Can Hediste Diversicolor Speed Up the Breakdown of Cigarette Butts in Marine Sediments?

Citation: Conradi, M.; Sánchez-Moyano, J.E.; Rodríguez-Martín, F.J.; Bayo, J. Can Hediste diversicolor Speed Up the Breakdown of Cigarette Butts in Marine Sediments? Appl. Sci. 2024,14, 4409. https://doi.org/10.3390/ app14114409 Academic Editor: Franco Mutinelli Received: 11 April 2024 Revised: 14 May 2024 Accepted: 15 May 2024 Published: 23 May 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). applied sciences Article Can Hediste diversicolor Speed Up the Breakdown of Cigarette Butts in Marine Sediments? Mercedes Conradi 1, J. Emilio Sánchez-Moyano 1,* , Francisco J. Rodríguez-Martín1and Javier Bayo 2 1Department of Zoology, Faculty of Biology, University of Seville, Av. Reina Mercedes s/n, 41012 Seville, Spain; [email protected] (M.C.); [email protected] (F.J.R.-M.) 2Department of Chemical and Environmental Engineering, Technical University of Cartagena, Paseo Alfonso XIII 44, 30203 Cartagena, Spain; javier[email protected] *Correspondence: [email protected] Abstract: Cigarette butts (CBs) are non-biodegradable harmful residues of synthetic origin and are widespread in marine environments around the world. Although environmental factors are often primarily responsible for the fragmentation of microplastics in the marine environment, biotic factors have recently been shown to be equally important in plastic debris. This study evaluates the role of the Hediste diversicolor polychaete in the fragmentation of CBs in the marine environment. Polychaetes were exposed to three concentrations of CB (0 (as the control), 0.25, and 1 butt L −1 ) at two different temperatures (15 ◦ C and 23 ◦ C) for 28 days. At each temperature, aquaria without polychaetes were used to study the effect of the burrowing activity of the polychaete on CB fragmentation. Toxicants analysed from exposed sediments increased their concentration in a dose-dependent manner to the CB concentration at a temperature of 15 ◦ C but not at 23 ◦ C. CBs did not directly decrease Hediste survival, but prolonged elevated temperatures increased the polychaetes’ susceptibility. The negative effects of CBs on burial success and burrowing behaviour could not be offset by the reduced start time caused by elevated temperatures. Regardless of temperature, both the weight loss and physical fragmentation of CBs buried in polychaete-contaminated sediments were significantly higher than those without Hediste, with no differences between the two concentrations tested. FTIR-ATR analysis used to evaluate CB degradation in relation to cellulose acetate decomposition showed a greater degradation of this compound in treatments with Hediste than in those without polychaetes ( ~2.75 times ), but these differences were not significant. This study is a promising initial step for future research, as any factor that facilitates the fragmentation of this prevalent and hazardous waste must be carefully studied to extract the maximum benefit to help to reduce CBs in the marine environment. Keywords: cigarette butts; fragmentation; FTIR-ATR; cellulose acetate; polychaetes 1. Introduction Due to the difficulty in collecting and subsequently treating them, cigarette butts (CBs) are the most abundant and dispersed garbage in the world [ 1 ] and the most abundant type of litter on beaches [ 2 – 4 ]. CBs on beaches not only generate large economic costs but also serious environmental problems, which is why this waste is considered an important social problem [ 5 ]. The economic cost is determined by its impact on tourism, a value that can be reduced by up to 97% due to dirt [ 6 – 8 ]. Thus, most countries invest heavily in keeping their coasts clean. But CB cleaning is not only expensive, it is also ineffective (30% recovered) [ 9 , 10 ], with CBs remaining in the environment even after cleaning, especially automatic cleaning, of the beaches [ 11 ]. The environmental cost is more difficult to determine. CBs represent a unique combination of physical and chemical contamination [ 12 ]; physical contamination because this trash sheds 100 small microfibrils per day [ 13 ], which marine organisms can feed on and transfer throughout the food chain [ 14 – 16 ] and chemical contamination because CBs can filter more than 7000 toxins into the environment, many of which have been shown to be dangerous for marine organisms (e.g., [ 17 – 21 ]). The effects of Appl. Sci. 2024,14, 4409. https://doi.org/10.3390/app14114409 https://www.mdpi.com/journal/applsci Appl. Sci. 2024,14, 4409 2 of 15 this chemical pollution are difficult to estimate in the marine environment, since CBs can not only leach toxicants into the seawater but can also adsorb toxicants from the ocean floor, depending on the concentrations available and relative to its degradation level (e.g., [22]). Our current knowledge about CB biodegradation mechanisms and their efficiency is quite limited [ 23 ]. CBs are generally made of cellulose acetate (CA), a chemically modified natural polymer [ 24 ] widely used in the textile, plastic film, and packaging industries [ 25 ]. CA degrades slowly since its high degree of acetylation makes its degradation difficult in the natural environment [ 26 , 27 ]; thus, it remains in the environment for a long time. Although the decomposition of polymer material by physical forces of a mechanical nature is considered vital in the degradation process [ 28 ], recently, biotic factors have been considered equally important in this fragmentation [ 29 , 30 ]. For example, some isopods and polychaetes create thousands of microfibres when digging blocks of expanded polystyrene over a short period (days) [ 31 , 32 ]; thus, organisms can break down large polymers [ 33 – 35 ] at an even faster rate than abiotic processes [ 36 ] and/or can also alter their bioavailability by reducing their size and incorporating them into faeces and faecal matter [36–39]. Hediste diversicolor (O.F. Müller, 1776) is a common and ecologically important polychaete burrowing species in estuarine sedimentary habitats of European and North American coasts, where it plays an essential role as a bioturbation species that greatly influences the flux of organic matter, oxygen, and bacterial diversity in sediments [ 40 ]. For the first time, here, we tested whether the bioturbation created by this polychaete in marine sediments, the most likely final fate of CBs, can directly or indirectly favour mechanical fragmentation of this type of litter. To do this, we determined not only if Hediste can survive the different toxicants leached from CBs at summer and winter temperatures but also if CBs influence the burrowing activity of polychaetes and whether this activity influences directly or indirectly the mechanical fragmentation of CBs. We consider the broader definition of CB degradation that includes weight loss by the leaching of additives or part of the material with low molar mass [41]. 2. Material and Methods 2.1. Study Area and Sample Collection Natural sediment was collected at low tide from an intertidal zone in the Bay of Cádiz (southwestern Spain, 36 ◦ 29 ′ 20 ′′ N 6 ◦ 15 ′ 50 ′′ W) with well-identified sediment characteristics [ 42 ] and low contamination levels [ 43 ] to avoid collateral contamination. This sediment was sieved through a 0.5 mm mesh to remove fauna, stones, and large organic particles, transported to the laboratory, and stored at 5 ± 1 ◦ C (in the dark) until the time of the experiment. Hediste diversicolor individuals were collected at the beginning of October to avoid the reproductive season [ 44 ]. Specimens of similar weight (1.37 ± 0.24 g) and size ( 10.66 ±0.49 cm ) were selected to minimise differences in biological responses. In the laboratory, polychaetes (~400 individuals/m 2 ) were placed in different aquariums for a 10-day acclimatisation period with the selected sediment (1/3 of the height of the aquarium) and artificial seawater (ASW-filtered freshwater with Tropic Marin SEA SALT, salinity 35). During the acclimatisation period, the organisms were kept under a constant photoperiod (12 h light: 12 h dark), temperature (15 ±1◦C, a winter temperature close to values of the natural environment), continuous aeration, and ambient pH (pH 8.1). Polychaetes were fed with a commercial mixture every other day (Tropical Bionautic Flakes—protein 46.0%, fat 7.0%, fibre 3.5%, moisture 8.0%, phosphorus 0.4%) and at least 50% of the water was exchanged every three days. The leftover food was removed by a manual suction pump if it was seen on top of the sediment. 2.2. Experimental Design Polychaetes were exposed to three concentrations of CB (0 (as the control), 0.25, and 1 butt L−1equivalents, [14]) at 15 ◦ C and 23 ◦ C (mean seawater temperatures in winter and summer, respectively, in Cádiz; https://seatemperature.info/es/espana/cadiz-temperatura- Appl. Sci. 2024,14, 4409 3 of 15 del-agua-del-mar.html, access date: 1 September 2023) for a period of 28 days . Two different temperatures were used since this affects the burrowing activity of H. diversicolor sediments [ 40 ]. Therefore, there were six experimental conditions: (1) control ( 0 butt L−1 + winter temperature (15 ◦ C); (2) control (0 butt L −1 ) + summer temperature (23 ◦ C); (3) moderate CB contamination (0.25 butt L −1 ) + winter temperature (15 ◦ C); (4) moderate CB contamination (0.25 butt L −1 ) + summer temperature (23 ◦ C); (5) extreme CB contamination (1 butt L −1 ) + winter temperature (15 ◦ C); (6) extreme CB contamination ( 0.25 butt L−1 ) + summer temperature (23 ◦ C). For each condition, three aquaria (5 L in volume) with 600 g of sediment (1/3 of the height of the aquarium) were used with ten specimens per aquarium (density: ~400 individuals/m 2 ) to evaluate mortality, burrowing activity, and CB fragmentation. Furthermore, three aquaria without polychaetes added were used at each temperature to study the effect of the burrowing activity of the polychaete on CB fragmentation. To achieve the required temperature conditions, 18 aquaria were placed in a sealed thermostat-controlled 15 ◦ C room, and another 18 aquaria were placed in a 23 ◦ C water bath using an AquaMedic Titan 500 (Ab Aqua Medic GmbH, Bissendorf, Germany) closed cooling system connected to an AquaMedic Ocean Runner OR6500 pump. Once the sediment had settled in the experimental aquaria, the polychaetes were randomly placed in them and the temperature of those at 23 ◦ C was increased by 0.5 ◦ C per day. When this temperature was reached, the CBs were added by dropping them onto the surface of the water. The majority (~90%) fell immediately into the sediment, and only some remained floating for up to 2 h before sinking. The CBs were then manually buried in the sediment. The brand ”Malboro” was used since it is the most popular and most-sold cigarette in Spain (https://www.hacienda.gob.es/es-ES/Areas%20Tematicas/CMTabacos/Paginas/ EstadisticasCMT2023.aspx, access date: 10 January 2024). Cigarettes collected within one week of consumption were “naturally smoked” by 3 different individuals. The remaining tobacco was removed from these collected CBs, so the CBs consisted only of a filter wrapped in paper. Later, they were weighed, labelled, and individually stored at − 20 ◦ C until the start of the experiment [45]. Water quality was maintained by re-renewing the ASW twice a week, always replenishing less than 50% of the water to disturb as little as possible both the CBs and the polychaetes. During the first two weeks of the experiment, the temperature, pH, salinity, and level of dissolved oxygen (DO%) were measured 3 times a day (Hanna EDGE Multiparametric HI2020-02 (Hanna Instruments, Smithfield, VA, USA), accuracy ± 0.002 pH at 0.001 pH resolution; ± 0.01 to a resolution of 0.01) since previous work suggested that the toxicants leached from CB could change these characteristics of seawater [ 17 ]. After this time period, these parameters were measured twice a day, except for dissolved oxygen, which was measured once a week. 2.3. Sediment Contamination At the end of the experiment, sediment samples from each aquarium and condition were dried at 60 ◦ C until they reached a constant weight and were transported to the EU-accredited company INNOAGRAL (Seville, Spain) (https://www.innoagral.com, access date: 10 January 2024), where the most abundant alkaloids, nicotine [ 46 ], polycyclic aromatic hydrocarbons [ 17 ], and the heavy metals Cu and Zn [ 46 , 47 ] in the CB leachate were analysed. 2.4. Biological Parameters Mortality: Determined weekly by dividing the number of dead individuals at the end of a given week by the number of live individuals at the beginning of that week, within each condition (0 CBL −1 + 15 ◦ C; 0 CBL −1 + 23 ◦C ; 0.25 CBL −1 + 15 ◦ C; 0.25 CBL −1 + 23 ◦C ; 1 CBL −1 + 15 ◦ C; 1 CBL −1 + 23 ◦ C). The number of live specimens was determined by sieving the sediments of each aquarium. Mortality was expressed as a percentage. Appl. Sci. 2024,14, 4409 4 of 15 Burrowing activity: After 7, 15, and 28 days of exposure, thirty-six polychaetes (two per aquarium; six for each condition) were placed individually into a 150 mL beaker containing 5 cm of natural sediment from the sampling area and 100 mL of ASW to observe the burrowing activity. Burial was considered successful when the polychaetes were able to bury themselves completely within 45 min. The success rate was expressed as a percentage. The burrowing behaviour was considered as the time it took for individuals to start the burrowing process as soon as they were placed in the beaker, while the burial rate was considered as the total time it took the polychaetes to completely bury themselves in the sediment [40]. 2.5. Physical Degradation of CBs Physical degradation of the CBs was determined by the CB weight loss. The weight loss of each CB was calculated by the weight difference between day 0 and day 28: % weight loss: [(m0 −mt)/m0] ×100 where m0 is the initial mass of the material (day 0) and mt is the mass at the end of the experiment (day 28) in g [48]. The degradation level was visually classified into four states defined similarly as in [ 49 ] but with slight modifications: (I) preserved paper and filter; (II) coated paper but considerably worn and discoloured, without the manufacturer’s identification and preserved filter; (III) only the plug-wrapped paper and filter, and (IV) only the fibres of the fat and compact filter, without any paper. The CBs were always washed twice in distilled water for 5 min and kept in a light-protected desiccator at 30 ◦ C until a constant weight was reached. Each CB was weighed using a precision balance (±0.0001 g) [45]. 2.6. Cigarette Filter Characterization and Degradation Tracking The CBs were characterised by attenuated total reflexion Fourier-transform infrared spectroscopy (ATR-FTIR) [45]. To identify the functional groups and the molecular composition of the polymers, ATRFTIR was used. Samples were compressed in a diamond anvil compression cell, and spectra were acquired with a Thermo Nicolet 5700 Fourier-transformed infrared spectrometer (Thermo Nicolet Analytical Instruments, Madison, WI, USA) fitted with a deuterated triglycine sulphate (DTGS) detector and KBr optics. The spectra were then evaluated using the Thermo Scientific OMNIC Specta software. An average of 20 scans (resolution of 16 cm−1 in the range of 400–4000 cm −1 ) was performed for each sample. The identification of the polymers was carried out thanks to different libraries of reference polymers such as Hummel Polymer and Additives (2011 spectra), Polymer Additives and Plasticisers ( 1799 spectra ), Sprouse Scientific Systems Polymers from the ATR library ( 500 spectra ), rubber composite materials (350 spectra), and other literature [ 50 ]. The standard criteria of [ 51 ] were followed for the percentage match (>60%) between the CB degradation state and the reference spectra. 2.7. Statistical Analysis Differences between the concentrations of the selected CB-derived toxicants were statistically analysed using the PRIMER v6 software to perform univariate PERMANOVA based on Euclidean distance similarity matrices. Differences were considered significant for p-values less than 0.05. To test whether temperature and CB contamination affect biological traits tested over time, statistical comparisons of the survival and burrowing activity of H. diversicolor were determined by repeated measures of analysis of variance in IBM SPSS 21, using time as a within-subject factor and temperature and CB concentrations as factors between subjects. Pillai’s trace was applied except when the sphericity assumption was violated (in this case, the Greenhouse–Geisser correction was used). Homogeneous groups for the factors were separated by a Tukey test, while the factors were separated by the Bonferroni test. In the Appl. Sci. 2024,14, 4409 5 of 15 case of significant interactions, multiple comparisons between factors were made using the Bonferroni test with the probability of a Type I error. Posteriorly, a Spearman correlation analysis was performed between biological traits and the sediment contamination level of each selected toxicant. A two-way ANOVA was applied to establish whether the presence of H. diversicolor or temperature significantly affected the physical degradation of CB (weight and level of degradation), after verifying the homogeneity of variances (Levene test) and normality (Kolmogorov–Smirnov test). 3. Results 3.1. Physicochemical Parameters The seawater parameters and mean values for the different treatments at both temperatures (15 ◦C and 23 ◦C) are presented in Table 1. Table 1. Artificial seawater (ASW) conditions (mean ± SD) at 15 ◦ C and 23 ◦ C during the 30 days of the experiment in each treatment. Average pH (NBS), salinity (PSU), temperature ( ◦ C), dissolved oxygen (DO %). D, treatments without H. diversicolor. pH Temperature Salinity DO 15 ◦C Control 8.13 ±0.09 14.99 ±0.13 35.38 ±0.09 131.79 ±7.6 0.25 CBL−18.13 ±0.08 14.97 ±0.13 35.39 ±0.11 131.64 ±9.3 1 CBL−18.12 ±0.09 14.96 ±0.13 35.39 ±0.12 134.10 ±6.7 D-0.25 CBL−18.14 ±0.08 14.94±0.16 35.47 ±0.11 129.75 ±6.6 D-1 CBL−18.15 ±0.08 14.96 ±0.16 35.44 ±0.11 130.64 ±6.5 23 ◦C Control 8.11 ±0.08 23.15 ±0.31 35.42 ±0.12 126.62 ±11.4 0.25 CBL−18.12 ±0.10 23.15 ±0.36 35.45 ±0.08 125.60 ±10.1 1 CBL−18.11 ±0.10 23.15 ±0.36 35.44 ±0.09 127.29 ±10.6 D-0.25 CBL−18.11 ±0.09 23.17 ±0.34 35.50 ±0.11 128.36 ±10.4 D-1 CBL−18.13 ±0.09 23.15 ±0.36 35.49 ±0.10 129.40 ±9.8 All toxicant concentrations showed significant differences in the sediments of the tested treatments, and there was an interaction between temperature and the concentration of CB. Regarding temperature, only Zn concentrations were different between those sediments exposed to 15 ◦ C (winter temperature) compared with those exposed to 23 ◦ C (summer temperature) (Table 2). Table 2. PERMANOVA results for the Cu, Zn, naphthalene and nicotine concentrations in the sediments exposed to different CB concentrations (0 CBL −1 , 0.25 CBL −1 , 1 CBL −1 with Hediste diversicolor, and 0.25 CBL −1 , 1 CBL −1 without polychaetes) at the two temperatures studied (15 ◦ C, 23 ◦ C) for 28 days. Sum square (SS); mean square (MS); degrees of freedom (df); ns = nonsignificant. df SS MS Pseudo-F p Cu CB 4 30.41 7.62 31.86 0.001 Temperature 1 0.30 0.30 1.26 ns CB ×Temperature 4 23.77 5.94 24.81 0.001 Res 62 14.82 0.24 Total 71 71 Zn CB 4 38.78 9.69 253.2 0.001 Temperature 1 6.88 6.88 179.7 0.001 CB ×Temperature 4 25.41 6.35 165.91 0.001 Res 62 2.37 3.828 ×10−2 Total 71 71 Appl. Sci. 2024,14, 4409 6 of 15 Table 2. Cont. df SS MS Pseudo-F p Naftalene CB 4 37.23 9.31 26.56 0.001 Temperature 1 0.45 0.45 1.28 ns CB ×Temperature 4 11.44 2.86 8.16 0.001 Res 62 21.73 0.35 Total 71 71 Nicotine CB 3 29.15 9.72 38.45 0.001 Temperature 1 6.32 ×10−26.32 ×10−20.25 ns CB ×Temperature 3 26.45 8.82 34.89 0.001 Res 52 13.14 0.25 Total 59 69.14 Sediments of the two CB concentrations tested without Hediste diversicolor (0.25 and 1 CBL−1 D treatments) had similar concentrations of heavy metals (Cu and Zn) and naphthalene at both temperatures, but Cu and Zn sediment concentrations at 0.25 CBL −1 were higher at 23 ◦ C than at 15 ◦ C (Figure 1). Similarly, the Zn concentration from the sediment without H. diversicolor at 23 ◦ C was also higher from that at 15 ◦ C. These treatments also had similar nicotine concentrations at 23 ◦ C but not at 15 ◦ C. Nicotine concentrations in sediments exposed to 0.25 CBL −1 were lower at the winter temperature than at the summer temperature (Figure 1C). Appl.Sci.2024,14,xFORPEERREVIEW6of16  Res622.373.828×10−2  Total7171   Naftalene CB437.239.3126.560.001 Temperature10.450.451.28ns CB×Temperature411.442.868.160.001 Res6221.730.35  Total7171   Nicotine CB329.159.7238.450.001 Temperature16.32×10−26.32×10−20.25ns CB×Temperature326.458.8234.890.001 Res5213.140.25  Total5969.14   SedimentsofthetwoCBconcentrationstestedwithoutHedistediversicolor(0.25and 1CBL−1Dtreatments)hadsimilarconcentrationsofheavymetals(CuandZn)andnaphthaleneatbothtemperatures,butCuandZnsedimentconcentrationsat0.25CBL−1were higherat23°Cthanat15°C(Figure1).Similarly,theZnconcentrationfromthesediment withoutH.diversicolorat23°Cwasalsohigherfromthatat15°C.Thesetreatmentsalso hadsimilarnicotineconcentrationsat23°Cbutnotat15°C.Nicotineconcentrationsin sedimentsexposedto0.25CBL−1wereloweratthewintertemperaturethanatthesummer temperature(Figure1C).  Figure1.Meanvalues(+SD)ofthetoxicantanalysedinthesedimentexposedtothedifferentconcentrationsofCB(0CBL−1,0.25CBL−1,1CBL−1withHedistediversicolor,and0.25CBL−1,1CBL−1withoutpolychaetes)atthetwotemperaturesstudied(15°C,23°C)for28days.(A)Cuconcentration; (B)Znconcentration;(C)Nicotineconcentration;(D)Naphthaleneconcentration.SignificantdifferencesamongtheCBconcentrationsarerepresentedbydifferentletters(uppercaselettersforCB concentrationswithpolychaetes;lowercaselettersforCBconcentrationswithoutpolychaetes;letterswithout′for15°Ctreatment;letterswith′for23°Ctreatment).Significantdifferences(p<0.05) Figure 1. Mean values (+SD) of the toxicant analysed in the sediment exposed to the different concentrations of CB (0 CBL −1 , 0.25 CBL −1 , 1 CBL−1 with Hediste diversicolor, and 0.25 CBL −1 , 1 CBL−1 without polychaetes) at the two temperatures studied (15 ◦ C, 23 ◦ C) for 28 days. (A) Cu concentration; (B) Zn concentration; (C) Nicotine concentration; (D) Naphthalene concentration. Significant differences among the CB concentrations are represented by different letters (uppercase letters for CB concentrations with polychaetes; lowercase letters for CB concentrations without polychaetes; letters without ′ for 15 ◦ C treatment; letters with ′ for 23 ◦ C treatment). Significant differences (p< 0.05) between the same concentration of CB at the two temperatures tested are represented by asterisks (*). Appl. Sci. 2024,14, 4409 7 of 15 The Cu concentrations of the CB-contaminated sediments with polychaetes were significantly different from the control at 15 ◦ C. However, only the control sediments and 0.25 CBL −1 had significantly different Cu concentrations at summer temperatures. Higher Cu and Zn concentrations were found in sediments exposed to 1 CBL −1 at 15 ◦ C compared with those exposed at 23 ◦ C (Figure 1A,B). However, the Zn concentration of the sediments containing 0.25 CBL −1 at 23 ◦ C was higher than at 15 ◦ C (Figure 1B). Except for the control and the 1 CBL −1 condition at 23 ◦ C, the Zn concentrations in the sediment were different in all CB treatments tested at both temperatures. Nicotine concentrations were different between all H. diversicolor treatments at both temperatures and between CB concentrations at 15 ◦ C compared with those at 23 ◦ C (Figure 1C). A difference was found between the naphthalene concentrations of the two CB-contaminated sediments tested (0.25 and 1 CBL −1 ) at both temperatures (Figure 1D), and there were also differences between those two concentrations at 15 ◦ C compared with those at 23 ◦ C. According to the Spearman correlation analysis, nicotine concentrations showed a correlation with those of naphthalene ( ρ = 0.68). None of the toxicants studied had different concentrations between the controls at 15 ◦C compared with those at 23 ◦C. 3.2. Biological Parameters Although the two CB concentrations tested did not affect the survival of H. diversicolor, there was an interaction between this factor and time (p< 0.05, Table 3). As shown in Figure 2, the mortality of polychaetes exposed to CB increases with time, especially in those exposed to summer temperatures (23 ◦ C). Indeed, both temperature (p< 0.001) and time (p< 0.0001) significantly reduced polychaete survival, with an interaction between these two factors. The survival of control polychaetes at 15 ◦ C is significantly different from that of CB treatments after 21 days of exposure (p< 0.05). In contrast, the differences between control survival at 23 ◦ C and the lower CB concentration tested are significant after 7 days of exposure. Hediste survivals of all treatments at 15 ◦ C are significantly different from their respective treatments at 23 ◦C. Table 3. Results of the three-factor repeated-measures ANOVA for the mortality, burrowing behaviour, and burial rate of Hediste diversicolor exposed to the different CB concentrations (0 CBL −1 , 0.25 CBL −1 , 1 CBL −1 ) and at the two temperatures studied (15 ◦ C, 23 ◦ C) for 28 days. Sum square (SS); mean square (MS); degrees of freedom (df); ns = nonsignificant. df SS MS F p Mortality rate Time 1.32 8496.72 8496.70 21.91 0.0001 Time ×CBs 2.63 1324.99 1324.99 3.42 0.0440 Time ×Temperature 1.32 6205.38 6205.38 16.00 0.0001 Error (Time) 42 387.77 170.20 - - CBs 2.00 7430.71 3715.36 3.500 ns Temperature 1.00 19,181.87 19,181.87 18.10 0.0010 Error 14 14,843.52 1070.25 - - Burrowing behavior Time 2.45 24,140 24,140.29 1.65 ns Time ×CBs 4.40 67,741.38 13,010.80 2.30 ns Time ×Temperature 2.40 28,048.55 11,436.80 1.90 ns Error (Time) 56 205,169.38 5975.56 - - CBs 2.00 58,063.40 29,031.70 4.70 0.0270 Temperature 1.00 36,200.28 36,200.28 5.90 0.0290 Error 14 85,485.62 6106.18 - - Burial rate Time 2.00 119,836.15 57,548.40 3.60 0.0370 Time ×CBs 4.20 54,932.31 54,932.31 0.84 ns Time ×Temperature 2.10 42,614.38 20,464.52 1.30 ns Error (Time) 56 458,695.55 8190.99 - - CBs 2.00 23,835.47 11,917.73 0.82 ns Temperature 1.00 77,088.40 77,088.40 5.32 0.0370 Error 14 20,2705.33 14,478.95 - - Appl. Sci. 2024,14, 4409 8 of 15 Appl.Sci.2024,14,xFORPEERREVIEW8of16  Time×Temperature2.1042,614.3820,464.521.30ns Error(Time)56458,695.558190.99-- CBs2.0023,835.4711,917.730.82ns Temperature1.0077,088.4077,088.405.320.0370 Error1420 , 2705.3314 , 478.95--  Figure2.Survival(%)ofHedistediversicolorexposedfor28daystodifferentCBconcentrations(0 CBL−1,0.25CBL−1,1CBL−1)atthetwotemperaturesstudied:15°C(lightcolor),23°C(darkcolor). Significantdifferences(p<0.05)betweenCBconcentrationsandthecontrolonthesamedateare representedbyasterisks(*). H.diversicolorexposedtoCBconcentrationsat23°Creducedtheirburialsuccessby 33%comparedwithcontrolsafteronly7daysofexposure.Thisreductionwasmaintained untilday21oftheexperimentinpolychaetesexposedto0.25CBL−1,whilethedifference betweenthoseexposedto1CBL−1andthecontrolsincreasedto66%at14and21daysof exposure.Indeed,regardlessoftime,boththeCBconcentrationandtemperaturesimilarly affectedtheburialbehaviourofHediste,asthereisnointeractionbetweenthesetwofactors(Table3).PolychaetesfrombothCBconcentrationsbegantoburrowintothesediment significantlylaterthanthosefromthecontrolthroughouttheexperiment(Figure3A).Exceptforthosepolychaetesthatwereunsuccessfulinburrowing,themeantimeneededto startdiggingwasshorterforpolychaetesat23°C.Temperaturealsoaffectedtheburial rateofH.diversicolor,asdidexposuretime(Table3).Onthecontrary,CBconcentrations didnotaffecttheburialrate,althoughthereisatrendforthetimeittakesforpolychaetes toburythemselvestoincreaseastheCBconcentrationinthewaterincreases(Figure3B). Figure 2. Survival (%) of Hediste diversicolor exposed for 28 days to different CB concentrations ( 0 CBL−1 , 0.25 CBL −1 , 1 CBL −1 ) at the two temperatures studied: 15 ◦ C (light color), 23 ◦ C ( dark color ). Significant differences (p< 0.05) between CB concentrations and the control on the same date are represented by asterisks (*). H. diversicolor exposed to CB concentrations at 23 ◦ C reduced their burial success by 33% compared with controls after only 7 days of exposure. This reduction was maintained until day 21 of the experiment in polychaetes exposed to 0.25 CBL−1, while the difference between those exposed to 1 CBL −1 and the controls increased to 66% at 14 and 21 days of exposure. Indeed, regardless of time, both the CB concentration and temperature similarly affected the burial behaviour of Hediste, as there is no interaction between these two factors (Table 3). Polychaetes from both CB concentrations began to burrow into the sediment significantly later than those from the control throughout the experiment (Figure 3A). Except for those polychaetes that were unsuccessful in burrowing, the mean time needed to start digging was shorter for polychaetes at 23 ◦ C. Temperature also affected the burial rate of H. diversicolor, as did exposure time (Table 3). On the contrary, CB concentrations did not affect the burial rate, although there is a trend for the time it takes for polychaetes to bury themselves to increase as the CB concentration in the water increases (Figure 3B). Appl.Sci.2024,14,xFORPEERREVIEW9of16   Figure3.Theburrowingactivity(meanvalues+SD)ofHedistediversicoloratdifferentCBconcentrations(0CBL−1,0.25CBL−1,1CBL−1)andatthetwotemperaturesstudied(15°C,23°C)over28 days.(A)Burrowingbehaviour(seconds);(B)Burialrate(seconds).(*)Unsuccessfulburial. Spearmancorrelationsbetweenthebiologicaltraitsandchemicalparameterswere significantbetweentheburrowingbehaviourofpolychaetesandnaphthalene(−0.69). 3.3.PhysicalDegradationoftheCBs ThepresenceofH.diversicolorsignificantlydecreasedtheweightoftheCBssuchthat alltheCBs,regardlessoftreatment(0.25or1CBL−1),hadalowerfinalweightthanthose CBsfromthetreatmentswithoutpolychaetes(D-0.25CBL−1andD-1CBL−1)(Figure4,Table4).Tukey’stestclearlyshowstwohomogeneousgroups:oneformedbythetwoconcentrationsofCBswithoutpolychaetesandanotherbythetwoconcentrationswithpolychaetes(p<0.05).Onthecontrary,thetemperaturedidnotaffectthefinalweightofthe CBs,norwasthereanyinteractionbetweenthisfactorandtheCBs(Table4).  Figure 3. The burrowing activity (mean values + SD) of Hediste diversicolor at different CB concentrations (0 CBL −1 , 0.25 CBL −1 , 1 CBL −1 ) and at the two temperatures studied (15 ◦ C, 23 ◦ C) over 28 days. (A) Burrowing behaviour (seconds); (B) Burial rate (seconds). (*) Unsuccessful burial. Appl. Sci. 2024,14, 4409 9 of 15 Spearman correlations between the biological traits and chemical parameters were significant between the burrowing behaviour of polychaetes and naphthalene (−0.69). 3.3. Physical Degradation of the CBs The presence of H. diversicolor significantly decreased the weight of the CBs such that all the CBs, regardless of treatment (0.25 or 1 CBL −1 ), had a lower final weight than those CBs from the treatments without polychaetes (D-0.25 CBL −1 and D-1 CBL −1 ) (Figure 4, Table 4). Tukey’s test clearly shows two homogeneous groups: one formed by the two concentrations of CBs without polychaetes and another by the two concentrations with polychaetes (p< 0.05). On the contrary, the temperature did not affect the final weight of the CBs, nor was there any interaction between this factor and the CBs (Table 4). Appl. Sci. 2024, 14, x FOR PEER REVIEW 9 of 16 Figure 3. The burrowing activity (mean values + SD) of Hediste diversicolor at different CB concentrations (0 CBL−1, 0.25 CBL−1, 1 CBL−1) and at the two temperatures studied (15 °C, 23 °C) over 28 days. (A) Burrowing behaviour (seconds) ; (B) Burial rate (seconds). (*) Unsuccessful burial. Spearman correlations between the biological traits and chemical parameters were significant between the burrowing behaviour of polychaetes and naphthalene (−0.69). 3.3. Physical Degradation of the CBs The presence of H. diversicolor significantly decreased the weight of the CBs such that all the CBs, regardless of treatment (0.25 or 1 CBL−1), had a lower final weight than those CBs from the treatments without polychaetes (D-0.25 CBL−1 and D-1 CBL−1) (Figure 4, Table 4). Tukey’s test clearly shows two homogeneous groups: one formed by the two concentrations of CBs without polychaetes and another by the two concentrations with polychaetes (p < 0.05). On the contrary, the temperature did not affect the final weight of the CBs, nor was there any interaction between this factor and the CBs (Table 4). Figure 4. Weight (g; maximum, mean, and minimum) of the CBs without Hediste diversicolor (left) and with these polychaetes (right) exposed to two different CB concentrations: 0.25 CBL −1 , 1 CBL −1 and at the two temperatures studied: 15 ◦C (blue dots), 23 ◦C (red dots) for 28 days. Table 4. Results of two-factor ANOVAs for the CB weight loss and degradation level at the different CB concentrations (0 CBL −1 , 0.25 CBL −1 , 1 CBL −1 with Hediste diversicolor, and 0.25 CBL −1 , 1 CBL −1 without polychaetes), and at the two temperatures studied (15 ◦ C, 23 ◦ C) after 28 days. Sum square (SS); mean square (MS); degrees of freedom (df); ns = nonsignificant. df SS MS F p Weigth loss CB 3 16,426.65 5475.55 6.26 0.0001 Temperature 1 450.76 450.76 1.137 0.248 CB ×Temperature 3 597.90 199.30 1.137 0.298 Error 22 8719.28 396.33 - Total 30 26,412.62 - - Degradation level CB 3 24.53 8.18 38.55 0.0001 Temperature 1 0.19 0.19 0.89 0.345 CB ×Temperature 3 0.53 0.17 0.38 0.487 Error 22 4.67 0.21 Total 30 350 According to our classification of the different degradation states, the physical degradation of CB was significantly increased by polychaetes (Table 4). Therefore, 100% of the CBs of any treatment with H. diversicolor lacked both the paper coating and the plug wrap. In non-polychaete treatments, 66% of CBs still had all or most of the paper coating at 15 ◦C , while at 23 ◦ C, 80% of the butts had the coating paper partially removed, and only 20% still had it. Despite this difference, there were no significant differences in the CB degradation