Scotland's Marine Atlas: Information for The National Marine Plan

BIOLOGICAL EFFECT OF CONTAMINANTS - IMPOSEX

What is imposex?

Imposex is the development of male sexual characteristics (the formation of a vas deferens and growth of a penis) in female dog whelks. The common dog whelk, Nucella lapillus, is a marine snail. It is abundant on many rocky shores, where it feeds on barnacles.

Cause of imposex

Imposex is caused by exposure to Tri-Butyl Tin ( TBT). TBT entered the marine environment through its use as the active biocide in anti-fouling paints applied to boats' and ships' hulls from the 1970s. The TBT was released by leaching from the ships' hulls and in ports and harbours when old coatings were replaced with fresh paint.

How we measure imposex

The effect of TBT on the dog whelk population is assessed using the Vas Deferens Stage Index ( VDSI). This index is based upon the development of a vas deferens in females and relates directly to the reproductive capability of the dog whelk population. Low levels of imposex appear to cause no significant harm, but at higher levels females may become sterile. This in turn, could result in the loss of populations and seriously affect the shore communities of which they are part. The VDSI is used to classify dog whelk populations from A to F, where A is close to background, D indicates the presence of some sterile females, and F indicates that the dog whelks have died out.

Trends in imposex

TBT was initially banned for use on boats less than 25m in length in 1988, on all international shipping in 2003, and completely on all boats and ships in 2008.

Since the late 1980s, populations of dog whelks have been sampled, on an 'ad hoc' basis, at over 100 sites around the Scottish coast. In 2002 several sites were Class D but no sites were Class E or F. By 2008 the number of Class D sites had decreased and the number of Class A sites had increased. The overall picture is one of a decrease in the levels of imposex, showing that the ban on TBT has been effective. By the late 2000s, the levels of imposex at many sites had decreased to background so the monitoring programme was reduced. A few local 'hotspots' near point sources of TBT, which yielded high VDSI scores in the late 1980s and 1990s are still evident, but even in these hotspots the severity of imposex has decreased.

Imposex in Dog Whelks from 2000-2002

Imposex in Dog Whelks 2007-2009

Common dog whelk

© SEPA

BIOLOGICAL EFFECT OF CONTAMINANTS - EROD

What is EROD?

EROD [7-Ethoxyresorufin-O-deethylase] is an enzyme found in fish liver which is important in the metabolism of contaminants such as certain polycyclic aromatic hydrocarbons ( PAHs) and other structurally similar compounds.

Why do we measure EROD?

The expression and activity of EROD increases in fish exposed to these contaminants through the food they eat, contact with contaminated sediments or via the water column. Although the health effects of high levels of EROD are unknown, the mechanism of enzyme induction produces highly reactive intermediates of certain contaminants which may cause the onset of carcinogenesis, cell death and embryonic mortality.

EROD measurements

EROD in plaice and flounder has been measured annually since 2001 at potentially contaminated and uncontaminated (background) sites throughout Scotland.

As the health effects of EROD are unknown, the results were assessed from the range of data obtained from 466 observations in plaice (excepting values below the limit of detection and including data from reference sites). The lowest 10th percentile value of 5.26 pmol min ^-1 mg ^-1 (protein) was multiplied by 3.3, 10 and 33 to give the background response range ( BRR), close to background response range ( CBRR), elevated response range ( ERR) and high effect response range ( HRR) (see table below).

Due to the relatively small dataset available for flounder EROD activity and the fact that flounder and plaice respond similarly, the response ranges developed for plaice were applied for assessment of EROD data for both species.

Flounder

© Paul Kaye

EROD activity in plaice and flounder in 2007

EROD in plaice

EROD activity in plaice was at the background response range at the reference sites, Broad Bay, Colonsay and Pladda, and did not exceed the close to background range at Irvine Bay, Hunterston and Skelmorlie. The response range in plaice at the Garroch Head site was in the elevated response range between 2001 and 2005, but decreased to the close to background response range in 2006 and 2007.

ERODin flounder

EROD activity in flounder was in the effects response range in the Forth estuary in 2001, reducing to close to background then background response range between 2004 and 2007. EROD activity in flounder in the Clyde estuary (Bowling) did not exceed the close to background response range. The response range in flounder at the St Andrews Bay reference site was close to background in 2001 and 2002, and decreased to the background response range during the remainder of the sampling period.

Trends in EROD

The results show that EROD activity in flounder and plaice caught in the Clyde and Forth has decreased and is now in the close to background or background response range at all sites. This is particularly apparent at one of the most affected sites in the Clyde, Garroch Head where sewage sludge disposal ceased in 1998 and the evidence from EROD data suggests that exposure of plaice in this area to planar organic contaminants has been decreasing consistently since 2001.

There is a clear indication that the EROD response measured at Garroch Head and Holy Loch may be related to a decrease in contaminant availability at these sites. It would be of value to relate these responses to sediment contaminant data over the same sampling period. Furthermore the EROD responses reported here could be used in an integrated assessment framework with chemical contaminant concentration in sediment and biota and with other higher biological effect responses.

Response ranges used for classification of EROD activity in plaice and flounder

Criteria	Assessment calculation of upper range value for each criteria	Assessment criteria range; values as pmol/ min/mg protein ( LV = lowest value)
BRR	10th percentile value x 3.3	LV - 17
CBRR	10th percentile value x 10	18 - 52
ERR	10th percentile value x 33	53 - 174
HRR	> 10th percentile value x 33	> 175

BIOLOGICAL EFFECT OF CONTAMINANTS - ENVIRONMENTAL GENOMICS

What are environmental genomics?

Genomics is the study of gene sequences. Analytical techniques are being developed to assess the stress response of mussels exposed to contaminants in the marine environment from the expression of gene sequences. The results of a study of the stress response of mussels exposed to contaminants and three sites in the Clyde are described here.

Biochemical response of exposure to contaminants

When animals are exposed to harmful chemicals they respond by producing enzymes that can eliminate the chemical threat. The chemicals bind to a cellular protein known as a receptor. The receptor activates gene sequences that promote the synthesis of the protective enzymes to produce a messenger molecule, messenger RNA ( mRNA). The messenger RNA is subsequently translated into the protein sequence of the enzyme.

How can we measure this response?

The quantities of mRNAs are very low and chemically they cannot be distinguished one from another. Polymerase Chain Reaction or PCR is used to amplify the signal of specific mRNAs. This method has been described as "molecular photocopying" because it can start with complex mixtures of DNA and quickly produce millions of copies of a single component of that mixture. Monitoring the "molecular photocopying" process in real time allows quantification of the amount of DNA in the starting material. This adaptation is known as Quantitative, Real-time PCR or qPCR for short.

Responses measured

The levels of mRNAs (messenger RNAs) produced by several genes associated with adaptive stress responses to different contaminants were measured. The genes measured included metallothionein, which respond when exposed to metals in the environment, and others such as catalase and glutathione S-transferase, which respond to exposure to trace organics compounds.

Mussels were collected from three sites in the Clyde Sea Area in January 2009, as shown on the map. Tissue from half of the animals was used for chemical determination of metals and organic compounds (polycyclic aromatic hydrocarbons and polychlorinated biphenyls). Tissues from the other half were used to isolate mRNA which was converted by an enzymic method to DNA which in turn was used for qPCR. The levels of 8 different mRNAs or transcripts were measured either in all tissues or in selected tissues.

Location of sampling sites for environmental genomics

Comparison of results

Results are expressed as a ratio relative to the clean reference site at Colintraive. Significant differences were found in transcript levels at the other two sites relative to the Colintraive reference site, particularly at Woodhall where the gene transcripts HSP90 and MT10 in gill and digestive gland were higher.

Comparison of qPCR results of mRNA for each gene relative to Colintraive, showing levels of significance

Key: DG: digestive gland; NS: not significant; ND: not done
Key to gene transcripts: MT20 II, metallothionein-20; MT10, metallothionein-10; Vdg3, Veliger digestive gland 3; HSP90, Heat shock protein 90; GST, glutathione S-transferase; p53 protein 53kDa; Try, serine protease CFSP3. Source: Glasgow Caledonia University

Woodhall © Thomas Nugent

Colintraive © Kenneth Hall

What do the results mean?

Each of the genes studied are involved in adaptive stress responses to contaminants, so the implication of transcript values significantly higher than at Colintraive indicate higher levels of biological effects from contaminants than at Colintraive, while significantly lower values indicate less biological effect.

The most obvious finding from this study was that a number of genes in all three tissues were affected at Woodhall, whereas fewer genes and fewer tissues differ at Garnock relative to Colintraive. The higher levels of gene transcripts in metallothioneins at Woodhall suggest effects due to heavy metals, and since both MT10 and MT20 were affected this indicated exposure effects attributable to essential metals and heavy metals. This is consistent with the observation that mussel body burdens of zinc, chromium and lead at Woodhall were more than twice as high as in animals at Colintraive.

In contrast, the mussels collected from the Garnock Estuary showed no significant differences in MT10 or MT20 in any of the tissues compared to Colintraive, and body burdens of metals were also similar to those at Colintraive.

Elevated levels of catalase at Woodhall and HSP at Woodhall and Garnock may be indicative of oxidative stress resulting from exposure to heavy metals and/or to organic contaminants. Body burdens of several polycyclic aromatic hydrocarbons (chrysene and indeno (1, 2, 3-cd) pyrene) were more than 15 times higher at Woodhall compared to Colintraive. The gene transcript Trp was altered in the digestive gland at both Woodhall and Garnock. Elevated Trp may be associated with the presence of pathogens and mussels exposed to chemicals may be more susceptible to infections.

What do the findings of the study tell us?

The study showed that the new analytical technique was able to measure changes in stress-response genes that reflected the exposure of mussel populations to contaminants. The chronic effects of contaminants may have implications for the health of mussel populations due to stress on the animals, since physiological energy is required to protect themselves from the environmental pollutants.

The concentration (mg/kg) of cadmium, chromium, copper, nickel, lead, zinc and arsenic in mussels at Woodhall, Colintraive and Garnock Source: SEPA	The concentration (µg/kg) of silver and mercury in mussels at Woodhall, Colintraive and Garnock Source: SEPA
The concentration (µg/kg) of polycyclic aromatic hydrocarbon compounds at Woodhall and Colintraive Source: SEPA	Mussels Source: SEPA
Relative levels of transcripts at Woodhall Source: Glasgow Caledonia University	Relative levels of transcripts at Garnock Source: Glasgow Caledonia University