Three biomarkers of contaminant-induced stress (liver histopathology, the lipid peroxidation (LPx) assay and the acetyl-cholinesterase (AChE) assay) were adapted for application to the estuarine-dependent marine fish Rhabdosargus holubi (Steindachner, 1881). Specimens of R. holubi were collected using a seine net from 3 temporarily open/closed estuaries in the Eastern Cape, South Africa, each impacted by different anthropogenic activities. The East Kleinemonde estuary has a housing settlement on the banks in the lower reaches and some agriculture in its catchment. The Old Woman's estuary has a golf course adjacent to its lower and middle reaches and is crossed by a national road in its upper reaches. The Mtana estuary is virtually pristine, with limited cattle grazing occurring along the banks of the estuary and some subsistence agriculture in the catchment. According to the biomarker results from this study, R. holubi from the East Kleinemonde were in good health, as reflected by low LPx and high AChE levels. The liver histopathology did, however, suggest possible previous exposure to stress (increased melanomacrophage centres, increased perivascular connective tissue and severe vacuolation). Overall, liver histopathology results did not differ significantly between estuaries. Fish from the Old Womans recorded significantly inhibited AChE and increased LPx, while fish from the Mtana exhibited significantly increased LPx only, suggesting possible exposure to anticholinesterase contaminants in the former estuary and some form of oxidative stress in the latter. Although water samples were collected from each of the 3 estuaries and analysed for polychlorinated biphenyls, organochlorines, organophosphorous pesticides and pyrethroids, none of these chemicals were detected. As pesticide residues in water are highly variable, both temporally and spatially, future studies should focus on measuring tissue burdens of organisms in order to identify the contaminant stressor. This study has shown that while chemical analyses of water provide a 'snap-shot' of water quality at the time of sampling, biomonitoring can integrate past exposures to stress and is thus useful for identifying potential situations of concern that require further detailed investigation.

ABSTRACT1. A prototype Estuarine Fish Health Index (EFHI) using biological indicators from multiple levels of biological organization is described. Three test estuaries of similar size and morphology but with different development pressures were sampled for this purpose.2. The EFHI integrates the well established Estuarine Fish Community Index (EFCI) developed for South African estuaries, which uses fish community level metrics to assess estuarine condition, with stress responses measured at lower levels of biological organization.3. Four sub-organism level biomarkers were used: liver histopathology assessment, acetylcholinesterase and lipid peroxidase assays and an individual fish condition factor analysis. The data from these analyses and the results of the EFCI were integrated to determine if combining these inputs produces a more sensitive and predictive tool for assessing estuarine health than when used in isolation.4. Challenges and management applications of this new tool are explored and further studies to improve its prognostic capacity are suggested.

1. Crayfish are invasive polytrophic keystone species, which are phylogenetically unique on the African continent. The Australian redclaw crayfish Cherax quadricarinatus is invasive in southern African freshwater systems including the Zambezi River Basin. Surveys conducted across the Zambezi Basin (Botswana, Zimbabwe, Namibia and Zambia) between 2017 and 2019 showed that C. quadricarinatus is broadly distributed across the Upper and Middle Zambezi and is rapidly spreading through natural and human-mediated means across several ecoregions. 2. The probability of capture (P capture ), catch per unit effort (CPUE) and population characteristics of C. quadricarinatus from the recent Barotse floodplain invasion were compared with older invasions from Lake Kariba and Kafue River.3. The P capture and CPUE of C. quadricarinatus in the recently invaded region of the Barotse floodplain were similar to those of the older invasions. Mass and carapace length of C. quadricarinatus from the Barotse floodplain were significantly lower than those of C. quadricarinatus from the older invasions. Sex † Deceased

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