Improvements and cost-effective measures to the automated intermittent water renewal system for toxicity testing with sediments.

The push to make bioassays more sensitive has meant an increased duration of testing to look at more chronic endpoints. To conduct these longer bioassays through the use of traditional bioassay methods can be difficult, as many traditional bioassays have employed manual water changes, which take considerable time and effort. To that end, static-renewal systems were designed to provide researchers a technique to ease the manual water change burden. One of the most well-known static-renewal designs, the static intermittent renewal system (STIR) was produced by the United States Environmental Protection Agency in 1993. This system is still being used in laboratories across the globe today. However, these initial designs have become rather dated as new technologies and methods have been developed that make these systems easier to build and operate. The following information details changes to the initial design and a proof of concept experiment with the benthic invertebrate, Chironomus tepperi, to validate the modifications to the original system.

Spatially variable synergistic effects of disturbance and additional nutrients on kelp recruitment and recovery.

Understanding the impact of multiple stressors on ecosystems is of pronounced importance, particularly when one or more of those stressors is anthropogenic. Here we investigated the role of physical disturbance and increased nutrients on reefs dominated by the canopy-forming kelp Ecklonia radiata. We combined experimental kelp canopy removals and additional nutrient at three different locations in a large embayment in temperate southeastern Australia. Over the following winter recruitment season, Ecklonia recruitment was unaffected by increased nutrients alone, but tripled at all sites where the canopy had been removed. At one site, the combination of disturbance and increased nutrients resulted in more than four times the recruitment of the introduced kelp Undaria pinnatifida. Six months after disturbance, the proliferation of the Undaria canopy in the canopy-removal and nutrient-addition treatment negatively influenced the recovery of the native kelp Ecklonia. Given the otherwise competitive dominance of adult Ecklonia, this provides a mechanism whereby Undaria could maintain open space for the following recruitment season. This interplay between disturbance, nutrients and the response of native and invasive species makes a compelling case for how a combination of factors can influence species dynamics.

Monitoring biofouling communities could reduce impacts to mussel aquaculture by allowing synchronisation of husbandry techniques with peaks in settlement.

Fouling organisms in bivalve aquaculture cause significant economic losses for the industry. Managing biofouling is typically reactive, and involves time- and labour-intensive removal techniques. Mussel spat settlement and biofouling were documented over 20 months at three mussel farms within Port Phillip Bay (PPB), Australia to determine if knowledge of settlement patterns could assist farmers in avoiding biofouling. Mussel spat settlement was largely confined to a 2-month period at one farm. Of the problematic foulers, Ectopleura crocea settlement varied in space and time at all three farms, whilst Ciona intestinalis and Pomatoceros taeniata were present predominantly at one farm and exhibited more distinct settlement periods. Within PPB, complete avoidance of biofouling is impossible. However, diligent monitoring may help farmers avoid peaks in detrimental biofouling species and allow them to implement removal strategies such as manual cleaning, and postpone grading and re-socking practices, until after these peaks.

Effects of early recruits on temperate sessile marine community composition depend on other species recruiting at the time.

In many environments recruitment of dispersive propagules (e.g. seeds, spores and larvae) can vary from situations when particular taxa recruit in relative isolation to times when they recruit simultaneously with other, functionally quite different taxa. Differences in the identity and density of recruiting taxa can have important consequences on community structure, but it is still not clear how the effects of individual taxa on communities are modified when they recruit together with other species. Using an experimental approach we compared early development of a temperate marine sessile community after the recruitment of mixtures of botryllid ascidians and barnacles to that when barnacles or botryllid ascidians recruited alone. Communities exposed to recruitment of botryllid ascidians in isolation differed from those that received barnacles, a mixture of botryllids and barnacles or no recruitment in 2-week-old communities. These early differences were driven by higher abundances of the species that were present as initial recruits in experimental treatments. After 2 months communities also differed between barnacle and mixed recruitment treatments but not mixed and botryllid or botryllid and barnacle treatments. These differences were not directly due to differences in the abundances of our manipulated taxa but occurred because of two abundant arborescent bryozoans, Bugula dentata, which occupied more space in communities that initially received mixed recruitment than in those that received barnacle or no recruitment, and Zoobotryon verticillatum, which occupied more space in communities that initially received only barnacle recruitment than those that initially received botryllid or mixed recruitment. These effects did not persist, and communities did not differ after 6 months. These results suggest that, more generally, species may influence community dynamics differently when they recruit alongside other species than when they recruit in relative isolation.

Spatially variable effects of a marine pest on ecosystem function.

The broad spectrum of anthropogenic pressures on many of the world's coastal bays and estuaries rarely act in isolation, yet few studies have directly addressed the interactive effects of multiple pressures. Port Phillip Bay in southeastern Australia is a semi-enclosed bay in which nutrient management is a major concern. In recent years it has been heavily invaded by marine pests. We manipulated the density of one such invader, the European fanworm Sabella spallanzanii, and showed that it causes changes in the composition of macrofauna in the surrounding sediments, provides habitat for epibiota (both fauna and flora) on Sabella tubes, and reduces the biomass of microphytobenthos on the surrounding sediments. Of greatest concern, however, was the indirect impact on nutrient cycling. We suggest that the impacts on nutrient cycling are largely due to the feeding of Sabella and the epifauna on its tubes, capturing organic N before it reaches the sediment, excreting it back up into the water column as NH4, thereby bypassing sedimentary processes such as denitrification. Most notably, the efficiency of denitrification, the key ecosystem process that permanently removes N from the system, fell by 37-53 % in the presence of Sabella. Importantly though, this study also demonstrated significant spatial variability in fauna, geochemistry and the magnitude of Sabella effects. Given that the effect of Sabella is also likely to vary in time and with changes in density, all of these sources of variability need to be considered when incorporating the effects of Sabella in nutrient management strategies.

Ruinous resident: the hydroid Ectopleura crocea negatively affects suspended culture of the mussel Mytilus galloprovincialis.

Hydroids are major biofouling organisms in global aquaculture. Colonies of the hydroid Ectopleura crocea have recently established in Australian commercial mussel leases culturing Mytilus galloprovincialis. This study examined the impacts of E. crocea on mussel culture at two stages of the production cycle: spatfall and grow-out. Hydroids most commonly fouled the body, edge and dorsal regions of the mussel shell and cause a reduction in the length (4%) and weight (23%) of juvenile mussels. They also consumed mussel larvae in the field and in the laboratory. Prey numbers of many taxa, including mussel larvae, were consistent in natural hydroid diets regardless of the temporal variation in prey availability, implying some selectivity in hydroid feeding. In the laboratory, E. crocea consumed settling plantigrade mussel larvae more readily than trochophore or veliger larvae. Fouling by E. crocea is detrimental to mussel condition, and may affect the availability of wild mussel larvae in the commercial culture of M. galloprovincialis.

Biofouling leads to reduced shell growth and flesh weight in the cultured mussel Mytilus galloprovincialis.

Competitive interactions between cultured mussels and fouling organisms may result in growth and weight reductions in mussels, and compromised aquaculture productivity. Mussel ropes were inoculated with Ciona intestinalis, Ectopleura crocea or Styela clava, and growth parameters of fouled and unfouled Mytilus galloprovincialis were compared after two months. Small mussels (≈ 50 mm) fouled by C. intestinalis and E. crocea were 4.0 and 3.2% shorter in shell length and had 21 and 13% reduced flesh weight, respectively, compared to the controls. Large mussels (≈ 68 mm) fouled by S. clava, C. intestinalis and E. crocea were 4.4, 3.9 and 2.1% shorter than control mussels, respectively, but flesh weights were not significantly reduced. A series of competitive feeding experiments indicated that S. clava and C. intestinalis did not reduce mussels' food consumption, but that E. crocea, through interference competition, did. Fouling by these species at the densities used here reduced mussel growth and flesh weight, likely resulting in economic losses for the industry, and requires consideration when developing biofouling mitigation strategies.

Contrasting effects of variable species recruitment on marine sessile communities.

The species composition, density, and frequency of recruitment into any given habitat are highly variable in most biological systems that rely on dispersive propagules (larvae, seeds, spores, etc.). There are few direct experimental studies of how recruitment variation between single species influences the composition and assembly of whole communities in many of these systems. We manipulated recruitment of a variety of single taxa and followed their effects on the subsequent development of hard-substrate communities of sessile animals living in temperate marine waters. The effects of recruitment on communities were complex. Patterns of recruitment of individual species influenced community structure, but these effects varied greatly depending on the identity of species recruits, the time of community development, and location across three different sites. Variable recruitment of arborescent bryozoans and didemnid ascidians had little effect on community structure. At one site, recruitment of the colonial ascidian Botryllus schlosseri had short-lived effects on community structure, while barnacles had more persistent effects. At another site, recruitment of B. schlosseri and the bryozoan Watersipora subtorquata had strong persistent effects on community structure, dominating space where they recruited and influencing the abundances of a variety of different taxa. Differences in the effects of species recruitment on communities appear to be caused by differences between the ecology and life history of recruiting species as well as differences in background processes between sites. These results demonstrate that discrete recruitment events that vary between single species can be important drivers of community composition but are likely to be heavily influenced by the local environment, even within a single species.

Effects of pulse versus steady recruitment on sessile marine communities.

Variation in patterns of propagule establishment (recruitment) has important effects on population dynamics and the structure of some communities. Most experimental studies have varied recruitment by changing the nature of a single event early in community development, but recruitment can also vary from steady rates of arrival to highly episodic 'pulse' events, causing differences in the temporal spacing of individuals recruiting into patches. We examined whether two different temporal patterns of recruitment of sessile invertebrates affected temperate marine communities in southeastern Australia in two experiments that were run at different times at the same site and that manipulated several different species. Target species entered communities as either a single pulse of recruits within a 2-week period or steady input of the same total number of recruits over a longer time period (5-6 weeks). The pattern of recruitment had variable effects on community structure. The colonial ascidian Botryllus schlosseri did not have a strong influence on community structure whether it recruited in a single pulse or steadily. The cover of B. schlosseri was higher when recruitment occurred as a single pulse. In a second experiment, botryllid ascidians caused changes in the composition of communities when they recruited steadily compared to when they did not recruit or didemnids recruited, but caused no differences in communities when they recruited in a shorter pulse. In contrast, recruitment frequency of didemnid ascidians had little effect, though their presence/absence caused community differences. Though we found that different temporal recruitment patterns can alter community composition, the life history and ecology of particular taxa as well as differences in environmental background processes are likely to influence the strength of these effects.

Potamopyrgus antipodarum has the potential to detect effects from various land use activities on a freshwater ecosystem.

Identifying risks to ecosystems from contaminants needs a diversity of bioindicators, to understand the effects of these contaminants on a range of taxa. Molluscs are an ideal bioindicator because they are one of the largest phyla with extremely high ecological and economic importance. The aim of this study was to evaluate if laboratory bred Potamopyrgus antipodarum has the potential to show the impact of contaminants from various land use activities and degree of pollution on a freshwater ecosystem. We assessed the impact of contaminants arising from runoff and direct discharges in Merri Creek by measuring organism level responses (survival, growth, and reproduction), and sub-organism level responses (glutathione S-transferase (GST) activity, lipid peroxidation (LPO) activity and catalase (CAT) activity) in snails after 28-d of deployment at nine sites in Merri Creek and one site in Cardinia Creek. In Merri Creek, the top two sites were reference sites (with low impact from human activities), while the rest were impact sites (impacted by various anthropogenic land uses). Cardinia Creek (an additional reference site) had lower human activity. High concentrations of heavy metals, nutrients, and/or synthetic pyrethroids (bifenthrin) dominated these sites, which are likely to have contributed towards the negative responses observed in the snails. There was little influence from environmental conditions and site location on the endpoints because we found a similar response at an additional reference site compared to the reference sites in Merri Creek. At the organism level, reproduction increased and/or reduced, while CAT was affected at the sub-organism level. Potamopyrgus antipodarum has the potential to be a sensitive bioindicator for Australian conditions because the snails responded to varying concentrations of contaminants across different land use activities and showed similar sensitivity to P. antipodarum found in other regions of the globe and other bioindicators.

Metal forms and dynamics in urban stormwater runoff: New insights from diffusive gradients in thin-films (DGT) measurements.

Stormwater runoff typically contains significant quantities of metal contaminants that enter urban waterways over short durations and represent a potential risk to water quality. The origin of metals within the catchment and processes that occur over the storm can control the partitioning of metals between a range of different forms. Understanding the fraction of metals present in a form that is potentially bioavailable to aquatic organisms is useful for environmental risk assessment. To help provide this information, the forms and dynamics of metal contaminants in an urban system were assessed across a storm. Temporal patterns in the concentration of metals in dissolved and particulate (total suspended solids; TSS) forms were assessed from water samples, and diffusive gradients in thin-films (DGTs) were deployed to measure the DGT-labile time-integrated metal concentration. Results indicate that the concentrations of dissolved and TSS-associated metals increased during the storm, with the metals Al, Cd, Co, Cu, Pb and Zn representing the greatest concern relative to water quality guideline values (GVs). The portion of labile metal as measured by DGT devices indicated that during the storm a substantial fraction (∼98%) of metals were complexed and pose a lower risk of acute toxicity to aquatic organisms. Comparison of DGT results to GVs indicate that current GVs are likely quite conservative when assessing stormwater pollution risks with regards to metal contaminants. This study provides valuable insight into the forms and dynamics of metals in an urban system receiving stormwater inputs and assists with the development of improved approaches for the assessment of short-term, intermittent discharge events.

Resource distribution influences positive edge effects in a seagrass fish.

According to conceptual models, the distribution of resources plays a critical role in determining how organisms distribute themselves near habitat edges. These models are frequently used to achieve a mechanistic understanding of edge effects, but because they are based predominantly on correlative studies, there is need for a demonstration of causality, which is best done through experimentation. Using artificial seagrass habitat as an experimental system, we determined a likely mechanism underpinning edge effects in a seagrass fish. To test for edge effects, we measured fish abundance at edges (0-0.5 m) and interiors (0.5-1 m) of two patch configurations: continuous (single, continuous 9-m2 patches) and patchy (four discrete 1-m2 patches within a 9-m2 area). In continuous configurations, pipefish (Stigmatopora argus) were three times more abundant at edges than interiors (positive edge effect), but in patchy configurations there was no difference. The lack of edge effect in patchy configurations might be because patchy seagrass consisted entirely of edge habitat. We then used two approaches to test whether observed edge effects in continuous configurations were caused by increased availability of food at edges. First, we estimated the abundance of the major prey of pipefish, small crustaceans, across continuous seagrass configurations. Crustacean abundances were highest at seagrass edges, where they were 16% greater than in patch interiors. Second, we supplemented interiors of continuous treatment patches with live crustaceans, while control patches were supplemented with seawater. After five hours of supplementation, numbers of pipefish were similar between edges and interiors of treatment patches, while the strong edge effects were maintained in controls. This indicated that fish were moving from patch edges to interiors in response to food supplementation. These approaches strongly suggest that a numerically dominant fish species is more abundant at seagrass edges due to greater food availability, and provide experimental support for the resource distribution model as an explanation for edge effects.

More severe disturbance regimes drive the shift of a kelp forest to a sea urchin barren in south-eastern Australia.

Climate change is influencing the frequency and severity of extreme events. This means that systems are experiencing novel or altered disturbance regimes, making it difficult to predict and manage for this impact on ecosystems. While there is established theory regarding how the frequency of disturbance influences ecosystems, how this interacts with severity of disturbance is difficult to tease apart, as these two are inherently linked. Here we investigated a subtidal kelp (Ecklonia radiata) dominated community in southern Australia to assess how different disturbance regimes might drive changes to a different ecosystem state: sea urchin barrens. Specifically, we compared how the frequency of disturbance (single or triple disturbance events over a three month period) influenced recruitment and community dynamics, when the net severity of disturbance was the same (single disturbance compared to triple disturbances each one-third as severe). We crossed this design with two different net severities of disturbance (50% or 100%, kelp canopy removal). The frequency of disturbance effect depended on the severity of disturbance. When 50% of the canopy was removed, the highest kelp recruitment and recovery of the benthic community occurred with the triple disturbance events. When disturbance was a single event or the most severe (100% removal), kelp recruitment was low and the kelp canopy failed to recover over 18 months. The latter case led to shifts in the community composition from a kelp bed to a sea-urchin barren. This suggests that if ecosystems experience novel or more severe disturbance scenarios, this can lead to a decline in ecosystem condition or collapse.

Identification of microplastics in surface water and Australian freshwater shrimp Paratya australiensis in Victoria, Australia.

Compared to marine microplastics research, few studies have bio-monitored microplastics in inland waters. It is also important to understand the microplastics' uptake and their potential risks to freshwater species. The Australian glass shrimp Paratya australiensis (Family: Atyidae) is commonly found in fresh waterbodies in eastern Australia, and are sensitive to anthropogenic stressors but have a wide tolerance range to the natural environmental conditions. This study aimed to understand the microplastics' occurrence and types in water samples and the shrimp P. australiensis, and identify if the shrimp could be a suitable bioindicator for microplastic pollution. Surface water and P. australiensis across ten urban and rural freshwater sites in Victoria were sampled. In total, 30 water samples and 100 shrimp were analysed for microplastic content, and shrimp body weights and sizes were also recorded. Microplastics were picked, photographed and identified using FT-IR microscopy: in water samples, 57.9% of items including suspect items were selected to identify; all microplastics found in shrimp samples were identified. Microplastics were present in the surface waters of all sites, with an average abundance of 0.40 ± 0.27 items/L. A total of 36% of shrimp contained microplastics with an average of 0.52 ± 0.55 items/ind (24 ± 31 items/g). Fibre was the most common shape, and blue was the most frequent colour in both water and shrimp samples. The dominant plastic types were polyester in water samples, and rayon in shrimp samples. Even though results from this study show a relatively low concentration of microplastics in water samples in comparison with global studies, it is worth noticing that microplastics were regularly detected in fresh waterbodies in Victoria, Australia. Compared with water samples, shrimp contained a wider variety of plastic types, suggesting they may potentially behave as passive samplers of microplastics pollution in freshwater environments.

Does interspecific competition affect offspring provisioning?

Offspring size is one of the most well-studied life-history traits, yet it is remarkable that few field studies have examined the manner in which the relationship between offspring size and performance (and thus, optimal offspring size) is affected by the local environment. Furthermore, while offspring size appears to be plastic in a range of organisms, few studies have linked changes in offspring size to changes in the relationship between offspring size and performance in the field. Interspecific competition is a major ecological force in both terrestrial and marine environments, but we have little understanding of its role in shaping selection on offspring size. Here we examine the effect of interspecific competition on the relationship between offspring size and performance in the field for the marine bryozoan Watersipora subtorquata along the south coast of Australia. Both interspecific competition and offspring size had strong effects on the post-metamorphic performance of offspring in the field, but importantly, they acted independently. While interspecific competition did not affect the offspring size-performance relationship, mothers experiencing competition still produced larger offspring than mothers that did not experience competition. Because larger offspring are more dispersive in this species, increasing offspring size may represent a maternal strategy whereby mothers produce more dispersive offspring when they experience high competition themselves. This study shows that, while offspring size is plastic in this species, post-metamorphic factors alone may not determine the size of offspring that mothers produce.

Does size predict demographic fate? Modular demography and constraints on growth determine response to decreases in size.

The modular construction of many plants and animals defies conventional approaches to the study of life histories and population dynamics. An important complication of modular construction is that individuals can rapidly decrease in size when some modules are removed or die or when an individual fragments. Most attempts to describe life histories and population dynamics of modular organisms classify individuals according to their size. This approach relies on the fundamental assumption that fragmentation and module loss have no consequences for an individual apart from a simple decrease in size. Here we experimentally test this assumption. Using a modular marine invertebrate, the encrusting bryozoan Watersipora subtorquata, as a model species, we manipulated colony size and then assessed performance against three potential explanatory models based on size, age, and damage. In a second experiment we disrupted the internal modular demography of colonies to determine whether the performance of a fragment is influenced by the type of modules that remain. Finally, we investigated how constraints on growth in modular organisms uniquely influence growth after module loss. We found that single-state variables such as size or age do not describe performance in our species. Internal constraints substantially reduce growth after a decrease in size, and the age of modules that remain determines the timing of reproductive onset and fecundity. A knowledge of the size history of individuals, including any decreases in size, is necessary to accurately describe life histories and population dynamics in this modular organism. Our results have major consequences for established methods for modeling the demography of modular organisms.

Fish responses to experimental fragmentation of seagrass habitat.

Understanding the consequences of habitat fragmentation has come mostly from comparisons of patchy and continuous habitats. Because fragmentation is a process, it is most accurately studied by actively fragmenting large patches into multiple smaller patches. We fragmented artificial seagrass habitats and evaluated the impacts of fragmentation on fish abundance and species richness over time (1 day, 1 week, 1 month). Fish assemblages were compared among 4 treatments: control (single, continuous 9-m(2) patches); fragmented (single, continuous 9-m(2) patches fragmented to 4 discrete 1-m(2) patches); prefragmented/patchy (4 discrete 1-m(2) patches with the same arrangement as fragmented); and disturbance control (fragmented then immediately restored to continuous 9-m(2) patches). Patchy seagrass had lower species richness than actively fragmented seagrass (up to 39% fewer species after 1 week), but species richness in fragmented treatments was similar to controls. Total fish abundance did not vary among treatments and therefore was unaffected by fragmentation, patchiness, or disturbance caused during fragmentation. Patterns in species richness and abundance were consistent 1 day, 1 week, and 1 month after fragmentation. The expected decrease in fish abundance from reduced total seagrass area in fragmented and patchy seagrass appeared to be offset by greater fish density per unit area of seagrass. If fish prefer to live at edges, then the effects of seagrass habitat loss on fish abundance may have been offset by the increase (25%) in seagrass perimeter in fragmented and patchy treatments. Possibly there is some threshold of seagrass patch connectivity below which fish abundances cannot be maintained. The immediate responses of fish to experimental habitat fragmentation provided insights beyond those possible from comparisons of continuous and historically patchy habitat.

Evaluating freshwater mining sediment toxicity in Tasmania: Achieving strong multiple lines of evidence.

Mining-impacted aquatic systems could be at risk from an assortment of pollutants. The present study evaluated toxicity of mining site sediments from western Tasmania by conducting bioassays with two Australian freshwater species (Chironomus tepperi and Austrochiltonia subtenuis). The present study used multiple lines of evidence (LoE) to assess risk to aquatic biota and the potential sources of that risk at these sites using a sediment quality guideline (SQG) comparison approach (i.e. comparing chemical concentrations at the site (in this case metals, sulfate, and acidic pH) to sediment guideline values) as well as a statistical approach (principle component analysis). Five of the nine mining site sediments showed significant toxicity to both species using survival (A. subtenuis and C. tepperi) and emergence (C. tepperi) as endpoints. Each LoE (SQG comparison and PCA analysis) provided a list of possible contaminants of concern for toxic sites, each list differing from one another. Evaluating these LoE collectively resulted in a stronger characterization of causality and reduced the potential contaminants of concern to a select few, including mainly: copper, sulfate, and acidic pH. Although using multiple lines of evidence reduced the number of potential contaminants of concern, the causality results were still not entirely conclusive, thus we also conducted preliminary investigations using toxicity identification evaluations (TIEs). These TIE investigations, showed the overall importance of acidic pH in these sediments, but also show the need for further work to improve the TIE technique for these types of sediment. The present study illustrates the strengths of using multiple LoE in assessing aquatic risk, especially in the assessment of complex sediments such as those in mining areas of Tasmania. The study, perhaps more importantly, also provides the foundation for more focused work to be conducted in the future to better understanding the implications of mining in western Tasmania.

The relationship between offspring size and performance in the sea.

The historical focus on offspring size has been to explain variation among populations, but there have been few attempts to determine whether variation is greatest at population scale. Offspring size variation is typically viewed as an adaptive response to changes in the relationship between offspring size and performance, yet direct tests remain elusive. We partitioned natural variation in offspring size for a marine invertebrate, Watersipora subtorquata, at a range of spatial and temporal scales across southeastern Australia, and we estimated the relationship between offspring size and performance at each population and time. There was significant variation in offspring size among populations, but regional differences explained only approximately 25% of the observed variation, suggesting that there should be a greater focus on small-scale variation in offspring size. We used our data to parameterize an optimality model to generate predictions of offspring size among different populations and times. Differences in the relationship between offspring size and postmetamorphic performance (and therefore changes in size of offspring that were predicted to maximize maternal fitness) among populations and times were associated with differences in offspring sizes among those populations and times. We suggest that interpopulation variation in offspring size can be an adaptive response to local conditions, but the optimal offspring size is surprisingly dynamic.

Resolving the false-negative issues of the nonpolar organic amendment in whole-sediment toxicity identification evaluations.

Three common false-negative scenarios have been encountered with amendment addition in whole-sediment toxicity identification evaluations (TIEs): dilution of toxicity by amendment addition (i.e., not toxic enough), not enough amendment present to reduce toxicity (i.e., too toxic), and the amendment itself elicits a toxic response (i.e., secondary amendment effect). One such amendment in which all 3 types of false-negatives have been observed is with the nonpolar organic amendment (activated carbon or powdered coconut charcoal). The objective of the present study was to reduce the likelihood of encountering false-negatives with this amendment and to increase the value of the whole-sediment TIE bioassay. To do this, the present study evaluated the effects of various activated carbon additions to survival, growth, emergence, and mean development rate of Chironomus tepperi. Using this information, an alternative method for this amendment was developed which utilized a combination of multiple amendment addition ratios based on wet weight (1%, lower likelihood of the secondary amendment effect; 5%, higher reduction of contaminant) and nonconventional endpoints (emergence, mean development rate). This alternative method was then validated in the laboratory (using spiked sediments) and with contaminated field sediments. Using these multiple activated carbon ratios in combination with additional endpoints (namely, emergence) reduced the likelihood of all 3 types of false-negatives and provided a more sensitive evaluation of risk. Environ Toxicol Chem 2018;37:1219-1230. © 2017 SETAC.