In the present study, A. simplex s.s. was the only species molecularly identified from the body cavity of C. harengus membras. Nematodes isolated from G. morhua fillets represented P. decipiens s.s., P. krabbei and A. simplex s.s. This is in line with previously published genetic data according to which A. simplex s.s. is the only Anisakis species recorded in the Baltic (Mattiucci et al. 1997). According to Gay et al. (2018), the major species isolated from the fillets and the viscera of G. morhua from the Northern North Sea was A. simplex s.s. (99.8%), while only three individuals of A. pegreffii were identified in fillets. Two sibling species of P. decipiens complex (P. decipiens s.s. and P. krabbei) were identified in fillets of G. morhua from the Northern North Sea (Gay et al. 2018).
The freezing rate is considered the most important factor influencing the size and location of ice crystals during freezing (Dalvi-Isfahan et al. 2017). Fast freezing produces small ice crystals, resulting in less tissue damage than a slow freezing rate, which usually results in the formation of more damaging, larger ice crystals (Erikson et al. 2016). The process of ice crystal formation is important not only for the quality of the fish product but also for the survival of anisakid nematodes during freezing (Wharton and Aalders 2002).
The results of our experiment did not demonstrate a difference in freezing tolerance of A. simplex s.s. and Pseudoterranova spp. All anisakid larvae in G. morhua fillets died at a temperature of − 15 °C or lower. However, this lack of difference in freezing tolerance requires further confirmation through future studies specifically designed for that purpose. According to Stormo et al. (2009), P. decipiens larvae may have a freeze tolerance similar to that of A. simplex. In contrast, the study of Lanfranchi and Sardella (2010) revealed that 100% of Anisakis sp. larvae survived 5 h at temperatures in the range of − 18 to − 22 °C, whereas larvae of Pseudoterranova sp. died within the first 3 h, which may suggest a lower resistance to freezing of the latter species.
Interpretation and comparison of results obtained by other authors are often difficult due to the unavailability of detailed data on the conditions of the freezing process used. Even if the same temperature and holding time are used in separate experiments, the results obtained can vary depending on the type of freezer, the freezing rate, and the nature of the frozen sample. In our approach, we assessed the impact of the following parameters on the survival of anisakid nematodes: temperature set in the freezer, time for the internal temperature of the sample to reach the set temperature, type of freezer (single- vs double-compressor), and type of raw material (skinless fillets vs whole fish; fatty vs lean fish). We demonstrated that A. simplex s.s. larvae survived in C. harengus membras held in a single-compressor freezer at − 20 °C for 24 h. In this freezing device, the time to reach target temperature in the sample was over 23 h. Under the same time-temperature conditions, but in a double-compressor freezing device (with a freezing rate twice as high), no viable parasites were recovered from C. harengus membras. In this case, the time needed to achieve the target temperature was much shorter (10 h 15 min). The freezing rate depends also on how full the freezer is, the mass of the fish, and the sample size. Wharton and Aalders (2002) found that the core of 20-kg containers of fish did not achieve ambient temperatures of − 35 °C after 28-h exposure, whereas C. harengus membras samples frozen during the present study reached − 35 °C after 6 h 45 min, which resulted in an effective freezing time lasting more than 15 h. No A. simplex larvae were viable after freezing under these time-temperature conditions. This result is in accordance with the finding of Deardorff and Throm (1988) that blast freezing to at least − 35 °C for 15 h effectively killed larval A. simplex. Other factors can affect the survival of anisakid nematodes, such as species of fish or type of raw material. Whole fish, still containing their viscera, might offer better physical protection to nematodes during freezing than gutted and headed fish (Deardorff and Throm 1988; Adams et al. 2005). Our results revealed that in the same (single-compressor) freezer, the effective freezing time for skinless G. morhua fillets at − 15 °C was almost twice as long (17 h 45 min) as for whole (ungutted) C. harengus membras (8 h 45 min).
EU Regulation No. 1276/2011 recommends freezing at − 20 °C or below for 24 h, or − 35 °C or below for 15 h, to kill parasites. During our investigation, several time-temperature conditions were tested, including − 25 °C, which is commonly used in fish processing plants, as well as − 15 and − 18 °C, which are typical of domestic freezers. Some A. simplex s.s. larvae subjected to freezing in the single-compressor device survived at − 15 and − 18 °C, which has implications for domestic freezer use. According to Sanchez-Alonso et al. (2018), the ability of some Anisakis larvae to survive freezing at these temperatures poses a risk to households, because a significant percentage of domestic freezers cannot attain the minimum temperature of − 20 °C recommended by the EU.
Live and dead anisakid larvae can be distinguished by observation of parasite motility (EFSA 2010), by the fluorescence of dead larvae excited by UV radiation (Rodriguez-Mahillo et al. 2008; Vidacek et al. 2010), and by staining with different dyes (Leinemann and Karl 1988). However, the utility of some of these procedures is questionable. In our study, spontaneous movement was observed in some A. simplex s.s. larvae immediately after thawing, and therefore clearly, these individuals were viable. Staining with malachite green is also useful for preliminary screening of apparently dead larvae. However, some experimentally frozen A. simplex s.s. larvae remained colorless after staining with malachite green despite being motionless. The majority of these were intact and without apparent damage to the body structure, as viewed under the light microscope. The highest percentage (16%) of unstained larvae was reported in C. harengus membras samples frozen at − 20 °C for 24 h in the single-compressor freezer. In this case, the limited time of exposure to the target temperature (only 15 min) might be insufficient to cause the structural damage necessary for the malachite green stain to penetrate the parasite tissues.
A high proportion of unstained larvae was also observed in C. harengus membras samples frozen in the double-compressor freezer, and the uptake of dye by A. simplex s.s. decreased with decreasing freezing temperature. The highest percentage of partly stained individuals (78%) occurred in larvae frozen at − 35 °C. Consequently, rapidly frozen parasites are likely to suffer less cellular damage and would be expected to absorb malachite green to a lesser extent than larvae frozen slowly. Lesion of the ventriculus (bloating) was the most commonly reported damage in partly stained nematodes. Moreover, the ventriculus was the only part of the body that was stained in the majority of larvae held in the double-compressor freezer. These findings suggest that this part of the A. simplex s.s. body is the most sensitive to freezing.
Among the experimentally tested time-temperature conditions (− 15, − 18, or − 20 °C for 24 h, − 20 °C for 48 h in the single-compressor freezer, and − 20, − 25, or − 35 °C for 24 h in the double-compressor freezer), only two met the criteria listed in the EU regulations: freezing of C. harengus membras samples in the single-compressor freezer at − 20 °C for 48 h, where the effective freezing time at the target temperature lasted > 24 h, and freezing in the double-compressor device at − 35 °C for 24 h, with an effective freezing time of > 15 h. It is important to note that the holding time of the product at the required freezing temperature should be sufficiently long to kill all viable anisakid nematodes.
The freezing process in the laboratory, which of necessity takes place on a small scale (e.g., single fillets), clearly differs from freezing on an industrial scale, where fish are often frozen in blocks. Nevertheless, it should be possible to monitor the freezing process and its effectiveness in almost every processing plant. By recording the parameters of the freezing of fish products (e.g., using loggers or thermocouples), it is possible to assess under which conditions the required temperature will be reached in all parts of the product and maintained for a sufficient length of time. As a result, the freezing process can be optimized to ensure that products are safe for consumers.