Industrial Cigarette Butts (ICBs) exposure to mussels
(Mytilus galloprovincialis)

In this experiment, we exposed mussels to industrial cigarette butts in different quantities for 14 days. We have analyzed some biological responses and the analysis is still ongoing.

The aim of this study is to see if there is a correlation between chemicals eluted by industrial cigarette butts in sea water and biological effects on the mussel Mytilus galloprovincialis after 14 days of exposure.

Experimental set-up

Control
(0 CBs/L)

Low concentration
(0.5 CBs/L)

Medium concentration
(1 CBs/L)

High concentration
(5 CBs/L)

Tanks containing 60 mussels and 17 litres of artificial seawater
(4 tanks with all different concentrations of cigarette butts)

38 mussels for biochemical analyses
22 mussels for PAHs and trace metal analyses

Exposure time: 14 days, mussels sampled at t0 and t14

Water and ICBs changed every 72 hours

Trace metal analysis

Sample preparation
- Homogenization of tissue
- Drying of samples
- Trituration
- Acid microwave mineralization
Flame atomization
- For copper, manganese, zinc, and iron
- Sample solution is atomized by a flame
Electrothermal atomization
- For aluminium, arsenic, cadmium, nickel, chromium, lead, and vanadium
- Small aliquots of samples put into a micro-furnace with a graphite tube
- Heating:
  1. Drying (80-120 °C)
  2. Ashing (400 - 800 °C)
  3. Atomization (2300 - 3000 °C)
Cold vapor atomization
- For mercury
- Mercury is irradiated by a hollow cathode lamp for mercury to measure its absorption

We expected mussels that were exposed to cigarette butts to have a higher concentration of trace metals present in their tissue. For this reason we analyzed the bioaccumulation of trace metals in the mussels sampled both at t0 and t14. We did this using Atomic Absorption Spectroscopy (AAS), which is an analytical technique for quantitative and qualitative determination of elements' ions in solutions. It uses the wavelength of light specifically absorbed by an element.

Sample preparation

Flame atomization

Electrothermal atomization

Cold vapor atomization

Total Oxyradical Scavenging Capacity Assay (TOSCA)
in digestive gland

Digestive gland homogenization

- With potters on ice
- 100 mM K-phosphate buffer (pH 7.5), 2.5% NaCl and protease inhibitors
Centrifugion
- Supernatants subdivided into aliquots
- Stored at -80 °C
10 mL vials in a shaking water bath of 35 °C for 103 min
- Vials with a final volume of 1 mL
TOSCA reaction
- ROO● is artificially produced by the thermal homolysis of ABAP
- HO● is generated from a Fenton driven reaction of Fe-EDTA and ascorbate
- KMBA (the substrate) is oxidized to ethylene measured by GC analysis

TOSCA can be used to characterize the total oxyradical scavenging capacity of cellular antioxidants to neutralize different forms of reactive oxygen species in marine organisms from different environments or exposed to chemical pollutants under field or laboratory conditions. In this case, the pollutants are the cigarette butts that we added to the tanks. We would thus expect a difference in the TOSC to neutralize reactive oxygen species.

Homogenization

Centrifugion

TOSCA reaction

Total glutathione concentration (TGSH) in digestive gland

Digestive gland homogenization
- With a potter on ice
- 5% sulphosalicylic acid with 4 mM EDTA at 4 °C
Centrifugion
- Before maintained on ice 45 min for deproteinization
- Supernatants subdivided into aliquots
- Stored at -80 °C
Spectrophotometric analysis
- λ = 412 nm, T = 18 °C
- Blank and GSH standards for calibration line

GSH is the most abundant cellular thiol involved in metabolic and transport processes, and in cell protection against the toxic effects of a variety of endogenous and exogenous compounds, including trace metals and ROS. GSH plays multiple protective roles against oxidative stress, acting as a direct scavenger of ROS and as a cofactor of several antioxidant enzymes.
Total GSH concentration (GSH + 2GSSG) is measured in a colorimetric reaction using DTNB (Ellman's Reagent), which reacts with thiolic compounds.
In this experiment, we were expecting a lower concentraiton of GSH for the exposure conditions, seeing as exposure to toxins would most likely mean some cellular components are damaged quicker or have already been damaged.

Acetylcholinesterase activity in hemolymph and gills

Gills homogenized
- In TrisHCl 0.1 m (pH 7.2) and sucrose 0.25 M at 4 °C
- With potter on ice
Both gills and hemolymph:
- Centrifuged
- Supernatants collected
- Stored in -80 °C
Spectrophotometric analysis
- λ = 412 nm, T = 18 °C

Acetrylcholinesterase (AChE) is a cholinergic enzyme and serine hydrolase essential for transmission of nerve signals. It breaks down or hydrolyzes acetylcholine (ACh), a naturally occurring neurotransmitter, into acetic acid and choline. The primary role of AChE is to terminate neuronal transmission and signaling between synapses to prevent ACh dispersal and activation of nearby receptors. AChE is inhibited by organophosphates and carbamate pesticides (and thus also possibly by cigarette butts). The inhibition of AChE may persist for days to weeks, and thus, the measurement of AChE activity has been established as biomarker of exposure to these compounds in aquatic environments.

Protein concentration

Standard solution and sample preparation
- Samples with diluted digestive gland and gills 1:100 and hemolymph 1:25 in a final volume of 500 microlitres
- Standard solution using a bovine serum albumin as protein standard
  - 5 serial dilutions at 400, 200, 100, 50, and 20 microgram/mL
Adding reagent C and reagent D
- Reagent C: CuSO4, Na citrate, Na2CO3, NaOH
- Regaent D: Folin-ciocolteau and water
Spectrophotometric analysis
- Measured absorbance of standard
- Measured absorbance and concentrations of samples
- λ = 750 nm

We measured protein content in samples following Lowry's method in order to normalize TOSC and Acetylcholinesterase results.

Results

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Industrial Cigarette Butts (ICBs) exposure to mussels (Mytilus galloprovincialis)