To assess whether depuration may provide a cost-effective means for producing a safe, live,
whole product we will conduct a series of laboratory experiments evaluating the potential
acceleration of depuration through four parameters: 1) feeding; 2) temperature; 3) emersion; and
4) cumulative parameters. Our null hypothesis for each experiment is that the parameter will not
accelerate depuration. We will first test if feeding increases the depuration rate using three
treatments; no food, a frozen seafood mix that includes mussels and clams that we and others
often use when holding crabs and lobster, and a commercially available pelleted diet used in crab
aquaculture (Turbo® 49.7% crude protein, 6.7% lipid, CP Feeds Thailand; Truong et al., 2008).
The no food treatment will serve as an experimental control and as a baseline assessment for
depuration rates of each species. The feeding experiment will be followed by a second
experiment evaluating whether depuration can be accelerated by water temperature. For this
experiment, test animals will be exposed to three water temperatures appropriate for the test
species; red rock and Dungeness crab, 9°C, 14°C, 19°C; lobster, 12°C, 17°C, 22°C. Third, we
will determine the effect of emersion on excretion rate by exposing test animals daily to one of
three emersion periods (0, 30 and 60 minutes) followed by re-immersion. Our fourth experiment
will entail combining a subset of the most promising treatments for each species to identify
optimal (fastest and most cost-effective) depuration methods.
We will conduct these experiments using methods similar to those used by Lund et al. (1997).
Prior to each experiment, test animals will be dosed with DA by feeding them razor clam
(Siliqua patula) meat with quantified, high levels of DA. Razor clams containing high DA levels
are typically available along the west coast because they retain DA for very long periods of time.
Samples will be collected throughout the project period and frozen until used in the various
experiments. Intentional dosing of crabs and lobsters will ensure the animals have been exposed
to DA in a consistent manner, as opposed to collecting crabs and lobster from the wild during
and after DA-producing blooms and assuming they contain similar DA concentrations.
For the factorial experiments, individual test animals will be assigned randomly to a treatment
with a 6 replicates per treatment – the same number of individual sampled by the California
Department of Public Health (CDPH) when evaluating DA levels in crabs and lobsters to inform
seafood health advisories and fishery closures (and the same sample size used by Lund et al.
(1997)). That said, if variation in DA levels among animals fed DA-contaminated clams in the
laboratory necessitates increased replicate numbers, we will compensate by reducing the number
of treatment levels and experiments (e.g. only two temperature levels). In addition to the
treatments we will have two additional sets of animals that will be used to identify DA levels of
animals when brought into the laboratory (pre-holding) and DA levels after being fed DAcontaining
clams (post-fed). Test animals will be held in separate tanks with filtered, sterilized
(UV-treated) seawater, as is typical for depuration systems (Lee et al. 2008, 2010), and fasted for
3 days prior to initiating laboratory-induced DA accumulation to ensure that their digestive
systems are completely evacuated (Curtis and McGaw 2010).
DA will be analyzed among a group of replicate animals at specified intervals: pre-fed (1
sample), post-fed (1 sample), and at days 5 and 10 of the experiment (1 sample from each of 3
treatment groups on each day). We have chosen day 10 as our cut-off sampling period because
literature shows it to be a reasonable time to elicit significant DA reduction (Lund et al., 1997)
but short enough to be plausible as a commercial fishery mitigation method. The additional few
days will enable us to identify factors that look promising and that may be further accelerated
when used in combination with other promising conditions. Because our primary interest is in
developing methods to improve food safety, we will focus on quantifying DA load in the
hepatopancreas (viscera); for the optimized final depuration methods we will measure DA in the
meat as well. At the appropriate sampling time, and following ice bath immersion, crabs and
lobsters will be sacrificed and their viscera and body/tail (meat) removed.
We will use CDPH protocols and associated methods (Quilliam et al. 1989, Quilliam 2003) to
determine DA levels in samples. Analyses will be done on raw rather than cooked
organisms/tissue, a deviation from CDPH protocols. (The use of cooked organisms is based on
the assumption that most people boil crab and lobster before eating it, where in fact there is a
range of preparation methods.) Analysis of raw tissue will 1) reduce loss of DA to the cooking
process (Hatfield et al. 1995; Costa et al. 2003) that could mask differences in depuration
efficacy reduce the effort and time required for processing, 2) reduce the effort and time required
for processing, 3) provide baseline information that can be used to estimate risks associated with
multiple cooking methods (e.g., grilling, steaming, stir-frying), and 4) provide information on
potential risk associated with consumption of raw product.
Once the tissues have been processed and DA extractions completed, presence and levels of DA
will be determined using similar CDPH HPLC-UV (DAD) and mass spectrometry analytical
methods (Dhoot et al. 1993), methods we have been using in our laboratory and that have been
validated through comparisons with CDPH DA-analyzed shellfish samples. DA calibration
curves will be created by running known dilutions of a DA standard and plotting against peak
area determined from the UPLC UV detection at 242 nm. Peak area calculation will be done
using peak integration with Waters MassLynx 4.2 software (Waters Corporation 2018). Samples
will be analyzed using the same method as the DA standard.
Our depuration experiments will be conducted for each species. This is necessary because
depuration of DA, and biotoxins generally, varies among species (see reviews by Shumway
1995; Trainer et al. 2012). Based on our field data of DA-producing HABs in the Santa Barbara
Channel, and data from CDPH and others, the California spiny lobster may be impacted for less
time than Dungeness crab, although both have experienced prolonged, high DA levels.
Depuration rates may explain, at least in part, differences among species. The levels of DA in the
viscera and, where appropriate, the body/tail will be compared using an analysis of variance and
post hoc univariate F-tests. Data will be transformed prior to analysis as needed to satisfy
assumptions of normality and homoscedasticity (Zar 1999).
Research results will be shared through publication of a summary report, a scientific manuscript,
and materials for fishing communities. The outreach materials will be distributed to interested
individuals and groups, such as CDFW, CDPH, OEHHA, the Legislature’s Joint Committee on
Fisheries and Aquaculture, the state’s Dungeness Crab Task Force, the California Ocean
Protection Council, and aquaculture and fishing communities.