The challenge of reducing the fecal indicator bacteria load and preventing spawning during depuration of Perna perna mussels

– Perna perna mussels commonly spawn during depuration under water temperatures above 18°C. A previous study conducted under experimental conditions (15L aquariums) has suggested reducing the temperature to decrease the odds of spawning. This study monitored the concentrations of Escherichia coli in mussels during three 48-hour and two 72-hour depuration cycles conducted in a small (600L) commercial depuration unit, under temperatures of 14°C and 17°C. The protocol prevented mussels from spawning, as this behavior was observed only in a few mussels in one out of the four depuration cycles. The reduction of fecal indicator bacteria loads, in turn, was unsatisfactory in all depuration cycles, with at least 67% of the samples from cycles 1, 3, 4, and 5 presenting E. coli concentrations above the end product testing limit (230MPN 100g −1 ) after 48 hours of depuration. Moreover, none of the sample cycles showed bacterial concentrations below that limit, even after 72 hours of depuration.


Introduction
Filter-feeding bivalve mollusks accumulate microorganisms, including human pathogenic bacteria and viruses when grown in sewage-polluted waters, posing a health risk when consumed raw or lightly cooked (Lees, 2000;Butt et al. 2004).Depuration is a postharvest treatment applied to reduce microbiological contaminants from commercially-harvested mollusks and consists of placing mollusks harvested from moderately polluted waters in tanks with clean seawater for a period of time (commonly at least 42 hours).This allows mollusks to cleanse or purge themselves of microbiological contamination while continuing their normal filter-feeding and digestive processes (Rees et al., 2010).This procedure can be applied to any species of mollusks traded worldwide, including mussels.
The South American rock mussel, Perna perna, is an important aquaculture resource in several countries and Brazil largely contributes to the production volume of this species, with a production of 7,000 tonnes in 2022 (available at: www.infoagro.sc.gov.br).
Microbiological results from research studies (Souza et al., 2022) and from the Santa Catarina state mollusk monitoring program (available at: www.cidasc.sc.gov.br)indicate that mollusks from most farming areas should undergo a post-harvest treatment to reduce microbiological contamination and ensure that the mollusks are safe to consumers.However, P. perna depuration is still incipient in Santa Catarina due to difficulties inherent to the depuration of this species and the weak law enforcement by sanitary authorities.Temperature variations and physical stress can trigger spawning in bivalve mollusks (Seed and Suchanek, 1992) and mussels such as P. perna are prone to undesirable spawning during depuration.Previous depuration studies at temperatures above 18°C resulted in widespread mussel spawning (Suplicy, 1999).A recent study (Suplicy et al., 2024) evidenced the high frequency of spawning during P. perna depuration, in which 21 out of 22 depuration assays resulted in spawning.Spawning reduces depuration efficiency since gametes in the water column make the water turbid, which, in turn, reduces the efficiency of the UV disinfection systems (Lees et al., 2010).Furthermore, spawning reduces the meat yield and increases the physiological stress of the animals, which can affect their pumping activity and impair the elimination of microbiological contaminants (Power and Collins, 1989).Suplicy et al. (2024) showed that, at experimental scale (15L-aquariums), conditioning the depuration water to temperatures 5°C lower than those in the harvesting area reduces the spawning frequency by more than 50%, and this chance drops to 8.2% when the temperature difference reaches 10°C.However, the authors did not investigate the implications of temperature modulation during depuration in the reduction of microbial loads and recommended further studies to "show how reducing water temperature during depuration influences its efficiency in terms of pathogen reduction."Following this recommendation, we investigated the impact of reducing the water temperature in the tanks on the efficiency of P. perna depuration in terms of reduction of E. coli (a species of bacterium that occurs in sewagepolluted waters and indicates the potential presence of pathogens in the mollusks), aiming to prevent the mussels from spawning.In the trials, we used commercial scale depuration units to ensure that the experimental conditions matched those in industrial premises.

Experimental setup and sampling
The study monitored the concentrations of the bacteria Escherichia coli in mussels during three 48-hour and two 72-hour depuration cycles conducted from February to October 2023 in a small-scale depuration unit.The unit was composed of a 600L depuration tank and a recirculation system containing a water pump (1hp and water flux adjusted at 2.000L hour −1 ), a chiller (1hp), and an ultraviolet disinfector (50WATTS lamp) (Fig. 1).The water temperature in the first four depuration cycles was maintained around 14°C, being increased to 17°C in the fifth experiment.
The mussels used in this study were obtained from a known sewage impacted aquaculture zone.The mussel density adopted was 90kg per batch, with animals accommodated in nine mesh trays containing 10kg of mussels each.Every 12 hours, water parameters were monitored and three groups with six mussels each were carefully collected from the upper trays, avoiding water disturbance that could resuspend feces or pseudofeces sedimented during depuration.The water parameters monitored included salinity and pH, obtained using a HI9829 multiparameter probe (Hanna, USA), and NH 3 concentration, using a photocolorimeter (Alfakit, AT100P, Brazil).The water temperature was measured every hour using a temperature logger (HOBO Pendant MX Water Temperature Data Logger, Onset, USA) deployed inside the tank.By the end of each depuration cycle, after draining the water from the tank, three additional mussel samples were collected from the trays positioned close to the bottom of the tank.
The mussel samples were immediately packaged in plastic bags, placed in styrofoam boxes and transported to the laboratory for microbiological analysis within 5 hours.
Concentrations of Escherichia coli in mussel flesh were estimated using the Most Probable Number (MPN) technique (ISO 16649-3 2015).The Condition Index (CI) of 24 randomly selected mussels was estimated before and after each depuration cycle.For this, the mussels were hand-picked from the trays and dried for 24 hours in an oven at 60°C and the weight of their dry flesh was divided by their total dry weight.

Data analysis
The E. coli and CI results did not meet the normality and homoscedasticity assumptions; thus, the analysis was performed using non-parametric methods.The CI of mussels used in the different depuration cycles was compared using the Kruskal-Wallis H test followed by the Dunn's test.The comparison of CI before and after depuration and the comparison of the final E. coli levels in samples collected from the upper and lower trays were both performed using the Mann-Whitney U test.
In total, two E. coli limits (230 and 700MPN 100g−1) were used as benchmarks in the data analyses since they are the legal limits established in the Codex Alimentarius (Fao and Who, 2008) for live or raw mollusks that says that the legal limits are that in five (5) 100g samples of the edible parts none may contain more than 700 E. coli and not more than one (1) of five (5) samples may contain between 230 and 700 E. coli, or equivalent as decided by the competent authority having jurisdiction.

Results and discussion
Regarding water parameters, little variations on pH and salinity were recorded during the study.The maximum ammonia level was 0.35mg L −1 (Table 1).The water temperatures adopted prevented mussels from spawning, as this behavior was observed in only a few mussels and only in cycle 3 out of the four monitored depuration cycles.
The mussel CIs were significantly different between the depuration cycles (KW chi-squared=91.0,p-value<2.2e−16 ), with the highest indexes recorded in cycle 2 and the lowest in cycle 5 (Fig. 2).Analysis of all data combined, as well as of data from each depuration cycle separately, revealed virtually no difference of CIs before and after depuration, indicating that the animals did not lose weight during the process (Fig. 3).The exception was depuration cycle 5, which was conducted with the highest water temperature and showed lower CI after depuration compared to the initial values (W=431, p-value=0.003).
The reduction of fecal indicator bacteria concentrations, in turn, was unsatisfactory in all depuration cycles (Fig. 4).After 48 hours, at least 66.6% (two out of three) of the samples from cycles 1, 3, 4, and 5 presented E. coli levels above 230MPN 100g −1 .The cycle 2, which held the lowest initial E. coli levels, showed the best results after 48 hours; however, it still presented one sample recording an E. coli level higher than 700MPN 100g −1 .Prolonging the depuration period to 72 hours in cycles 4 and 5 did not help reduce E. coli concentrations.By the end of the 72 hours cycle, one sample was still above 230MPN 100g −1 in cycle 4 and all samples were above this limit in cycle 5.
The final E. coli levels in mussel samples obtained from the upper or lower layers of trays did not differ significantly (W=15.5, p-value=1).
The efficiency of depuration is influenced by the system design and by different parameters and practices (e.g.shellfish load, water flux and physicochemical parameters, physical shocks) during the process (Souza et al., 2021).The depuration system used in this study is similar to those used by the industry around the globe (Seafish, 2018) and its efficiency has been proven for oysters Crassostrea gigas in a study also conducted in Santa Catarina state (Bobermim, 2013)   process observed in the present study is probably related to the colder water used.
Water temperatures ranging from 5°C to 15°C are recommended to depurate two of the most produced species in the world, namely the Mediterranean mussel Mytilus galloprovincialis and the blue mussel Mytilus edulis, to ensure adequate mollusk filtration and prevent spawning (Lee et al., 2008).This study shows that such temperatures are too low for P. perna, which can be expected since this species has different water temperature requirements to grow and reproduce from those of M. galloprovincialis.In a monitoring study in Plettenberg Bay, South Africa, Zardi et al. (2007) evaluated the reproductive behavior of these coexisting species and observed that M. galloprovincialis spawning events always occurred at temperatures ranging from 16.4 to 19.5°C, whereas P. perna spawned at the highest and lowest temperatures recorded in the 18 months of the survey (~14.5°C and ~24.2°C, respectively).
Further than impairing an efficient reduction of microbial loads, the water temperatures used in this study might have affected the mussel's post depuration survival.This aspect was not specifically addressed by the study.However, the mussels used in the trials were returned to the marine farm after depuration and the farm manager reported large mortalities.Further studies are needed to check whether the observed mortality is related to the temperatures adopted during depuration or if it is common occurrence for mussels submitted to depuration in general.
Based on the results, we can state that reducing the fecal indicator bacteria load and preventing P. perna mussels from spawning during depuration remains a challenge.In Brazil, these mussels are traditionally sold and consumed cooked (Furlan et al., 2007).However, to date, there is no specific protocol that ensures the effectiveness of heat treatment for minimizing the microbiological risks of P. perna mussels.Moreover, different studies provide evidence that the light cooking practices (steaming, searing) usually adopted by final consumers and restaurants do not necessarily provide the temperature/time combination required for the efficient inactivation of pathogens in bivalves (Souza et al., 2022).We highlight that there is still a high demand for live mussels in local markets or specific niches, such as haute cuisine restaurants.Therefore, future studies could try different approaches to allow microbial load reduction without mussel spawning during depuration.However, it is also urgent to validate a protocol that ensures the effectiveness of heat treatment for minimizing the microbiological risks of P. perna mussels produced in Brazil.

Conclusion
This research confirms that maintaining water temperature from 14°C to 17°C is an effective strategy to prevent mussels from spawning during depuration on a commercial scale, as previously observed on an experimental scale.
Depuration is not capable of efficiently reducing the fecal bacteria load.Even with extended depuration periods of up to 72 hours, E. coli levels were not reduced to within internationally established legal limits (Fao and Who, 2008).
We recommend that future studies try different approaches to prevent P. perna spawning during depuration.Moreover, we also suggest validating heat treatment protocols, as well as relocating mussels to approved areas to ensure the safety of P. perna mussels cultivated in Brazil., n.1, p.315-321, 1979

Figure 1 .
Figure1.Small-scale depuration unit used in the study.The unit was composed of a 600L depuration tank and a recirculation system containing a water pump, a chiller, and an ultraviolet sterilizer Figura 1. Unidade de depuração de pequena escala utilizada no estudo.A unidade era composta por um tanque de depuração de 600L e um sistema de recirculação contendo bomba d'água, chiller e esterilizador ultravioleta

Figure 2 .
Figure 2. Condition index of mussels before the five depuration cycles monitored.The letters indicate homogeneous groups according to the Dunn's Test Figura 2. Índice de condição dos mexilhões antes dos cinco ciclos de depuração monitorados.As letras indicam grupos homogêneos segundo o Teste de Dunn

Figure 4 .
Figure 4. Evolution of Escherichia coli levels in mussels during the five depuration cycles monitored.Symbols indicate raw values and lines indicate the summary results in terms of geometric mean.The black horizontal dashed line indicates the legal limit of 230MPN 100g −1 Figura 4. Evolução dos níveis de Escherichia coli nos mexilhões durante os cinco ciclos de depuração monitorizados.Os símbolos indicam valores brutos e as linhas indicam os resultados resumidos em termos de média geométrica.A linha tracejada horizontal preta indica o limite legal de 230MPN 100g -1

Table 1 .
Physical-chemical parameters of the seawater during the different depuration cycles Tabela 1. Parâmetros físico-químicos da água do mar durante os diferentes ciclos de depuração