The accumulation and increase of plastics in the marine ecosystem have become a significant issue in recent years, contaminating from coastal regions (^{Chenillat et al., 2021}) to oceanic areas (Lebreton et al., 2018). Due to erosive effects, plastics break down into smaller particles known as microplastics (MPs: 1 µm-5 mm) (^{Frias and Nash, 2019}). Microplastics (MPs) are known to be present throughout the global ocean and can be found by many species, including zooplankton. Although they fall within the size range of zooplankton prey, few in situ studies on MPs ingestion have been conducted (^{Zavala-Alarcón et al., 2023}), requiring digestion protocols to determine abundance and characteristics of microplastics (^{Alfonso et al., 2021}). Various studies have reported MPs ingestion by fish (Calderón et al., 2019; ^{Zitouni et al., 2021}; ^{Garcés Ordóñez et al., 2022}), bivalves (^{Lo and Chan, 2018}; ^{Liu et al., 2021}), crustaceans (^{Cau et al., 2019}; ^{Yin et al., 2022}), and zooplankton communities (^{Lee et al., 2013}; ^{Cole et al., 2015}; ^{Jeong et al., 2017}, Alfonso et al., 2021; Zavala Alarcón et al., 2023). Animals may ingest MPs by mistaking them for prey or passively while filtering seawater (^{Md Amin et al., 2020}). Ingesting plastic particles has triggered adverse effects, including reduced food consumption, incorporation of MPs into body tissues, physical damage to the digestive tract, and endocrine disruptions in organisms (de Sá et al., 2018; Lo and Chan, 2018; ^{Yu et al., 2020}; Zitouni et al., 2021; ^{He et al., 2022}).

Zooplankton generally feeds in surface waters where MPs abundance is higher than at mid-depth, increasing the chances of encounter and ingestion (^{Steinberg and Landry, 2017}; ^{Botterell et al., 2019}). In Colombia, the presence of MPs has been mainly reported in marine areas (^{Acosta-Coley et al., 2019}; Garcés-Ordóñez et al., 2021, 2022), and within the stomach contents of fish (^{Calderon et al., 2019}; ^{Tafurt-Villarraga et al., 2021}; ^{Jimenez-Cárdenas et al., 2022}). However, there is no published information on MPs ingestion by zooplankton in Colombia to date. Despite the current focus on MPs pollution, many questions remain about its effects on marine ecosystems, including potential impacts on the carbon cycle (^{Shen et al., 2020}) and secondary production through ingestion (^{Troost et al., 2018}). Zooplankton plays a crucial role in marine ecosystems, facilitating energy transfer through the food web and representing the early life stage of many marine species. Given their varied feeding habits and the size range of their prey, zooplankton is one of the most sensitive groups to MPs (Moore, 2008; ^{Zavala-Alarcón et al., 2023}).

Samples analyzed for MPs in marine ecosystems and zooplankton organisms are usually obtained with plankton nets, containing a significant amount of organic matter (OM) mainly represented by zooplankton and phytoplankton organisms. Digestion techniques are necessary to degrade the OM of organisms and to detect ingested MPs, and different protocols have been followed using various reagents (acids, bases, oxidants, enzymes), concentrations, digestion steps, duration, and temperature conditions depending on the type of organisms (^{Desforges et al., 2015}; ^{Cau et al., 2019}; ^{Md Amin et al., 2020}; ^{Zheng et al., 2020}; ^{Lusher et al., 2020}; ^{Alfonso et al., 2021}; ^{Aytan et al., 2022}). Considering the exponential increase in studies on MPs in the smaller fractions up to 100μm, which are ingested by zooplankton (Zhang et al., 2020; Alfonso et al., 2021), this study aims to explore the effects of digestion protocols on organisms and MPs, improving the efficiency of methods to investigate their presence and contributing to establishing an efficient and standardized methodology for research.

A literature review was conducted on Google Scholar, Web of Science, and Scopus using the keywords “microplastics,” “ingestion,” “aquatic organisms,” and chosen protocols were applied and adjusted based on a pilot sample collected on Isla Arena within Corales del Rosario and San Bernardo National Natural Park (PNNCRSB), Colombia, located at 10° 08’ 55.3” N 75° 43’ 45.1” W (Figure 1). Zooplankton organisms were collected with a 30 cm diameter mini bongo net equipped with two nets of 200 μm and 500 μm, and a General Oceanic mechanical counter to determine the filtered water volume. An oblique tow was conducted following the methodology of Smith and Richardson (1979), from the surface to the bottom with an approximate safety space of one meter above the bottom, for five minutes of bottom trawling at a speed of 5.6 km/h. The biological material was fixed in 4 % formaldehyde seawater neutralized with sodium tetraborate (Na2 B4 O7 / Na2 * 2 B2 O3 ) and transported to the hydrobiology laboratory at Universidad Industrial de Santander, Colombia, where the four most abundant zooplankton groups from the 200 μm net were observed and separated. Fifty (50) individuals from each zooplankton group were then separated and measured using a Discovery V.12 stereomicroscope with an Axiocam ERc 5s camera via ZEISS ZEN software (Blue edition). Finally, to validate the different protocols, the MPs ingestion rate was estimated.

Figure 1 Study area and sampling location in Isla Arena, Corales del Rosario and San Bernardo National Natural Park, Bolívar, Colombia. 

To avoid contamination in the laboratory, glass material was used, substances were filtered with a glass microfiber membrane (0.45 µm pore), cleaning with 70 % alcohol and distilled water, substance handling and filtrations in gas extraction hoods; organisms were rinsed with ultrapure deionized water (type I) to remove salts and impurities and deposited in glass jars. Tweezers were used to remove external material. To determine MPs integrity, control samples with MPs particles in a glass jar were used, evaluating the impact of the procedure for each of the employed protocols.

Four protocols were selected for organism degradation and MPs extraction: two of these protocols were developed by ^{Aytan et al. (2022)}, while the other two come from the research of ^{Cau et al. (2019)} and ^{Md Amin et al. (2020)} (Table 1). The protocols were chosen based on the results found in the databases, which show a wide diversity of digestion techniques for PM analysis, with only a few digestion techniques for MPs analysis, with only a few studies evaluating the efficiency of OM digestion and zooplankton organisms of Taxons that constituted the object of our study; additionally the ease and availability of reagents was considered (^{Li et al. 2016}; ^{Sun et al., 2017}; ^{Kosore et al. 2018}; ^{Botterell et al. 2019}; Calderón et al., 2019; Cau et al., 2019; Garcés Ordóñez et al., 2020; Md Amin et al., 2020; ^{Zheng et al., 2020}; Aytan et al., 2022; Jiménez-Cárdenas et al., 2022). The selected protocols were applied to samples composed of 50 organisms from the four most abundant zooplankton groups, corresponding to the Paracalanidae, Corycaeidae, Oncaeidae, and Chaetognatha families.

Table 1 Characteristics and generalities of the selected protocols aimed at achieving the degradation of zooplanktonic organisms. 

The protocol by ^{Aytan et al. (2022)} employs the Fenton reaction, combining FeSO4 · 7 H2 O + H2 O2 at 30 %, for OM degradation in water samples. This methodology is used by other authors for removing OM from larger organisms (^{Tagg et al., 2017}; ^{Al-Azzawi et al., 2020}; Lucher et al, 2020; ^{Schrank et al., 2022}). However, the laboratory results with this technique did not achieve complete degradation of organisms. Modifications were made to this protocol by doubling the H2 O2 at 30 % volume in the Fenton formula and extending the heating phase by two additional hours. Temperature conditions were kept constant because the Fenton reaction is exothermic, and an increase in temperature could pose risks in the laboratory while trying to maintain MPs integrity, as authors suggest not exceeding 60 °C when using oxidative and alkaline reactions (^{Lusher et al., 2020}; Alonso et al., 2021).

The protocol by Cau et al. (2020) uses 15 % hydrogen peroxide for the degradation of Nephrops norvegicus and Aristeus antennatus crustaceans; crustaceans, being a dominant taxonomic group in plankton, which present greater difficulty in degrading OM due to the presence of chitin in exoskeletons (^{Souza et al., 2011}). H2 O2 volumes at 2 mL were modified to adapt to zooplankton OM. The temperature and time set by the authors in the protocol did not degrade the organisms, especially copepods and chaetognaths. To degrade zooplankton organisms, especially crustaceans like copepods and chaetognaths, the heating time was extended to 12 h at the same temperature (50 °C), as reported in Table 2, without obtaining 100 % effective degradation and possibly affecting MPs integrity (Alonso et al., 2021).

In ^{Aytan et al.’s (2022)} protocol, H2 O2 at 30 % was also used for zooplankton OM degradation, with a controlled temperature of 45 °C over a period of 4 h to 6 h. This protocol also failed to achieve total degradation of zooplankton organisms. To digest organisms, the H2 O2 volume was adjusted to 2 mL, and the temperature was increased to 75 °C for 12 h. Despite suggestions that high temperatures can damage MPs integrity (^{Alfonso et al., 2021}), our results did not even achieve OM degradation (Table 2, Figure 2).

Table 2 Selected and adapted protocols for achieving marine zooplankton degradation. 

Figure 2 Partially degraded organisms; a) Chaetognatha. d,g) Oncaeidae. b,e,h) Paracalanidae. c,f,i) Corycaeidae. 

In ^{Md Amin et al.’s (2020)} protocol, between 17 µl - 20 µl of 65 % nitric acid (HNO3 ) was used for 30 min to degrade zooplankton organisms. The proposed acid volume was not sufficient to cover the zooplankton sample, so it was increased to 2 mL. Additionally, the author’s specified heating time did not fully degrade the chaetognath hooks, so the time was increased to 1 h. To avoid physical damage to MPs, the temperature was not modified, and MPs integrity was maintained compared to the control sample ^{Alfonso et al. (2021)}. After the procedures, the solutions were filtered with a cellulose nitrate ester membrane (0.45 µm pore) with an area of 5 cm², and the retained material on the filter was observed under the stereomicroscope.

Protocols based on oxidation and acidification (^{Cau et al., 2019}; ^{Md Amin et al., 2020}; ^{Aytan et al., 2022}) only achieved partial degradation in some zooplankton groups under certain conditions. Regarding oxidation, hydrogen peroxide is efficient in removing OM, as reported in sediments, water samples (Aytan et al., 2022), soft tissues of Mytilus edulis (^{Li et al., 2016}; ^{Kolandhasamy et al., 2018}), and the partial digestion of fish and lobster intestinal tracts (^{Avio et al., 2015}; Cau et al., 2019). However, the organisms used in our research, having chitinous structures, were not fully degraded (Figure 2), similar to findings by ^{Cole et al. (2014)}, where hydrogen peroxide partially removed zooplankton samples for up to seven days.

The Fenton reaction is a process that combines oxidation and bases when combining hydrogen peroxide (H2 O2 30 %) with iron sulfate (II) (FeSO4 ). This solution has proven efficient in decomposing OM in sludge samples from wastewater (^{Al-Azzawi et al., 2020}) and degrading dissolved OM in marine water samples (^{Aytan et al., 2022}). In several studies, the Fenton reaction has been considered a key method for standardizing the digestion of organic matter (OM) (^{Tagg et al., 2017}; Al-Azzawi et al., 2020; ^{Schrank et al., 2022}) due to its effectiveness in a shorter exposure time and its lack of adverse effects on the physical and chemical composition of plastics (Al-Azzawi et al., 2020; Schrank et al., 2022). However, in our research, the structures of the zooplankton organisms used were only partially degraded.

The Fenton methodology involves an exothermic reaction, reaching temperatures up to 90 °C (^{Al-Azzawi et al., 2020}), requiring conical flasks of at least 100 mL to prevent content spillage, making it challenging to extract microplastics (MPs) from small-sized samples. ^{Möller et al. (2020)} reported that the Fenton reaction alone is insufficient for complete OM removal, as observed in our study. A complementary step, such as the use of digestive enzymes, is necessary for total digestion, increasing sample processing costs. No alterations in the integrity of MPs were observed, similar to the findings of ^{Hurley et al. (2018)}, who reported that MP forms maintain their integrity between 86 % and 90 %, depending on the shape and polymer composition.

Acidification has proven useful for removing organic material from marine samples, facilitating MPs isolation (^{Avio et al., 2015}). Nitric acid has been used in the digestion of zooplankton samples (^{Sun et al., 2017}; Desforges et al., 2019; ^{Zavala-Alarcón et al., 2023}) and shrimp digestive tracts (^{Gurjar et al., 2021}). In this research the exposure time of 65 % nitric acid was modified from 30 minutes to 1 hour, resulting in positive outcomes with complete degradation of zooplankton organisms and preserved physical characteristics of MPs.

Some authors suggest that nitric acid, under high pressures and temperatures, can oxidize, destroy, and damage polymer MPs intolerant to low pH, such as polystyrene, polyethylene, and polyamides (^{Cole et al., 2014}; ^{Schrank et al., 2022}). ^{Desforges et al. (2015)} used 100 % nitric acid for 30 minutes, finding MPs in samples of the copepod Neocalanus cristatus and krill Euphausia pacifica, reporting that acid impact on MPs depends on exposure time and concentration. ^{Md Amin et al. (2020)} and ^{Zavala-Alarcón et al. (2023)} used 65 % and 55 % nitric acid, respectively, modifying Desforges et al.’s (2015) methodology, showing no impact on the physical and chemical integrity of MPs.

^{Schrank et al. (2022)} reported the degradation of PET polymers using 69 % nitric acid for two hours, while ^{Zavala-Alarcón et al. (2023)} found that this polymer degrades using 55 % nitric acid for 30 minutes, demonstrating that modifications in exposure time and concentration of nitric acid remain favorable for preserving MP integrity and achieving complete digestion of zooplankton organisms. In each protocol, the ingestion rate per taxonomic group was calculated. However, only the modified protocol of ^{Md Amin et al. (2020)} yielded results (Figure 3), as other protocols failed to degrade OM, making it impossible to observe the presence of MPs. The MPs ingestion rate using the modified protocol found the following values for each zooplankton group: Paracalanidae 16 MPs/50 (0.31), Corycaeidae 8 MPs/50 (0.16), Oncaeidae 8 MPs/50 (0.16), and Chaetognatha 12 MPs/50 (0.24). Fragments were the only MPs form detected for copepods (Paracalanidae, Corycaeidae, Oncaeidae); their ingestion is associated with environmental availability, and their omnivorous nature makes them more susceptible to MPs ingestion (Zavala Alarcón et al., 2023), supported by ^{Garcés-Ordoñez et al.’s (2022)} findings of higher fragment occurrence in surface waters of coastal areas in the Colombian Caribbean. Filaments, the second form found, were ingested by the Chaetognatha group, consistent with other authors’ findings (^{Kosore et al., 2018}; Md Amin et al., 2020).

Figure 3 Examples of MPs ingested by marine zooplankton; a,b) Chaetognatha. c) Corycaeidae. d) Oncaeidae. e,f) Paracalanidae. (Scale bar = 200 µm).