Degree Thesis
HALMSTAD
UNIVERSITY
Master’s Program in Applied Environmental Science
Copper bioaccumulation in blue mussels and periwinkles from marinas
Environmental Science, 15 credits
Tomas Sjökvist
Copper bioaccumulation in blue mussels and periwinkles from marinas
-Tomas Sjökvist-
Supervisor: Antonia Liess Examiner: Stefan Weisner
Halmstad University, School of Business, Engineering and Science, Master’s Program in Applied Environmental Science
Abstract
Copper (Cu) is a heavy metal that is essential for life but toxic at high concentrations. This toxic effect is used on boats to prevent biofouling on boat hulls by painting the hulls with antifouling paint that contain high levels of Cu. The Cu is slowly diffused out in the water and accumulated by animals higher up in the food chain. In order to test the effects of marinas on Cu bioaccumulation in invertebrates, I sampled molluscs at seven marinas and seven shore sites on the Swedish west coast. Two molluscs with different feeding behaviours, one grazer, the periwinkle (Littorina littorea) and one filter feeder, the blue mussel (Mytilus edulis) were used as study organisms. Both species were sampled at each location within 50 m from each other. Body Cu concentration of both species was measured with a Flame Atomic Absorption Spectrometer (FAAS). Cu concentration of periwinkle soft body tissue was generally higher than in mussel soft body tissue. In addition, periwinkle tissue Cu concentration reacted strongly positively to the presence of marinas, whereas mussel tissue Cu concentrations did not. This shows that contamination from marinas affects the grazing periwinkle but not the filter feeding blue mussel. Thus, for biomonitoring purposes, grazers may be more suited as indicator organisms.
Keywords
Littorina littorea, Mytilus edulis, antifouling paint, invertebrates, biomonitoring
1. Introduction
Marinas and boats are a well-known contamination source of a variety of different metals that are used as a pesticide in anti-fouling paint to prevent fouling by algae and barnacles on the boat hull (Neira et al., 2013). Fouling increases the resistance of the boat in the water so that the fuel consumption increases and the boats manoeuvrability is decreased (WHOI, 1952).
These antifouling paints contain different toxic compounds, mainly the metals copper (Cu) or zinc. Cu is the most common metal used today as a biocide in antifouling paints (Kemi.se, 2019). At the moment, 28 antifouling paints containing Cu are allowed to be sold in Sweden and used on the Swedish west coast for boats in private use(Kemi.se, 2019). Cu paint is constructed so that Cu slowly diffuses out to the water and thus prevents biofouling. After Cu has leaked out to the water it sinks to the bottom and accumulates in the sediments(Karadede and Ünlü, 2000, Champ, 2000, Neira et al., 2013). Therefore sediments in marinas are often Cu contaminated and contain much higher concentrations of Cu than sediments in coastal areas without boat activity. A recent Swedish study found Cu concentration per dry weight sediment of over 55 mg/kg in small marinas (Sjökvist and Kvibling, 2018).
Cu exist in the environment at low concentrations and is vital for life but can be toxic to biota at higher concentrations (for lethal concentration of aquatic biota see Jin et al., 2015). High levels of Cu in the body can contribute to physiological stress and lead to damage of the DNA structure of invertebrate animals such as mussels and snails (Troncoso, Galleguillos and Larrain, 1998, Hu et al., 2014, Van den Broeck et al., 2010). There are two uptake pathways of Cu into marine animals (Figure 1). The first pathway is via bioaccumulation, where algae take up Cu from the water or sediment (Baumann et al., 2009). The second pathway is biomagnification, where contaminated algae are eaten by invertebrate animals, which
accumulate Cu over their lifetime and transfer it further to fish (Brooks et al., 2015, Guthrie et al., 1979), birds and even to humans. Even though Cu biomagnifies, the effect is strongest on benthic species, living and grazing directly on or in contaminated sediments, such as molluscs and Annelida (Rygg, 1985). In Cu polluted areas with high levels of sediment contamination (Cu in sediment > 200 mg/kg dry weight), many benthic species decline in number or go locally extinct due to acute poisoning. Therefore Norway has the following thresholds for Cu contamination (Norwegian guidelines for Cu contaminated sediments, expressed as Cu per dry weight sediment): < 35 mg/kg naturally occurring, 35-51 mg/kg nontoxic effects, 51-55 mg/kg chronic effects, 55-220 mg/kg acute toxic and >220 mg/kg widely spread toxicity (Statens Forurensningstilsyn, 2007).
Figure 1: Pathways of Copper into the food chain.
Different species are affected differently by sediment contamination, because of differences in feeding behaviour. Species ingesting contaminated sediments (Annelida), or species feeding on algae that grow on contaminated sediments (certain mollusc, especially
gastropods), will be more exposed to contaminants accumulated in the sediment, compared to for example filter feeders (Rygg, 1985). On the West Coast of Sweden, common benthic invertebrates are molluscs, represented by both gastropods (snails) and bivalves (mussels).
Snails and mussels have fundamentally different feeding behaviours; snails mainly graze on benthic algae, growing e.g. on sediments, whereas mussels are often filter feeders, filtering the water column for phytoplankton. Snails and mussels may therefore differ in their exposure to sediment bound contaminants (Figure 1).
The aim of this study is to examine if small marinas lead to copper contamination of the benthic biota, and weather sediment-living grazers (snails) are more exposed to the contamination and thus more suitable as bioindicators than sediment-living filter-feeders (mussels).
Study questions
1. Do benthic invertebrates have higher copper levels in their body tissue if they live on contaminated sediments in bays with small marinas compared to those living on sediments in bays free of boat traffic?
2. Do benthic invertebrates with different feeding behaviours differ in copper
bioaccumulation so that grazers (snails) accumulate higher amounts of copper than filter-feeders (mussels)?
2. Material and Methods
Study species
My study species are the filter feeder blue mussel (Mytilus edulis) and the grazer periwinkle (Littorina littorea). Periwinkles eat algae that are growing on stones and other hard surfaces under the water while the blue mussels filtrate the water that are passing theme through their gills (Tang and Riisgård, 2017, Seuront et al., 2018, Storey et al., 2013). These two species were chosen because of the separate ways they collect their food: periwinkles graze and blue
mussles filter feed. Both species have a cleaning ecosystem service on eutrophicated seas (Díaz, Kraufvelin and Erlandsson, 2012, Wollak, Forss and Welander, 2018). Both periwinkles and blue mussels are common species in this area (Tomas sjökvist, personal observation).
2.1 Study site
The studied marinas and reference sites are located on Sweden’s West Coast (Bohuslän) around Orust, including its surrounding islands and the south shoreline of the fjord Gulmarn. I chose this location because the region has a long history and tradition in boat activity with lots of small marinas (Valinder et al., 2005). The samples were taken in the early spring when the boat activity is generally low in the area and most of the boats are still laid up on land.
2.2 Study design
To test if the presence of marinas increases the copper concentration in periwinkles and blue mussels, I collected invertebrate samples from 7 marinas and 7 reference shore sites. Marinas (with a capacity of 100-400 boats) and reference sites with no boat activity were used. I used similarly sized marinas, because too big a range in boat capacity could lead to high variation between samples from marinas. Only sites were both test species were present were selected.
Animals were collected at a maximum of 50 meters from each other in every shore site or marina.
2.3 Sampling and preparing samples for analyses methods
The samples were collected with a D-net or by hand and wading-trousers were used to get access to deeper parts. 15-20 periwinkles and 4 blue mussels were taken from each location.
The samples were then put into a plastic bag, marked with which species and location, and then stored in a portable freezer at a temperature of -18°C.
After collection all samples were prepared for analysis first by freeze drying the sample to get rid of all the moisture. The freeze dryer was set to -50 to -60°C with a pressure of 0.049 mbar for 3 days. Further the dried samples were weighed (dry weight) before ashing to take away all the carbon and nitrogen by an oven that holds 550°C for 2.5 hours (Schneider et al., 2017).
After ashing the samples were weighed again. Then 20 ml 7 mol nitric acid was added to each sample to solve all copper in the sample, and samples were then put into an autoclave set to 120°C over night to increase the effect of the acid (Hervé-Fernández et al., 2010). Later the samples was filter with a gf/f. After filtration each sample had a volume of 22-23 ml. To avoid damaging the Flame Atomic Absorption Spectrometer (FAAS), samples were then diluted with 77-78 ml distilled water. The final volume of the more neutralised solutions were 100 ml.
2.4 Analyses
For the analysis, a Varian spectraAA 100 a FAAS was used to measure the Cu concentration in the samples. First a detection curve with 0.5, 1.00 and 2.00 mg/L as known Cu
concentrations was made. For those samples with higher concentration than the first detection curve, a separate curve was made with 1.00, 2.00 and 5.00 mg/L as known concentration. The FAAS measured concentration (C) in mg/L. Because the sample volume of 100 ml is divided with analyse answer in mg/1000ml, a factor of 0.1 is inserted into the following equation, used to calculate the dry weight Cu concentration.
(𝐶 𝑥 0.1) 𝐷𝑊
Where C is Cu concentration (mg/L) and DW is the dry weight (mg) of invertebrate soft body tissue.
2.5 Ethical aspects
To reduce the suffering of these invertebrates as much as possible, all animals were transported in a cooler and later killed by freezing. No ethical permits are necessary for animal experiments with invertebrates and the degree of suffering was deemed low.
2.6 Statistics
Data was not normal distributed even after it was log-transformed with Log10(C+1). There are no non-parametric tests comparable to two-way ANOVAs, but ANOVAs are generally robust against the violation of the assumption of normal distribution. Therefore I conducted a two-way ANOVA on body tissue copper content with the factors site (marina/shore site) and species (blue mussel/periwinkle) as fixed factors in SPSS IBM 24.
3. Results
Invertebrates in marinas had significantly higher Cu concentrations in body tissue than invertebrates on shore sites, but it was only the periwinkles that had significant higher Cu concentration among the two species (statistics are presented in table 1).
The Cu concentration in body tissue also differed between species, and there was a significant interaction between species and site with Cu as depending variable. The Cu concentration in blue mussel was not affected by marinas. The Cu concentration in periwinkles was higher in both sites and periwinkles was affected by marinas (figure 2).
Figure 2: Differences in Cu concentration between blue mussels and periwinkles depending on site.
(error bras +/− 1 SE)
Table 1: Results of 2-way ANOVA on tissue Cu concentration with the factors site (marina [M]/ shore [S]), species (Periwinkle [P]/ Mussels [M]) and their interaction. df= degrees of freedom, F= F-value, P= P-value.
Factor df F P
Site 1 7.142 0.013 M > S
Species 1 38.178 < 0.005 P > M
Site × Species 1 7.021 0.014
4. Discussion
Benthic invertebrates living on contaminated sediments in bays with small marinas have higher Cu levels in their body tissue, compared to those living on sediments in bays free of boat traffic. This shows that Cu from boat activities in marinas is diffused and spread into the environment and later entering the food chain. High levels of Cu affect the feeding behaviour of invertebrates, leading to decreased appetite, fitness and losses of biota (De Wolf, Backeljau and Blust, 2000). These effects might be even bigger in larger marinas (>400 boat capacity) since sediments in larger marinas tend to be more Cu contaminated (Sjökvist and Kvibling, 2018). Therefore, if larger marinas had been included in this study the differences between shore sites and marinas might have been bigger.The samples were taken during a period of
low boat activity in the area. However, that should not have any effect on the result, as the Cu has accumulated in invertebrate soft body tissue over time (Roesijadi, 1992).
The difference in invertebrate body tissue Cu concentration between shore sites and marinas was only found in periwinkles, not in blue mussels. Periwinkles in marinas contained more than double the amount of Cu compared to periwinkles in shore sites. In both marinas and shore sites, periwinkles contained more Cu than blue mussels. In shore sites the concentration was 7 times higher and in marinas 15 times higher. Therefore, it seems periwinkles aremore exposed to Cu in the environment.
According to Nakajima, Horikoshi and Sakaguchi (1979), there are two main factors that affect algal Cu uptake capacity: species and time. Algae start to take up Cu two hours after exposure and have the highest uptake rate after 20 hours. Because Cu is a heavy metal it may sink to fast for the algae in the free water to have time to accumulate it. When Cu reaches the bottom, it is accumulated in sediments and after that taken up by phytoplankton living on the bottom (Chapman et al., 2009). Even though there was a lot of Cu in the marinas (Sjökvist and Kvibling, 2018), blue mussels in marinas did not contain more Cu than blue mussels in shore sites.
One explanation for higher Cu concentration in periwinkles compared to blue mussels could be their diet, since periwinkles graze on sediment-living algae and blue mussel filtrate free- water algae. Therefore, periwinkles are more suitable for biomonitoring Cu contaminations in marinas than blue mussels (Rainbow, 1995). The periwinkle is also a common species in Europe, North America in both the Atlantic and the Pacific Ocean, which make it easy to monitor regions against each other (Johannesson, 1988).
The difference in Cu concentration between periwinkles in shore sites and periwinkles in marinas was much smaller than the previously shown differences between sediments in shore sites and marinas (Sjökvist and Kvibling, 2018). In shore sites the concentration in
periwinkles was much higher than in the sediment. But in the marinas the Cu concentration in periwinkles was much lower. This could be because of the effect shown by (De Wolf,
Backeljau and Blust, 2000): when affected by Cu the grazers stop feeding and therefore stop accumulating Cu.
5. Conclusion
Invertebrates in marinas contain more copper than invertebrates in shore sites. Thus, copper released in to an aquatic environment can travel up the food chain and be stored in animal soft body tissue. However, this was only observed in grazing benthic invertebrates and not in filter feeding benthic invertebrates. It therefore seems feeding behaviour affects the level of copper exposure, and that grazers are especially exposed.
6. Acknowledgements
I want to send special thanks to my supervisor Antonia Liess who has supported and helped me in this work in full. I also want to thank Per Magnus Ehde who has provided me
information on how the machines in the lab work, and Josefin Nilsson for spellcheck and good advice. Finally, I want to thank Fredrik Lindgren who also was helpful in the lab.
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