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Ocean Acidification and Artificial Seawater - The comet assay in an Arenicola marina study

Ocean acidification, the change in pH and carbonate chemistry of the world’s oceans as a result of increasing atmospheric concentrations of carbon dioxide, is now broadly considered to represent a major threat to global marine biodiversity. While copper is a naturally occurring trace element essential for some biological functions, elevated levels can be toxic to a range of marine organisms. Metals, such as copper, exert toxicity via the production of reactive oxygen species. At elevated levels these can overwhelm an organism’s antioxidant defences and induce oxidative damage of cellular components such as proteins, lipids, and DNA. 

The lugworm or sandworm, Arenicola marina, is a large marine worm of the phylum Annelida.  A. marina is an ecologically important benthic species inhabiting intertidal sediments across Northern Europe with important roles as a sediment engineer and prey species for wading birds and fish.

Here, the researchers investigated the response of the early life history stages of A. marina to combined exposure to ocean acidification conditions and copper in order to test the hypothesis that copper toxicity will be enhanced at reduced seawater pH.

Worms were collected from Mothecombe beach, Devon, UK during November 2013 and assessed for sex and maturity.  Their tanks were filled with natural sediment collected from Mothecombe and well-aerated artificial seawater made up to the correct salinity.

The comet assay was utilized to measure DNA strand breaks in sperm exposed to seawater pH and copper treatments. A 10 μL aliquot of undiluted sperm from each male was added to individual vials containing 1.85 mL of treatment seawater. Sperm-seawater solutions were incubated at 12 ± 0.1 °C for 1 h before being centrifuged for 4 min at 7826 g. The excess fluid was removed and the cell concentrate gently mixed with 1% low melting point agarose heated to 37 °C and dropped onto slides previously coated in 1% high melting point agarose. Briefly, cells were subjected to 2 h of lysis in alkaline conditions at 5 °C, followed by 45 min of denaturation in electrophoresis buffer (0.3 M NaOH and 1 mM EDTA), 30 min of electrophoresis at 25 V and a final neutralization step. Slides were stained with SYBR Safe DNA Gel Stain and examined using fluorescence microscopy (excitation: 502 nm; emission: 530 nm). One hundred cells per slide were analyzed using Comet Assay IV (Perceptive Instruments Ltd.) to quantify the percentage of DNA in the comet tail (resulting from DNA strand breaks) to approximate the percentage of DNA damage induced in each treatment.

The researchers found that there was an increase in spermatozoan DNA damage in the presence of copper across all pH treatments from an average of 14% damage in the controls to 26% in copper treatments (a figure of this data can be seen in the original publication). The researchers concluded that seawater pH and copper interactively induced sperm DNA damage and subsequent analysis identified that this interaction took place between copper and our lowest seawater pH. Medium-term ocean acidification significantly influenced sperm DNA damage.

Additional findings:

  • Larval survival was reduced by 24% when exposed to both ocean acidification and copper combined compared to single ocean acidification or copper exposures.
  • Sperm motility was negatively affected by both ocean acidification and copper singularly with additive toxicity effects of the two stressors when combined.
  • Fertilization success was also negatively affected by both ocean acidification and copper individually, but no additive effects when exposed as combined stressors were present for this stage.

The scientists believe that these findings add to the growing body of evidence that ocean acidification will act to increase the toxicity of copper to marine organisms, which has clear implications for coastal benthic ecosystems suffering chronic metal pollution as carbon dioxide levels rise and drive a reduction in seawater pH.

If you are interested in reading more, please take a look at the original publication:

Ocean Acidification Increases Copper Toxicity to the Early Life History Stages of the Polychaete Arenicola marina in Artificial Seawater. Anna L. Campbell , Stephanie Mangan , Robert P. Ellis , and Ceri Lewis. Environ. Sci. Technol., 2014, 48 (16), pp 9745–9753