites (all treatments combined for each site) (F = 6.11, P = 0.003, d.f. = 2228) and treatments (all sites combined for each treatment) (F = 3.50, P = 0.016, d.f. = 3228). The mean gonadal DA content of scallops from sites 1 and 3 were not significantly different (P > 0.05) and exhibited significantly higher gonadal DA levels than scallops from site 2. The mean DA toxicity of gonads in treatment 3 was significantly greater than that in treatment 2 (mucus contaminated) (P < 0.05). In treatments 1 and 4, the mean gonadal DA content was not significantly different from all other treatments (P > 0.05) (Table 3), demonstrating that professional processing methods can reduce gonad DA contamination to levels comparable with those produced by a rigorous laboratory technique and that scallop mucus did

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Table 3 Mean and standard error (S.E.) domoic acid levels (␮g DA g−1 wet weight) in the gonad of P. maximus for each treatment and site (back transformed from the log10 scale) Variable

n

Treatment 1 2 3 4

60 60 60 60

Site 1 2 3

80 80 80

Mean 6.74 5.72 10.42 6.72

ab a b ab

8.02 b 5.17 a 9.04 b

S.E.

Minimum

Maximum

95% CI

1.143 1.152 1.188 1.124

0.46 0.12 0.32 0.61

89.36 43.07 91.17 49.06

5.15–8.80 4.31–7.60 7.38–14.70 5.32–8.49

1.121 1.127 1.143

0.46 0.12 0.32

91.17 42.07 89.36

6.39–10.07 4.07–6.56 6.93–11.80

Number >20 (␮g g−1 ) 9 8 25 6 18 6 24

Means with different alphabets in the same column are significantly different (P < 0.05, two-factor ANOVA and Tukey’s test on log10 transformed data). Minimum and maximum levels of DA obtained (␮g DA g−1 ), 95% confidence intervals (CI) and number of individuals with toxin burdens >20 ␮g DA g−1 are given.

not contain sufficient levels of toxicity to increase gonadal DA toxicity. The interaction term was also significant (sites × treatments, F = 2.26, P = 0.038, d.f. = 6228), indicating different responses, with respect to of gonadal toxicity, between the sites and the treatments. A Kolmogorov Smirnov Z-test was used to compare the frequency distributions of DA levels (␮g DA g−1 ) yielded by the different treatments (sites pooled). The results indicated that no significant differences were found among treatments 1, 2 and 4. However, treatment 3 was isolated as yielding significantly more extreme values of gonadal DA toxicity compared to all other treatments, at the 95% confidence level. These results established gonads from treatment 3 to have had greater levels of DA, resulting from the DA toxicity of the digestive loop contents. From the 95% confidence intervals of gonadal toxicities for scallops from site 1 in treatment 2 (3.6–8.0 ␮g DA g−1 ) and treatment 3 (12.7–28.3 ␮g DA g−1 ), the variation in mean gonadal DA toxicity, as a result of the differing gonad preparation treatments, ranged between 3.6 and 28.3 ␮g DA g−1 . Thus the mean gonadal DA burden in scallops from site 1 was reduced by 4.7–24.7 ␮g DA g−1 by treatment 2. This equates to a decrease in gonadal toxin loading of 87–40%, through washing and removing the digestive loop content. On removal of treatment 3 from the data set, no significant differences in gonadal toxicities among the sites (F = 2.73, P = 0.07, d.f. = 2, 171) and treatments (F = 0.51, P = 0.60, d.f. = 2, 171) were observed. Thus, different preparatory methods of gonads

prior to toxin testing can significantly alter gonad toxicities in scallops from the same site. Fig. 1c and d illustrate the sources of variation inherent in the mean gonadal DA toxicities with respect to the treatment and sites given in Fig. 1a and b. Treatment 3 gave the widest variation in gonadal DA toxicities among sites, and gonads from treatment 2 showed the least variation in DA toxicities among sites. Gonadal toxicities in scallops from site 2 showed the least variation among the treatments and did not exhibit elevated gonadal toxicities in treatment 3 as observed in individuals from sites 1 and 3, which were significantly higher (P < 0.05). This suggests that the toxicity of the digestive loop content in scallops from site 2 was lower, and was not at a level sufficient to increase gonadal DA toxicity, in contrast to individuals from sites 1 and 3.

4. Discussion The levels and variability of DA found in the gonad tissue of P. maximus in the present study are consistent with those of previous studies (Arevalo et al., 1998; Campbell et al., 2001; Hess et al., 2001). The results demonstrate that DA contamination can be derived from the digestive loop contents. The mean level of gonadal toxicity attributed to the digestive loop contents was estimated at 4.7–24.7 ␮g DA g−1 . The digestive loop descends from the stomach sac and passes through the digestive gland immediately

220 D.A. Campbell et al. / Harmful Algae 2 (2003) 215–222 Fig. 1. Overall mean and standard error domoic acid toxicity levels (␮g DA g−1 wet weight) in the gonad of P. maximus for each treatment (a) and site (b) (back transformed from the log10 scale). Individual mean and standard error DA toxicity levels (␮g DA g−1 wet weight) in the gonad of P. maximus with respect to treatments (c) and sites (d).

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before entering the gonad (Purchon, 1977). As a consequence, the toxicity of the digestive loop contents can correspond directly to the toxicity of the stomach and digestive gland. Given that during ASP events, mean digestive gland DA concentration routinely exceeds 200 ␮g DA g−1 and values above 1000 ␮g DA g−1 have been reported (Campbell et al., 2001), the gut contents present a considerable source for potential contamination, which is reflected by the high variation observed among DA levels in gonads with loop contents included. Individual variation may also result from variable feeding activity of scallops resulting in differing fullness of the digestive loop and variation in the seasonal relationship between gonad volume and the loop volume (Mackie, 1986). Potentially toxic, viable cells of Pseudo-nitzschia spp. have been observed intact, within the contents of the digestive loop, when routine plankton monitoring did not detect cells in the water column (Campbell et al., unpublished). Work to determine the source and temporal variation of the toxic digestive contents may prove useful to assess the potential of the constituents to contaminate over time, and give inference to the role of Pseudo-nitzschia spp. ingestion and DA retention within the scallop. In scallops with elevated gonadal toxicities resulting from higher digestive loop content toxicity, the effect of flushing out the contents of the digestive loop significantly reduced the toxin burden of the tissue and lowered the frequency of individuals harbouring levels above the 20 ␮g DA g−1 statutory limit. Furthermore, the methods applied by the processor, i.e. of washing and agitating an adductor muscle and gonad “roe on” product, was able to remove the contamination effect caused by contents of the digestive loop contents. This demonstrates that professional processing methods can reduce gonadal DA contamination to a standard comparable with that of a laboratory technique. Within the current study, scallop mucus did not increase gonadal DA toxicity. However, studies such as analysis of tissue cores taken from the center of lyophilised gonad, to eliminate the surface contamination (Cembella et al., 1994), are needed to determine more accurately natural toxin variation and to establish the level attributed to contamination. This will help develop effective tissue preparation protocols with known cleaning efficiencies. Interestingly, individuals subjected to mucus showed the least variation in DA

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toxicities among sites of all the treatments. The mucus may have shielded the gonad surface by providing a less attractive medium for further DA contamination and/or have contained properties that directly reduced DA concentrations; alternatively, the addition of mucus may have merely diluted the concentration of DA. These inferences do, however, suggest alternative solutions to reduce surface contamination of tissues at the processing stage. The potential to develop chemical washes that either directly break down DA or possess a greater affinity to bind and remove DA more efficiently than water, and the practical use of autochthonous DA-utilizing bacteria in bivalves to eliminate the toxin (Stewart et al., 1998) should be ascertained. Furthermore, such applications may prove a more viable approach than to depurate DA-contaminated scallops under controlled conditions. The extent of toxin variation resulting from differing gonad preparatory methods emphasizes the need to standardize scallop tissue preparation techniques during ASP events. Consequently, detailed protocols aimed at minimizing the contamination of edible components, for both processing facilities and monitoring bodies, should be developed. All individuals involved in handling toxic scallops should be informed of the anatomical distribution of DA within the scallop and the potential sources of and procedures used to minimize contamination. It is imperative that processors and monitoring bodies avoid rupturing the digestive sac and exposing the edible portion, or component for testing to highly toxic exudates. Furthermore, these toxic tissues must be immediately discarded upon removal. During the process of “trimming”, all traces of digestive gland from the gonad digestive gland interface and digestive tract, such as the anal tube around the adductor muscle, should be removed. The kidneys of P. maximus have been shown to sequester paralytic shellfish poisoning (PSP) toxins at markedly higher levels than in the digestive gland (Lassus et al., 1996). Therefore, as ASP and PSP toxins attain proportionally similar anatomical distributions within scallops (Shumway and Cembella, 1993), the kidneys should also be removed. The roe-on product should not be allowed to soak, but be washed in continuously running water and sufficiently agitated to aid clearance of the digestive loop contents. Protocols should also be implemented to minimize the risk of cross contamination, in order to maintain the integrity of the

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batch product and samples for end product testing. These measures are closely in line with current industry practices and their implementation should prove both straightforward and inexpensive. Furthermore, standards should be set for the interval between harvesting scallops and their preparation for market and end product testing, to achieve consistency between processors and monitoring bodies and prevent contamination by digestive gland leakage through cell degradation. This interval would be dependent upon harvest method (dredged versus diver collection) and handling factors post-harvest. Reducing contamination will obviously deliver a better estimate of biological variability among DA levels of scallop populations for spatio-temporal comparisons. Moreover, standardization between industry and regulatory bodies will allow for a more confident comparison between processor produced end product samples and samples collected as part of the biotoxin monitoring program. Public knowledge that all processors meet an approved standard, with regard to handling toxic scallops, will ultimately support consumer confidence in the scallop product. Furthermore, if contamination rates can be minimized to a known efficiency, this would facilitate the development of risk assessment models to assess scallop toxicity with respect to the rate of consumption by humans.

Acknowledgements We thank Colin Campbell and Mull Marine Services for the sample collection and Loch Fyne Seafarms for processing the scallops. This study was funded by The Highlands and Islands Enterprise and The Highland Council. References Arevalo, F.F., Bermudez de la Puente, M., Salgado, C., 1998. ASP toxicity in scallops: individual variability and tissue distribution. In: Reguera, B., Blanco, J., Fernandez, M.L., Wyatt, T. (Eds.), Harmful Algae. Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, pp. 499– 502. Campbell, D.A., Kelly, M.S., 2001. ASP in king scallops. Shellfish News 11, 24–25.

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