Detection of DNA damage in mussels and sea urchins exposed to crude oil using comet assay

The single-cell microgel electrophoresis assay or the comet assay was used to evaluate DNA damage of dispersed crude oil on sea urchins (Strongylocentrotus droebachiensis) and mussels (Mytilus edulis L.). Sea urchins were exposed to 0.06 and 0.25 mg/L dispersed crude oil in a continuous flow system, while the mussels were exposed to 0.015, 0.06 and 0.25 mg/L dispersed crude oil. Sea urchin coelomocytes and mussel haemocytes were sampled after 4 and 5 weeks exposure, respectively. In the sea urchin coelomocytes, there was a significant concentration-related increase in the percentage of DNA in comet tail. In mussel haemocytes, there was a significantly higher percentage of DNA in comet tail for all treatments compared to the control. The responses were concentration-related up to 0.06 mg/L oil. The two highest exposure concentrations of mussels were not significantly different from each other. These results indicate that the comet assay can be used for biomonitoring of DNA damage in marine invertebrates following oil contamination.     

Different levels of mussel (Mytilus edulis) DNA strand breaks following chronic field and acute laboratory exposure to polycyclic aromatic hydrocarbons

Levels of polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) were at least seven-fold higher in mussels sampled from a polluted site (Loch Leven, in Scotland, UK) compared to a nearby clean reference site (Loch Etive) throughout the year 2000. Levels of DNA strand breaks (alkaline COMET assay) using both gill and digestive gland nuclei were similar at both sites despite the difference in contaminant load (total PAH). In contrast, mussels collected from a reference site (Port Quin, Cornwall, UK) had an increase in DNA strand breaks in digestive gland cells following laboratory exposure to B[a]P-dosed Isochrysis galbana. However, after 14 days high dose (20 ppb-exposed diet) animals had returned to levels similar to the controls. There was no evidence of increased necrosis or apoptosis after treatments. The results from these two studies suggest that an adaptive response may prevent ongoing DNA damage in mussels exposed to high levels of B[a]P and PAH contamination.     

DNA strand breakage in mussels (Mytilus edulis L.) deployed in intertidal and subtidal zone in Reykjavík harbour

DNA single-strand breaks were analysed in the blue mussel (Mytilus edulis L.) deployed in intertidal and subtidal zones in the PAH contaminated Reykjavík harbour and at a reference site, Hvalfj?rethur, Iceland. DNA strand breaks were analysed by Comet assay in isolated gill and haemocyte cells from six mussels from each site and depth. Increased DNA damage in both gill cells and haemocytes were observed in mussels deployed in Reykjavík harbour compared to the reference site. Intertidal mussels from Reykjavík harbour had higher DNA damage in haemocytes compared to subtidal mussels. The Comet assay seems to be useful for measuring genotoxic exposure in mussels from the field, and that DNA damage might be higher in the intertidal zone either due to higher exposure to contaminants or because of physiological and biochemical responses to variations in oxygen availability.     

Implications of seasonal priming and reproductive activity on the interpretation of Comet assay data derived from the clam, Tapes semidecussatus Reeves 1864, exposed to contaminated sediments

We explore the use of the clam Tapes semidecussatus Reeves 1864 as an indicator for the presence of potentially genotoxic substances in estuarine sediments. The limitations associated with the interpretation of Comet assay data (expressed as % DNA in tail) in terms of clam reproductive state, size (age) and thermal exposure history following laboratory acclimation are discussed. Hatchery-reared clams, subjected to ambient temperature fluctuations during growth, were exposed in vivo under laboratory conditions for three weeks to sediment samples collected from a polluted site and a "clean" reference site. The DNA damage observed in haemocytes, gill and digestive gland cells was significantly higher in animals exposed to contaminated sediment compared to those exposed to sediment from the reference site. The extent of DNA damage recorded was not correlated with size (age). Spawning was not observed during the experiment. Nevertheless, clams with well-developed gonads showed a statistically higher degree of DNA damage in gill and digestive gland cells- but not haemocytes, demonstrating an increased sensitivity to potential genotoxic compounds, possibly caused by impaired DNA repair capacity due to reproductive activity. Furthermore, the degree of DNA damage in clams exposed to contaminated sediments was higher in autumn and winter compared to spring and summer, suggesting an effect of seasonal priming.     

Accumulation and depuration of petroleum hydrocarbons by black mussels. 1. Laboratory exposure trials

Laboratory experiments were conducted to measure the rates of accumulation and subsequent depuration of hydrocarbons by black mussels after exposure to various concentrations of water-soluble fractions of crude oil. The initial rate of uptake was related to the concentration in the water, but a maximum level of accumulation was found after long-term exposure. The rate of depuration was closely related to the time of exposure prior to depuration. The depuration profile consists of an initial rapid loss followed by a slower loss of the remaining hydrocarbons. Increased exposure time decreases the relative quantity of hydrocarbons lost in the initial rapid depuration. The results indicate that the use of mussels as petroleum hydrocarbon input "monitors" might have limitations.     

Polycyclic aromatic hydrocarbons in bivalves from the San Francisco estuary: Spatial distributions, temporal trends, and sources (1993-2001)

Bivalve tissue samples were examined over a range of spatial and temporal scales (1993-2001) to determine PAH distributions, trends, and possible sources. Mussels (Mytilus californianus), oysters (Crassostrea gigas), and clams (Corbicula fluminea) were deployed for three months in the estuary at stations remote from known point source discharges. The range of summation operatorPAH detected in bivalves was oysters 184-6899 microg/kg dry wt (mean 678 microg/kg dry wt), mussels 21-1093 microg/kg dry wt (mean 175 microg/kg dry wt), and clams 78-720 microg/kg dry wt (mean 323 microg/kg dry wt). Linear regression analysis showed no statistically significant (p>0.05) temporal trends in clam and mussel summation operatorPAH at any of the deployment stations or estuary segments. On the other hand, a statistically significant (p<0.05) decreasing trend was found in summation operatorPAH in oysters at the Petaluma River station, and in the North Estuary segment. PAH isomer pair ratios applied as diagnostic indicators suggested that the bioaccumulated PAH were derived primarily from petroleum combustion, with lesser amounts derived from biomass and coal combustion, and unburned petroleum.     

DNA adducts and polycyclic aromatic hydrocarbon (PAH) tissue levels in blue mussels (Mytilus spp.) from Nordic coastal sites

DNA adducts in gills and digestive gland, as well as polycyclic aromatic hydrocarbon (PAH) tissue levels were analysed in blue mussels (Mytilus spp.) from Nordic coastal areas (Iceland, Norway and Sweden) with diffuse or point sources of PAHs of various origins. Both DNA adduct and PAH tissue levels were generally low, indicating low PAH exposure to the mussels in the areas studied. DNA adducts were found to be higher in gills than in digestive gland of the mussels at all sites studied. Elevated DNA adduct levels in gills were found at 6 sites out of 18 compared to reference sites in respective coastal zones. Adduct levels ranged from 0.5 to 10 nmol adducts/mol normal nucleotides, being highest in mussels from Reykjavík harbour, Iceland (intertidal mussels), and from Fiskaatangen, Norway (subtidal mussels). Total PAH tissue levels in the mussels ranged between 40 and 11,670 ng/g dry wt., and were significantly correlated with DNA adduct levels (r(2)=0.73, p<0.001). PAH ratio values indicated that the PAHs were in most cases of pyrolytic origin, but with petrogenic input near harbours and an oil refinery.     

Oil well produced water discharges to the North Sea. Part I: comparison of deployed mussels (Mytilus edulis), semi-permeable membrane devices, and the DREAM model predictions to estimate the dispersion of polycyclic aromatic hydrocarbons

The oil companies operating in the Norwegian sector of the North Sea have conducted field studies since the mid-1990s to monitor produced water discharges to the ocean. These studies have been used to refine monitoring methods, and to develop and validate a dispersion and impact assessment model. This paper summarizes monitoring data from surveys conducted in two major oil and gas production areas, and compares the results to concentrations of polycyclic aromatic hydrocarbons (PAH) in surface waters predicted by the dose-related risk and effect assessment model (DREAM). Blue mussels and semi-permeable membrane devices (SPMDs) were deployed in the Ekofisk and Tampen Regions and analyzed for more than 50 PAH. PAH concentrations in ambient seawater were estimated based on the mussels and SPMD concentrations, and compared to model predictions. Surface water total PAH concentrations ranged from 25 to 350 ng/L within 1 km of the platform discharges and reached background levels of 4-8 ng/L within 5-10 km of the discharge; a 100,000-fold dilution of the PAH in the discharge water. The PAH concentrations in surface water, predicted by three methods, compared well for the Ekofisk Region. The model predicted higher concentrations than the field-based methods for parts of the Tampen Region; particularly the most tidally influenced areas. Tidally-mediated fluctuations in PAH concentrations in surface water must be considered because they affect the estimation of PAH concentrations from mussel and SPMD residue data, and the predictions by the DREAM model. Predictions using mussels, SPMDs, and modeling support and complement each other; all are valuable tools for estimating the fate and impact of chemical contaminants in produced water that are discharged to the ocean.     

Polycyclic aromatic hydrocarbon (PAH) burden of mussels (Mytilus sp.) in different marine environments in relation with sediment PAH contamination, and bioavailability

Sediments and mussels (Mytilus edulis, Mytilus galloprovincialis) were sampled in different European coastal environments (Germany, France, Spain) and analysed for their polycyclic aromatic hydrocarbon (PAH) content by Gas Chromatography/Mass Spectrometry (GC-MS). Bioaccumulation factors of individual compounds from the sediment were calculated and discussed according to the compound solubility. The mussels showed different accumulation patterns according to the pollution source they were exposed to (dissolved fraction of PAHs, particulate fraction, petroleum present in the water column). The exposure source also depends on the geographical location of the mussels. In the Mediterranean Sea, the bivalves were mainly exposed to the dissolved fraction of PAHs, while in the Baltic Sea and in the Atlantic Ocean, the PAHs associated to the particles were significant sources.     

Effects of toxic dinoflagellate Alexandrium tamarense on the energy budgets and growth of two marine bivalves

Harmful algal blooms (HAB) may impose a serious threat to aquatic lives and human health. We determined the effects of a toxic dinoflagellate Alexandrium tamarense (clone ATCIO1, isolated from Hong Kong coastal waters) on the energy budget, quantified as scope for growth (SFG), and the growth rate of the manila clam Ruditapes philippinarum and the green-lipped mussel Perna viridis. To quantify the SFG, clams and mussels were dosed with different amounts of toxic A. tamarense for 6 days, resulting in different toxin burdens in the tissues. Clearance rate, absorption efficiency, and respiration rate were subsequently measured in these toxin containing bivalves. Clearance rate significantly declined at the highest toxin burden in the clams only, while there was no significant difference in the clearance rate among different treatments for the mussels. The respiration rate of either bivalve was not significantly affected by toxin accumulation in the tissues. In contrast, the absorption efficiency of both clams and mussels decreased, in a concentration-dependent manner for mussels but not for clams, when the tissue accumulated the toxins. With an increase in paralytic shellfish poisoning (PSP) toxin burden, SFG in both clams and mussels was significantly reduced, primarily because of the decrease of absorption efficiency. The growth rate of juvenile clam R. philippinarum, measured as an increment in tissue dry weight over a 15 d exposure period, was significantly lower during their feeding on toxic dinoflagellate than the growth rate of clams feeding on the diatom Thalassiosira pseudonana. The juvenile mussel P. viridis, however, exhibited similar growth rates after feeding on the toxic dinoflagellates and the nontoxic diatom. This study showed that SFG can provide a sensitive and integrated measure of the effect of HAB on the physiology of bivalves. Clam R. philippinarum may be more sensitive, in terms of their energy budget, to PSP toxin accumulation than the mussel P. viridis.     

仿生人工贻贝在筼筜湖海水石油烃污染监测上的应用

仿生人工贻贝（Ｓｅｍｉ－Ｐｅｒｍｅａｂｌｅ Ｍｅｍｂｒａｎｅ Ｄｅｖｉｃｅｓ）是近年来海洋污染监测中新技术,其制作简单,吊挂取样方便,在筼筜湖纳潮进水渠和排污出水口进行夏、冬应用实验,结果显示筼筜湖内石油烃污染仍然较严重,其每ｇ甘油三油酸酯中石油烃浓度夏季可达２．１～７．８ｍｇ。退潮排污出水口人工贻贝石油烃浓度为纳潮进水口的２倍,冬季人工贻贝石油烃浓度又为夏季的１．５倍。同时也说明仿生人工贻贝（ＳＰＭＤ）可克服天然贻贝因性别、生长期、健壮程度和海区条件引起的差异,应用前景良好。     

Exposure of pink salmon embryos to dissolved polynuclear aromatic hydrocarbons delays development,prolonging vulnerability to mechanical damage

Exposure to dissolved polynuclear aromatic hydrocarbons (PAHs) from crude oil delays pink salmon (Oncorhynchus gorbuscha) embryo development, thus prolonging their susceptibility to mechanical damage (shock). Exposure also caused mortality, edema, and anemia consistent with previous studies. Hatching and yolk consumption were delayed, indicating the rate of embryonic development was slowed by PAH exposure. The net result was that exposed embryos were more susceptible to shock than normal, unexposed embryos. Susceptibility to shock was protracted by 4–6 d for more than a month in embryos exposed to exponentially declining, dissolved PAH concentrations in water passed through oiled rock; the initial total PAH concentration was 22.4 μg L⁻¹ and the geometric mean concentration was 4.5 μg L⁻¹ over the first 20 d. Protracted susceptibility to shock caused by exposure to PAHs dissolved from oil could potentially increase the reported incidence of mortality in oiled stream systems, such as those in Prince William Sound after the Exxon Valdez oil spill, if observers fail to discriminate between direct mortality and shock-induced mortality.     

Biota: sediment partitioning of aluminium smelter related PAHs and pulp mill related diterpenes by intertidal clams at Kitimat, British Columbia

The question of polycyclic aromatic hydrocarbon (PAH) bioavailability and its relationship to specific PAH sources with different PAH binding characteristics is an important one, because bioavailability drives PAH accumulation in biota and ultimately the biochemical responses to the PAH contaminants. The industrial harbour at Kitimat (British Columbia, Canada) provides an ideal location to study the bioavailability and bioaccumulation of sediment hydrocarbons to low trophic level biota. Samples of soft shell clams (Mya arenaria) and intertidal sediment collected from multiple sites over six years at various distances from an aluminium smelter and a pulp and paper mill were analysed for 106 PAHs, plant diterpenes and other aromatic fraction hydrocarbons. Interpretation using PAH source ratios and multivariate data analysis reveals six principal hydrocarbon sources: PAHs in coke, pitch and emissions from anode combustion from the aluminium smelter, vascular plant terpenes and aromatised terpenes from the pulp and paper mill, petroleum PAHs from shipping and other anthropogenic activities and PAHs from natural plant detritus. Harbour sediments predominantly contain either pitch or pyrogenic PAHs from the smelter, while clams predominantly contain plant derived PAHs and diterpenes from the adjacent pulp mill. PAHs from the smelter have low bioavailability to clams (Biota-Sediment Accumulation Factors; BSAFs <1 for pitch and coke; <10 for anode combustion, decreasing to ∼0.1 for the mass 300 and 302 PAHs), possibly due to binding to pitch or soot carbon matrices. Decreases in PAH isomer ratios between sediments and clams likely reflect a combination of variation in uptake kinetics of petroleum PAHs and compound specific metabolism, with the importance of petroleum PAHs decreasing with increasing molecular weight. Plant derived compounds exhibit little natural bioaccumulation at reference sites, but unsaturated and aromatised diterpenes released from resins by industrial pulping processes are readily accumulated by the clams (BSAFs >500). Thus while most of the smelter associated PAHs in sediments may not be bioavailable to benthic organisms, the plant terpenes (including retene, totarol, ferruginol, manool, dehydroabietane and other plant terpenes that form the chemical defence mechanism of conifers) released by pulp mills are bioavailable and possess demonstrated toxic properties. The large scale release of plant terpenes by some of the many pulp mills located in British Columbia and elsewhere represents a largely undocumented risk to aquatic biota.     

消油剂处理120#燃料油对海水青鳉(Oryzias melastigma)胚胎抗氧化酶活性影响的研究

以海水青鳉(Oryzias melastigma)胚胎为研究对象,比较了120#燃料油分散液(water-accommodated fractions,WAFs)与乳化液(biologically enhanced water-accommodated fractions,BE-WAFs)的急性毒性效应,并研究了不同浓度(40、100、250 mg/L)下WAFs、BE-WAFs对胚胎内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽硫转移酶(GST)活性的影响。结果表明:在受到石油烃的氧化胁迫后,海水青鳉胚胎内3种抗氧化酶活性变化明显。随着石油烃浓度的升高和暴露时间的延长,3种酶表现出程度不同的诱导效应和抑制效应。其中受石油烃污染影响最为明显的为SOD酶;而GST酶则对消油剂单独暴露表现较为敏感。实验证明,海水青鳉体内SOD酶活性对石油烃污染反应最为敏感,适合作为监测石油烃污染程度的生物标志物。     

Reproductive life history stages sensitive to oil and naphthalene in Atlantic croaker

The reproductive effects of chronic exposure to water-soluble fractions (WSF) of diesel fuel oil and naphthalene in female Atlantic croaker during critical periods of their reproductive life history were investigated. Fish were repeatedly exposed to WSF of oil (2.5 or 5%) or naphthalene (0.5 or 1.0 ppm) for 5 or 8 weeks. Both pollutants blocked sexual maturation in some fish and impaired ovarian recrudescence in others. The majority of the oocytes in exposed fish were undeveloped and widespread oocyte atresia was evident at higher concentrations of these polycyclic aromatic hydrocarbons (PAH). The disruption of ovarian growth and oocyte development in croaker after PAH exposure was associated with alterations in reproductive endocrine function at multiple sites on the hypothalamus-pituitarygonadal axis. A third period of increased sensitivity to xenobiotics was observed during final oocyte maturation.     

Immune inhibition in marine mussels by polycyclic aromatic hydrocarbons

Polycyclic aromatic pollutants produced by a range of industrial processes and the combustion of fossil fuels. They eventually enter the marine environment, and are bioaccumulated in the tissues of sessile, filter-feeding bivalves, causing a variety of sublethal effects. Few studies, however, have examined immune inhibition in molluscs by these xenobiotics. Here, the immunotoxic effects of PAHs were investigated in marine mussels (Mytilus edulis) by means of in vivo exposure experiments. Animals were exposed to a PAH cocktail of anthracene, fluoranthene and phenanthrene for 2 or 4 weeks. The haemolymph was then extracted, and phagocytosis and lysosomal neutral red retention assays performed to examine the effects of PAHs on particle uptake by haemocytes and lysosome integrity, respectively. PAHs were found to inhibit phagocytosis and damage lysosomes. It is hypothesized that PAHs exert their toxicity directly on the lysosomes. A possible consequence of such lysosomal damage will be immune impairment in mussels leading to reduced resistance to infectious diseases.     

Oil well produced water discharges to the North Sea. Part II: comparison of deployed mussels (Mytilus edulis) and the DREAM model to predict ecological risk

Large volumes of water often are produced with oil and gas from offshore platforms. The produced water is separated from the oil and gas and either reinjected into a deep formation or discharged to the ocean. The Norwegian oil and gas industry advocates ecological risk assessment as the basis for managing produced water discharges to the North Sea. In this paper, we compare estimates of ecological risks to water-column communities based on data on hydrocarbon residues in soft tissues of blue mussels deployed for a month near offshore platforms and based on predictions of the Dose related Risk and Effect Assessment Model (DREAM). The study was performed near produced water discharges to the Tampen and Ekofisk Regions of the Norwegian Sector of the North Sea. Because polycyclic aromatic hydrocarbons (PAH) are considered the most important contributors to the ecological hazard posed by produced water discharges, comparisons made here focus on this group of compounds. The mussel approach is based on predicted environmental concentrations (PECs) of individual PAH, estimated from PAH residues in mussels following deployment for a month near several produced water discharges, and predicted no effects concentrations (PNECs) based on a K(ow) regression model. In the DREAM method, PECs for three PAH fractions are estimated in the three-dimensional area around produced water discharge with the DREAM model. PNECs for each fraction are based on the chronic toxicity of a representative PAH from each fraction divided by an assessment factor to account for uncertainty in the chronic value. The mussel method gives much lower estimates of ecological risk than the DREAM method. The differences are caused by the much lower PNECs used in DREAM than derived from the regression model, and by the lower concentrations of aqueous PAH predicted by DREAM than estimated from PAH residues in mussel tissues. However, the two methods rank stations at different distances from produced water discharges in the same order and both identify 2- and 3-ring PAHs as the main contributors to the ecological risk of produced water discharges. Neither method identifies a significant ecological risk of PAH in the upper water column of the oil fields. The DREAM model may produce an overly conservative estimate of ecological risk of produced water discharges to the North Sea.     

Hydrocarbons in bivalve mollusks of Port Valdez, Alaska: Consequences of five years' permitted discharge

Petroleum hydrocarbons were investigated in Mytilus edulis and Macoma balthica at six intertidal sites in Port Valdez, Alaska after three to five years of oil terminal operation. Information about the concentrations of total hydrocarbons, unresolved complex mixture, normal alkanes, isoprenoids, olefins, hopanes and polycyclic aromatic hyrocarbons shows that petroleum accumulation in these bivalve mollusks is greatest at the Valdez municipal boat harbor and a crude oil terminal which has been permitted to discharge 170 kg oil day⁻¹. Accumulation two- to ten-fold less were observed at 3 km from the terminal and boat harbor. The temporal trends in petroleum hydrocarbon concentrations in M. edulis and M. balthica reflect the effects of selective retention and depuration of different hydrocarbon classes as well as variation in hydrocarbon sources. It is not clear whether the petroleum concentrations in Port Valdez bivalves had reached steady state by 1982, the final sampling time reported here.     

柴油水溶性成分胁迫下菲律宾蛤仔溶酶体生物标志物的应答

溶酶体生物标志物是环境污染引起生物效应的早期预警信号。石油类污染物对菲律宾蛤仔(Ruditapes philippinarum)溶酶体完整性的影响尚未研究。本研究以0号柴油的水溶性成分(WSF)为污染物,以养殖的菲律宾蛤仔为受试生物,测试3种溶酶体生物标志物对柴油WSF胁迫应答的剂量-效应关系和时间-效应关系。采用室内模拟暴露实验,将菲律宾蛤仔置于添加不同浓度(0.1~1.6 mg/L)柴油WSF的海水中5 d,结果显示:不同浓度的暴露组血细胞溶酶体中性红保留时间(NRRT)值比对照组低23.1%~48.5%(p<0.05或p<0.01)。而各浓度暴露组的鳃和内脏团酸性磷酸酶(ACP)活性均与对照组无显著差异。选取柴油WSF为0.8 mg/L进行污染的时间-效应实验,于第0、1、3、7、11天取样测定,结果显示:暴露组NRRT值始终低于对照组,且在第1、3天差异显著(p<0.05)。暴露组鳃和内脏团ACP活性都是先逐步升高到第7天达最大值后迅速下降,鳃ACP活性从第3天到第11天与对照组差异显著(p<0.01),内脏团ACP活性从第7天到第11天与对照组差异显著(p<0.05)。本研究未能获得基于N-乙酰-β-氨基己糖苷酶的组织化学染色反应上的溶酶体膜稳定性(LMS)应答。研究表明,菲律宾蛤仔血细胞NRRT和鳃ACP活性可作为海洋污染生物效应监测的潜在生物标志物。     

Mussels document loss of bioavailable polycyclic aromatic hydrocarbons and the return to baseline conditions for oiled shorelines in Prince William Sound, Alaska

Polycyclic aromatic hydrocarbons (PAH) were measured in mussels (Mytilus trossulus) collected between 1990 and 2002 from 11 sites on the shores of Prince William Sound (PWS), Alaska, that were heavily oiled by the 1989 Exxon Valdez oil spill (EVOS). This study, utilizing the methods of the NOAA Status and Trends Mussel Watch Program, found that concentrations of PAH released from spill remnants have decreased dramatically with time and by 2002 were at or near the range of total PAH (TPAH) of 3-355 ng/g dry weight obtained for mussels from unoiled reference sites in PWS. Time-series TPAH data indicate a mean TPAH half-life in mussel tissues of 2.4 years with a range from 1.4 to 5.3, yielding an annual mean loss of bioaccumulated TPAH of 25%. The petroleum-derived TPAH fraction in mussel tissues has decreased with time, reflecting the decreasing release of EVOS residues in shoreline sediments. These results show that PAH from EVOS residues that remain buried in shoreline sediments after the early 1990s are in a form and at locations that have a low accessibility to mussels living in the intertidal zone.