Baseline survey of marine sediments collected from the Kingdom of Bahrain: PAHs, PCBs, organochlorine pesticides, perfluoroalkyl substances, dioxins, brominated flame retardants and metal contamination.

A baseline survey of sediment contamination was undertaken at 14 locations around the coastline of Bahrain in May 2017, followed by a focused survey of 20 sites, in November 2019. Samples were assessed for industrial pollutants, including metals, PAHs and a suite of organohalogen compounds. The data generated indicated that levels of chemical contaminants were generally low and did not pose a toxicological risk when assessed against commonly applied sediment quality guidelines (SQG). The highest concentrations of PAHs and PCBs were identified in samples collected at coastal sites adjacent to a refinery area known to contain a diverse mix of industry. Tubli Bay, a heavily stressed small bay receiving high loads of sewage effluent, was also identified as an area warranting further investigation with elevated concentrations of BDE209, PFOS and metal contamination. Such data provides a useful baseline assessment of sediment contamination, against which management control measures can be assessed.

Histopathological survey of potential biomarkers for the assessment of contaminant related biological effects in species of fish and shellfish collected from Kuwait Bay, Arabian Gulf.

The marine environment in Kuwait is dominated by Kuwait Bay, a shallow, depositional habitat vital for the breeding and propagation of marine organisms. The bay receives effluent inputs from industrial centres, ports, sewage outflows along with discharges from power and desalination plants. The major classes of pollutant discharged into the bay include petroleum hydrocarbons, metals, nutrients, cooling water and hyper-saline water. Further, the bay has been historically impacted by a deliberate release of oil and contamination with ordnance and shipwrecks during the 1991 Gulf war. With an aim to establish an integrated pollution effects monitoring programme in Kuwait, this paper describes the application of a quality assured approach to conduct a histopathology baseline survey in oriental sole (Synaptura orientalis) and the large-toothed flounder (Pseudorhombus arsius), which are two potential sentinel flatfish species present in the Arabian Gulf. Liver and gonadal histopathology revealed a range of pathologies similar to those previously observed in European and American pollution effects surveys that utilise flatfish (including pathology markers indicative of possible carcinogenesis and endocrine disruption). Further, we extended these studies to invertebrates (Jinga prawn, Metapenaeus affinis and the grooved tiger prawn, Penaeus semisulcatus) found within the Arabian Gulf. Such baseline data is essential before attempts are made to develop integrated monitoring programmes that aim to assess the health of fish and shellfish in relation to chemical contamination.

Laser-assisted microdissection: a new tool for aquatic molecular parasitology.

Laser-assisted microdissection (LMD) has been developed to isolate distinct cell populations from heterogeneous tissue sections, cytological preparations, or live cell samples. Downstream applications typically include gene expression studies using real-time PCR and array platforms, diagnostic PCR, and protein expression studies. LMD techniques are now commonplace in mainstream biological research and clearly have suitable applications in the field of aquatic pathology and parasitology. The present study used LMD to isolate 2 dinoflagellate parasites (Hematodinium spp.) from formalin-fixed paraffin-embedded tissue sections from 2 crustacean hosts, Cancer pagurus and Portunus trituberculatus. DNA was isolated from LMD parasite preparations, and partial regions (up to 300 bp) of the small subunit and the first internal transcribed spacer region of the rRNA gene complex from the Hematodinium spp. were PCR amplified using diagnostic primers. The amplification products were sequenced to confirm the identity of the targeted regions. The techniques, applications, and limitations of LMD to address questions in aquatic molecular pathology and parasitology are discussed.

Liver tumors in wild flatfish: a histopathological, proteomic, and metabolomic study.

Fish play host to viral, bacterial, and parasitic diseases in addition to non-infectious conditions such as cancer. The National Marine Monitoring Programme (NMMP) provides information to the U.K. Government on the health status of marine fish stocks. An aspect of this work relates to the presence of tumors and other pathologies in the liver of the offshore sentinel flatfish species, dab (Limanda limanda). Using internationally agreed quality assurance criteria, tumors and pre-tumors are diagnosed using histopathology. The current study has expanded upon this work by integrating these traditional diagnostic approaches with ones utilizing modern technologies for analysis of proteomic and metabolomic profiles of selected lesions. We have applied SELDI and FT-ICR technologies (for proteomic and metabolomic analyses, respectively) to tumor and non-tumor samples resected from the liver of dab. This combined approach has demonstrated how these technologies are able to identify protein and metabolite profiles that are specific to liver tumors. Using histopathology to classify "analysis groups" is key to the success of such an approach since it allows for elimination of spurious samples (e.g., those containing parasite infections) that may confuse interpretation of "omic" data. As such, the pathology laboratory plays a central role in collating information relating to particular specimens and in establishing sampling groups relative to specific diagnostic questions. In this study, we present pilot data, which illustrates that proteomics and metabolomics can be used to discriminate fish liver tumors and suggest future directions for work of this type.

DNA adduct analysis and histopathological biomarkers in European flounder (Platichthys flesus) sampled from UK estuaries.

The presence of genotoxic and potentially carcinogenic chemical contaminants in the estuarine and coastal marine environment is well documented. In this study, European flounder (Platichthys flesus) sampled from eight UK estuaries were analysed for hepatic DNA adducts, using the 32P-postlabelling assay and liver histopathology as part of an on going survey to establish the health status of UK estuaries. Fish were collected from the estuaries Tyne, Mersey, Thames, Alde (reference site), Belfast, Forth, Clyde and Southampton. At the majority of contaminated sites (Southampton, Thames, Clyde, Tyne and Mersey) the predominant DNA adduct profile consisted of diagonal radioactive zones (DRZs). In contrast, flounder collected from the Forth, Alde and Belfast lacked DRZs with only background levels of DNA damage being observed. Statistically significant differences were observed between several of the sites sampled with the hepatic DNA adduct levels detected in flounder from Southampton, Thames and Clyde statistically elevated (P < 0.05) over those levels detected at the Tyne (Southampton and Thames only), Forth, Alde and Belfast. Histological analysis of these samples revealed a range of lesions including foci of cellular alteration, hepatocellular fibrillar inclusions, nuclear pleomorphisms along with non-toxicopathic changes/alterations, such as those associated with cell turnover (apoptosis, necrosis, regeneration) and immune-related functions (melanomacrophage aggregates, inflammation). Although it is difficult to associate higher prevalence of these lesion types with specific sites, generally, the lowest prevalence was seen in flounder captured from the Alde estuary, with higher prevalence (particularly of melanomacrophage aggregates, inflammation and necrotic foci) seen in fish from the contaminated sites.

Histopathological biomarkers in estuarine fish species for the assessment of biological effects of contaminants.

The increasing emphasis on the assessment and monitoring of estuarine ecosystems has highlighted the need to deploy appropriate biological indices for these locations. Fish diseases and histopathology, with a broad range of causes, are increasingly being used as indicators of environmental stress since they provide a definite biological end-point of historical exposure. This study reports on the histopathological alterations observed in selected organs and tissues of three species of estuarine fish (Platichthys flesus, Pomatoschistus minutus and Zoarces viviparus), captured from four British estuaries (the Tyne, Tees, Mersey and Alde), differently impacted by contaminants, including PAHs. A biannual sampling regime was used to identify the important seasonal variations that occur in terms of the observed biological effects. Inflammatory lesions and hepatocellular fibrillar inclusions attained their highest prevalence in P. flesus captured from the Tyne, Tees and Mersey. The presence of pre-neoplastic and neoplastic toxicopathic lesions was highest in P. flesus captured from these sites, when compared to fish from the Aide reference site. In particular, the prevalence of hepatic foci of cellular alteration (up to 43.3%) and hepatocellular adenoma (up to 10%) were highest in P. flesus captured from the Mersey estuary. Intersex (ovotestis) was only recorded in male P.flesus captured from the Mersey estuary (up to 8.3%) and from male Z. viviparous captured from the Tyne estuary (25%). Pathologies associated with the gill and the kidney were also most prevalent in fish captured from the Tyne, Tees and Mersey estuaries. This study has successfully applied histopathology to an estuarine monitoring program, both for the recording of toxicopathic lesions in the liver and other organs, and for the detection of the endpoint of endocrine disruption, intersex. As such, it provides a powerful integrative tool for the assessment of biological effects of contaminants in these environments.

Genotypic mutation analysis in the p53 gene of benzo[a]pyrene-treated European flounder (Platichthys flesus).

We have applied a genotypic mutation detection system (the Restriction Site Mutation (RSM) assay) to detect mutations in the marine teleost flounder (Platichthys flesus). The aim of this study was to evaluate this species as an environmental indicator of genotoxic exposure. We have used the model genotoxin benzo[a]pyrene (B[a]P) to determine the limits of mutation detection in the p53 gene of flounder liver DNA. This study has revealed two important findings. Firstly, we were able to demonstrate that a polymorphism exists in the TaqI restriction site of exon 8 of the flounder p53 gene at codon 243. This polymorphic allele was present as a heterozygote at a mean frequency of 15%, whereas 85% carried the homozygous wild type sequence. Secondly, we established that B[a]P treatment resulted in specific mutational events at the adenine base of the same TaqI site, contrasting previous reports stating that there was a guanine preference for this chemical in mammalian DNA. This difference in mutation specificity may possibly be accounted for by sequence specific factors or by species differences in metabolic activation and/or DNA repair and are worthy of further study.

An assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in selected invertebrate and vertebrate species.

The grounding of the Sea Empress oil tanker resulted in the release of 72,000 tonnes of crude oil into Milford Haven, Wales, UK. Our initial studies indicated that this contamination resulted in elevated levels of DNA adducts in one of the area's native marine species Lipophrys pholis [B.P. Lyons, J.S. Harvey, J.M. Parry, An initial assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in the intertidal teleost Lipophrys pholis, Mutat. Res. 390 (1997) 263-268]. These original studies were extended and the genotoxic impact of the oil contamination was investigated in the invertebrates Halichondria panicea and Mytilus edulis, along with the vertebrate fish species L. pholis, Pleuronectes platessa and Limanda limanda. DNA adduct levels were assessed in these species over a period of 2-17 months after the incident. The studies indicate differences in the impact of acute oil contamination upon vertebrate and invertebrate species. The oil contamination did not induce any detectable elevations in adduct levels in the invertebrate species H. panicea and M. edulis. In contrast, the oil contamination did appear to induce adducts in the vertebrate teleost species L. pholis, P. platessa and Lim. limanda. Despite some difficulties in sampling, the data obtained 12-17 months after the spill suggested that the affected species recovered from the oil contamination. While the studies indicate that the genetic impact of the oil contamination was less severe than might have been expected, it remains possible that the DNA adducts detected in the teleosts could lead to genetic changes in these species in the future.

An initial assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in the intertidal teleost Lipophrys pholis.

The Sea Empress oil spill resulted in the release of vast quantities of potentially genotoxic contaminants into the coastal environment of the county of Pembrokeshire (UK). We are at present attempting to determine the potential genotoxic impact of the incident upon the native marine species of the area. Here we describe the levels of DNA adducts in specimens of the intertidal teleost, Lipophrys pholis, exposed to extensive oil extensive oil contamination as an indication of exposure to potential genotoxins. We detected elevated levels of adducts in L. pholis specimens from an area that underwent heavy oil contamination as compared to specimens from a clean reference area devoid of oil contamination. These preliminary studies indicated that the oil contamination induced DNA adducts in the L. pholis specimens, which could potentially cause genetic damage in this native marine species. Further studies are now required to assess the full extent of the genotoxic impact of the oil spill upon the Pembrokeshire area's native marine life.