# Can _Hediste diversicolor_ Speed Up the Breakdown of Cigarette Butts in Marine Sediments?
## 2 Material and Methods
### Experimental Design  
Polychaetes were exposed to three concentrations of CB (0 (as the control), 0.25, and 1 butt L\({}^{-1}\) equivalents, [14]) at 15 \({}^{\circ}\)C and 23 \({}^{\circ}\)C (mean seawater temperatures in winter and summer, respectively, in Cadiz; [[https://seatemperature.info/es/espana/cadiz-temperatura](https://seatemperature.info/es/espana/cadiz-temperatura)]([https://seatemperature.info/es/espana/cadiz-temperatura](https://seatemperature.info/es/espana/cadiz-temperatura))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 \({}^{\circ}\)C); (2) control (0 butt L\({}^{-1}\)) + summer temperature (23 \({}^{\circ}\)C); (3) moderate CB contamination (0.25 butt L\({}^{-1}\)) + winter temperature (15 \({}^{\circ}\)C); (4) moderate CB contamination (0.25 butt L\({}^{-1}\)) + summer temperature (23 \({}^{\circ}\)C); (5) extreme CB contamination (1 butt L\({}^{-1}\)) + winter temperature (15 \({}^{\circ}\)C); (6) extreme CB contamination (0.25 butt L\({}^{-1}\)) + summer temperature (23 \({}^{\circ}\)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: \(\sim\)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 \({}^{\circ}\)C room, and another 18 aquaria were placed in a 23 \({}^{\circ}\)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 \({}^{\circ}\)C was increased by 0.5 \({}^{\circ}\)C per day. When this temperature was reached, the CBs were added by dropping them onto the surface of the water. The majority (\(\sim\)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%20](https://www.hacienda.gob.es/es-ES/Areas%20) Tematicas/CMTabacos/Paginas/EstadisticasCMT2023.aspx]([https://www.hacienda.gob.es/es-ES/Areas%20](https://www.hacienda.gob.es/es-ES/Areas%20) Tematicas/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 \({}^{\circ}\)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 \(\pm\)0.002 pH at 0.001 pH resolution; \(\pm\)0.01 to a resolution of 0.01) since previous work suggested that the toxicants reached 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.