Testing sediment biological effects with the freshwater amphipod Hyalella azteca: the gap between laboratory and nature

The freshwater amphipod, Hyalella azteca, is widely used in laboratory sediment toxicity and bioaccumulation tests. However, its responses in the laboratory are probably very different from those in the field. A review of the literature indicates that in its natural habitat this species complex is primarily epibenthic, derives little nutrition from the sediments, and responds primarily to contaminants in the overlying water column (including water and food), not sediment or porewater. In laboratory sediment toxicity tests H. azteca is deprived of natural food sources such as algal communities on or above the sediments, and is subjected to constant light without any cover except that afforded by burial into the sediments. Under these constraining laboratory conditions, H. azteca has been reported to respond to sediment or porewater contamination. In nature, contamination of overlying water from sediment is less likely than in the laboratory because of the large, generally non-static sink of natural surface water. H. azteca does not appear to be the most appropriate test species for direct assessments of the bioavailability and toxicity of sediment contaminants, though it is probably appropriate for testing the toxicity of surface waters. Toxic and non-toxic responses will be highly conservative, though the latter are probably the most persuasive given the exposure constraints. Thus H. azteca is probably a suitable surrogate species for determining sediments that are likely not toxic to field populations; however, it is not suitable for determining sediments that are likely toxic to field populations.     

An automated overlying water-renewal system for sediment toxicity studies

An automated water-renewal toxicity test system is described for exposing benthic invertebrates to whole sediments. The system will intermittently deliver laboratory or on-site water for overlying water replacement in sediment exposures. A range of cycle rates can be used to produce different volume additions of overlying water per day to exposure chambers. The system can be used with six different treatments and eight replicates per treatment producing 48 exposure chambers. Three formulated sediments with variable organic carbon (1.5%, 7.5%) and sand (14%, 63%) content were prepared to test the system exposing amphipods, Hyalella azteca and midges, Chironomus tentans in 10 day whole sediment tests. Intermittent water flow was used with a 90 min cycle time to create two volume additions of laboratory water per 24 h in exposure chambers (180 ml sediment, 320 ml water). Overlying water quality conditions, and survival and growth of both species were consistent and within acceptable limits for the testing requirements of the U.S. EPA guidelines for sediments with freshwater invertebrates.     

Sediment toxicity and stormwater runoff in a contaminated receiving system: consideration of different bioassays in the laboratory and field.

Several field and laboratory assays were employed below an urban storm sewer outfall to define the relationship between stormwater runoff and contaminant effects. Specifically, two bioassays that measure feeding rate as a toxicological endpoint were employed in the field and in the laboratory, along with bioassays measuring survival and growth of test organisms. In 7 to 10 d in situ exposures, amphipod leaf disc processing, growth and survival were monitored. Different exposure scenarios were investigated by varying the mesh size (74 microns or 250 microns mesh) and method of deployment (water column, sediment surface, or containing sediment) of in situ exposure chambers. Hyalella azteca, Daphnia magna, and Pimephales promelas survival were monitored in 48 h in situ exposures. Feeding inhibition was investigated via enzyme inhibition of H. azteca and D. magna and via leaf disc processing measurements of the detritivore H. azteca. Additionally, we investigated the extent of phototoxicity at this site via field exposures in sun and shade and laboratory exposures with and without UV light. The measurement of detritivore leaf disc processing, and thus its usefulness as an endpoint, was hindered by individual variability in the amount of leaf consumed and by leaf weight gain during the summer field exposures. For D. magna, enzyme inhibition measured in a laboratory exposure did not reveal the toxicity observed in field exposures. For H. azteca, enzyme inhibition measured in the laboratory indicated toxicity similar to that observed in short term chronic in situ exposures. Enzyme inhibition also did not detect differences in toxicity due to variations in flow conditions. There were no statistically significant effects of any exposure on P. promelas survival or H. azteca growth, and there were no statistically significant effects due to mesh size or sun exposure. Survival of H. azteca was the most sensitive and the least variable endpoint. Effects on survival were noted in the same treatments over short-term chronic exposures in the laboratory and in situ. Significant differences in survival were noted due to the method of deployment under low flow conditions. In situ chambers containing sediment resulted in greater mortality in the 10 d low flow in situ experiments. Under high flow conditions, significant reductions in survival and leaf disc processing were noted under all methods of deployment at the two impacted sites over a 7 d exposure. Also under high flow conditions, significantly greater mortality of H. azteca was reported at the downstream field site when sediment was included in the chamber at deployment. These results suggest that significant toxicity at this site is due to accumulation of contaminants in the sediment and the mobilization of these contaminants during a storm event. In situ exposures detected toxicity not observed in laboratory exposures. These results suggest that a combination of laboratory and field bioassays is most useful in defining field effects.     

Development and application of long-term sublethal whole sediment tests with Arenicola marina and Corophium volutator using Ivermectin as the test compound

Short-term whole sediment tests using the amphipod Corophium volutator and the polychaete Arenicola marina are now routinely used in Europe to assess the acute toxicity of marine sediments. However, there is still a need to develop longer-term assays which measure effects on sublethal endpoints that are more relevant to predicting impacts at the population level. The effect of increasing exposure times and measuring additional endpoints such as growth, on the sensitivity of these assays was investigated. The test compound used was the chemotherapeutant Ivermectin (IVM), used in aquaculture to treat sea lice infestations. IVM was found to be acutely toxic to both test organisms. Extending the lugworm test to 100 days increased sensitivity of survival by a factor of three; a significant reduction in casting rate was observed at concentrations an order of magnitude lower. This assay shows potential for detecting the sublethal effects of low concentrations of sediment contaminants. Increasing the exposure time did not seem to affect the sensitivity of the amphipod, but further method development is required.     

Passaic river sediment interstitial water phase I toxicity identification evaluation

A suite of tests was conducted to evaluate and identify the cause or causes of toxicity in Passaic River sediments. Sediment toxicity was measured with three types of bioassays: a whole sediment bioassay with the marine amphipod, Ampelisca abdita, and interstitial water bioassays with A. abdita and the bioluminescent bacterium Vibrio fisheri (Microtox((R))). In addition, a Phase I Toxicity Identification Evaluation (TIE) was conducted to elucidate the cause of observed toxicity. Analytical concentrations of selected residues in whole sediment and interstitial water from the five sampling stations were considered in conjunction with the conclusions drawn from the toxicity tests and Phase I TIE results. Finally, a toxic units approach was used to evaluate the predicted toxicity of measured interstitial water residue concentrations. There was a lack of toxic response in the short-term interstitial water bioassays, indicating that oxidants, soluble forms of metals, and dissolved phase neutral organics were not likely toxicants. However, there was significant toxicity indicated by the whole sediment A. abidita bioassays. After 10 days, there was complete or near complete mortality in amphipods exposed to all of the sediment samples tested. Removal of interstitial water toxicity by filtration was common to all four stations that exhibited measurable initial toxicity. The observed toxicity characteristics are consistent with particle associated neutral organics. This conclusion is supported by toxicity removal via filtration, lack of toxicity in the Microtox((R)) assays, and the fact that whole sediments were more toxic than was interstitial water.     

Phagocytic response of macrophages from the pronephros of American plaice (Hipoglossoides platessoides) exposed to contaminated sediments from Baie des Anglais, Quebec.

Sediments of Baie des Anglais on the St. Lawrence estuary have a history of environmental contamination, but little information exists regarding their toxicity. The purpose of the present study was to determine the effects of contaminated Baie des Anglais sediments on American plaice (Hippoglossoides platessoides) immune function. Three sites in Baie des Anglais were selected which vary in proximity to local industries and in their sediment contaminant load. Sites 1 and 2 (within the bay) are the closest to shore and most heavily contaminated while sediments at Site 3, which is outside the bay, are the least contaminated. In the first experiment, American plaice were placed in cages at each site for three weeks and immune function was assessed by measuring the phagocytic activity of pronephric macrophages. At the time of sampling, plaice displayed pronephros cell immune response disturbances indicating that Site 1 and 2 were most toxic and Site 3 the least toxic. The results obtained for phagocytosis revealed that contaminants present in the sediments are bioavailable to fish, which came in contact with them and significantly affected their immune system. In the second experiment, sediments from the most toxic site, Site 1, were collected for a laboratory controlled experiment in which plaice were exposed for up to 3 months to these contaminated marine sediments, while the control group was exposed to relatively uncontaminated beach sand. At the end of the exposure period, plaice were transferred from contaminated sediment to beach sand and sampled one month later in order to determine if immune function had returned to control levels. The total number of macrophages decreased following three months of exposure, while the active macrophages had already decreased after the first month of exposure. Following the rehabilitation period a significant trend toward normal response was noted. Sediments from Baie des Anglais contain primarily less highly chlorinated PCBs and lower concentrations of the intermediate and highly chlorinated PCBs. The total concentration of PCBs (sum of 20 congeners) in the contaminated sediments was 1500 ng/g while in the beach sand, the levels were 13.6 ng/g dry weight. Only the low chlorinated PCB congeners were efficiently transferred from the sediments to the plaice liver. Together, these results suggest that the effect of chemical exposure on the phagocytosis of plaice macrophages may be reversible if the fish are returned to a non-contaminated habitat.     

Limitations of reverse polyethylene samplers (RePES) for evaluating toxicity of field contaminated sediments

Passive samplers are used to measure dissolved nonionic organic contaminants (NOCs) in environmental media. More recently, reverse polyethylene samplers (RePES) have been used with spiked sediments to recreate interstitial water exposure concentrations and observed toxicity. In the present study, RePES were used with field contaminated sediments. The RePES was not capable of recreating the pattern of toxicity with the amphipod and mysid observed with intact field sediments. Decreased survival in the RePES exposures as compared to the whole sediment exposures was most likely caused by an overexposure to NOCs due to a lack of surrogate black carbon in the RePES system. As an alternative, aqueous phase studies were performed in which polyethylene was allowed to equilibrate with slurries of intact sediments for 3 weeks. Three weeks was found to be an insufficient amount of time for the polyethylene to equilibrate with the sediment. An additional study demonstrated 3 months was sufficient for lower contaminant concentrations, but might not be an adequate amount of time for more highly contaminated sediments. The aqueous phase transfer approach may be useful if equilibration is sufficiently long, although this length of time may be impractical for use in certain applications, such as toxicity identification evaluations (TIEs).     

Toxicity of sediment cores from Yangtze River estuary to zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) embryos

Toxicity evaluation is an important segment in sediment quality monitoring in order to protect aquatic organisms and human health. The purpose of this study is to assess the toxicity of sediments from three sediment cores in Yangtze River Estuary, China, using the zebrafish (Danio rerio) embryo tests. Fertilized zebrafish eggs were exposed to both whole sediments and sediment organic extracts prepared from collected sediments, in order to provide a comprehensive and realistic insight into the bioavailable toxicity potential of the sediments. As end points, development parameters (mortality, hatching rate, and abnormality) in the developing embryos were recorded during the 96-h exposure. The results showed that some samples increased mortality, inhibited the hatching of embryos, and induced morphological abnormalities. The embryonic toxicities presented serrated changes and irregular distribution with depth, which may be related to hydrodynamic effect and unstable environmental input. However, lethal and sub-lethal effects were more significant at the sub-surface sediments (10～40 cm), which indicated that the pollution is more serious in recent decades.     

Effects of storage method and duration on the toxicity of marine sediments to embryos of Crassostrea gigas oysters

The objective of laboratory sediment bioassays is to estimate in situ toxicity. This goal is difficult to achieve, as one of the main limitations of sediment toxicity tests is disruption of sediment geochemistry during sampling, handling and preservation. The effects of storage on the estimation of marine sediment toxicity to Crassostrea gigas embryos and larvae were investigated. Three storage methods and four storage periods were compared with three different sediment types contaminated by heavy metals, polycyclic aromatic hydrocarbons (PAHs) and both contaminants. Freezing and freeze-drying considerably increased the toxicity of decanted sediments and their elutriates as compared to the toxicity obtained with fresh sediments. Concerning the elutriates, the toxicity found with frozen and freeze-dried sediments was correlated with DOC, ammonia and PAH contents. However, the toxicity of fresh sediments kept at 4 degrees C increased with increasing duration of storage and was also correlated with the amount of ammonia in the elutriates.     

Seasonal variation of sediment toxicity in the Rivers Dommel and Elbe

Contaminated sediment in the river basin has become a source of pollution with increasing importance to the aquatic ecosystem downstream. To monitor the temporal changes of the sediment bound contaminants in the River Elbe and the River Dommel monthly toxicity tests were applied to layered sediment and river water samples over the course of 10 months. There is an indication that contaminated sediments upstream adversely affected sediments downstream, but this process did not cause a continuous increase of sediment toxicity. A clear decrease of toxic effects in water and upper layer sediment was observed at the River Elbe station in spring related to high water discharge and algal blooms. The less obvious variation of sediment toxicity in the River Dommel could be explained by stable hydrological conditions. Future monitoring programmes should promote a more frequent and intensive sampling regime during these particular events for ecotoxicological evaluation.     

Equilibrium partitioning theory to predict the sediment toxicity of the anionic surfactant C(12)-2-LAS to Corophium volutator

The study of the effect of the sorption of linear alkylbenzene sulfonates (LAS) on the bioavailability to marine benthic organisms is essential to refine the environmental risk assessment of these compounds. According to the equilibrium partitioning theory (EqP), the effect concentration in water-only exposure will be similar to the effect concentration in the sediment pore water. In this work, sorption and desorption experiments with two marine sediments were carried out using the compound C₁₂-2-LAS. The effect of the sediment sorption on the toxicity of benthic organisms was studied in water-only and in sediment bioassays with the marine mud shrimp Corophium volutator. In addition, three common spiking methods were tested for its application in the toxicity tests, as well as the stability of the surfactant during the water-only and sediment-water test duration. LC50 values obtained from water-only exposure showed a good correspondence with the pore water concentrations calculated from the sorption and desorption isotherms in the spiked sediments.     

Development of rearing and testing protocols for a new freshwater sediment test species: the gastropod Valvata piscinalis

This paper aimed at proposing rearing and testing protocols for Valvata piscinalis, a new potential species for sediment toxicity testing. Such tests were developed since this species reliably represents the bio/ecological characteristics of other gastropods. It may thus be representative of their sensitivity to chemicals. V. piscinalis was successfully cultured in our laboratory for six generations. Cultures provided a high productivity for a low working time and low costs. The tests conditions we proposed seemed to be relevant for the development of reliable tests with this species. Indeed, hatching probability of egg-capsules, as well as embryo, newborn and juvenile survival rates, were close to 100%. Moreover, growth rates and fecundity were significantly higher than in field and in other laboratory studies. Partial life-cycle tests on clean sediments were achieved for various feeding levels to determine survival, growth and reproduction patterns, ad libitum feeding level and life cycle parameters values. Ad libitum feeding levels for newborn, juveniles and adults were 0.1, 0.4 and 0.8 mg Tetramin/individual/working day. Growth tests with zinc-spiked sediments provided a no-effect concentration and a lowest effect concentration of respectively 200 and 624 mg zinc/kg dry sediment. Other growth tests on spiked sediments we ran at our laboratory with second, third and fourth instars larvae of Chironomus riparius pointed out that V. piscinalis was more sensible to zinc than the chironomid, which is a routine test species in ecotoxicology. According to these results, V. piscinalis is a promising candidate species for sediment toxicity testing.     

Comparison of test procedures for sediment toxicity evaluation with Vibrio fischeri bacteria

The use of bacterial luminescence assays is particularly effective in the contaminated sediment evaluation. In the present study, a comparison was made of different baterial luminescence assays including acute toxicity of elutriates with Microtox® and LUMIStox®; chronic toxicity of elutriates with LUMIStox®; and, acute toxicity of solvent extracts with both the tests and the Solid Phase test with Microtox. The toxicity assay procedures were utilised for the Po River sediment toxicity evaluation, testing samples with different physical and chemical characteristics. The results evidentiated that each sediment test procedures provided indipendent and complementary ecotoxicological responces usefull for a sediment classification of the most polluted samples.     

Influence of storage time on toxicity of freshwater sediments to benthic macroinvertebrates

Guidance concerning recommended storage times for sediments to be used in toxicity tests generally has not been based upon systematically collected experimental data. The objective of this study was to better define the effects of storage time on toxicity of a series of freshwater sediments. Sixteen sediments with varying types of contaminants were collected, homogenized and stored at 4 degrees C in 1 liter aliquots, which were periodically tested for toxicity to the amphipod Hyalella azteca and the midge Chironomus tentans after storage times of up to 101 weeks. The sediments ranged from non-toxic to extremely toxic (100% mortality) in 10-day assays, with several of the samples displaying an intermediate degree of toxicity (e.g. partial mortality, reduced growth). Biological responses in most of the samples did not vary with time relative to their statistical relationship to control values; samples identified initially as toxic (or non-toxic) tended to remain toxic (or non-toxic) regardless of when they were tested. The variations that were observed in biological responses over time generally were not systematic; that is, there were no apparent trends in samples becoming more (or less) toxic in the 10-day assays. This suggests that the source of at least some of the temporal changes in toxicity were due to inherent biological variability of the assays used to assess the sediments, rather than the effects of storage. In C. tentans tests with the least toxic sediments, among-replicate variability tended to be greater in initial assays than in tests with samples that had been stored for some period of time. This may have been due to the presence of indigenous competitive or predatory organisms that did not survive during prolonged storage.     

Sediment toxicity testing using large water-sediment ratios: an alternative to water renewal

Deterioration of overlying water quality during toxicity tests with benthic invertebrates is a serious problem with some sediments. One solution is periodic renewal of overlying water. However, this is either labour intensive or requires construction and maintenance of special equipment. Furthermore, water renewal has the potential for flushing toxic chemicals out of the test chamber and establishes nonequilibrium conditions between the water and sediment. An alternative is testing under static conditions using atypical test vessels (e.g. Imhoff settling cones) with a large water volume (1 l) overlaying a much smaller sediment volume (e.g. 15 ml). This results in dramatic improvement of overlying water quality compared to standard static toxicity tests. Compared to water renewal, the test method is much simpler, all toxic substances leached from the sediment are retained in the test vessel, and contaminant concentrations in water and sediment have more time to equilibrate. Chronic sediment toxicity tests (10-28 days) have been conducted successfully under these conditions with Chironomus riparius, Hexagenia sp., Hyalella azteca and Tubifex tubifex.     

Toxicokinetics,toxicity and lethal body residues of two chlorophenols in the oligochaete worm,Lumbriculus variegatus,in different sediments

Bioavailability, toxicokinetics and toxicity (LC(50)) of water- and sediment-associated 2,4,5-trichlorophenol (2,4,5-TCP) and pentachlorophenol (PCP) were measured in Lumbriculus variegatus Müller in a set of experiments. The critical body residue approach was applied by measuring also the lethal body residues (LBR(50)). Freshwater and three different sediments with various sediment organic carbon (SOC) concentrations were used as exposure media. SOC decreased the bioavailability of both chlorophenols, and the uptake rates decreased by 81% and 91% for 2,4,5-TCP and PCP, respectively, in the sediment with a SOC of 6.9% compared to those in sediment with a SOC of 0.5%. SOC appeared to be an important factor controlling the bioavailability as after the carbon normalisation the difference between the sediments was much smaller. The 96-h LC(50) values for instance for PCP were 145.3 microg/l in freshwater, and 6.8 and 8.1 microg/g dry weight in sediments with SOC concentrations of 0.5% and 2.4%, respectively. The LBR(50) values, were practically the same in freshwater and sediments: between 1.0 and 1.6 and from 0.4 to 0.9 micromol/g wet weight for 2,4,5-TCP and PCP, respectively, demonstrating the usefulness of this method for accurate, and more comparable, measurement of toxicity of chemicals with the same mode of toxic action in varying conditions. L. variegatus expressed a dose-response sediment avoidance behaviour but the PCP tissue concentrations were not affected by this behaviour.     

Impact of dredged urban river sediment on a Saronikos Gulf dumping site (Eastern Mediterranean): sediment toxicity,contaminant levels,and biomarkers in caged mussels

Impacts of chemical contaminants associated with dumping of dredged urban river sediments at a coastal disposal area in Saronikos Gulf (Eastern Mediterranean) were investigated through a combined approach of sediment toxicity testing and active biomonitoring with caged mussels. Chemical analyses of aliphatic hydrocarbons (AHs), polycyclic aromatic hydrocarbons (PAHs), Cu, and Zn in combination with the solid phase Microtox® test were performed on sediments. Concentrations of PAHs, AHs, Cu, and Zn as well as multiple biomarkers of contaminant exposure and/or effects were measured in caged mussels. Sediments in the disposal and neighboring area showed elevated PAHs and AHs concentrations and were characterized as toxic by the solid-phase Microtox® test during and after dumping operations. Biomarker results in the caged mussels indicated sublethal effects mainly during dumping operations, concomitantly with high concentrations of PAHs and AHs in the caged mussel tissues. Cu and Zn concentrations in sediments and caged mussels were generally not elevated except for sediments at the site in the disposal area that received the major amount of dredges. High PAHs and AHs levels as well as sublethal effects in the caged mussels were not persistent after termination of operations. The combined bioassay–biomarker approach proved useful for detecting toxicological impacts of dredged river sediment disposal in sediments and the water column. Nevertheless, further research is needed to evaluate whether sediment toxicity will have long-term effects on benthic communities of the disposal area.     

Inventory of marine biotest methods for the evaluation of dredged material and sediments

An inventory of marine biotest methods for the evaluation of dredged material and sediments was compiled on behalf of the Federal Environmental Agency of Germany. Relevant assays were identified from the literature and experts from several countries contributed to a questionnaire survey on established and developing procedures. The biotest methods are applicable to whole sediment, sediment suspension, sediment elutriate, porewater and/or sediment extract. The endpoints cover acute and long-term toxicity, bioaccumulation, endocrine effects, toxic effects on reproduction, carcinogenicity and mutagenicity. Comparative analyses and evaluation of the biotest methods were conducted with regard to their sensitivity, specificity, applicability (regional specificity, availability and suitability of the test organisms), variability (physicochemical factors, natural factors and factors related to sampling and testing), cost-effectiveness, aspects of animal ethics, standardization (guidelines, intercalibration) and application for monitoring purposes in the areas of the OSPAR and Helsinki Conventions. The available information was integrated to rate the validity of the methods, their relevance for assessing impacts on ecosystems and the suitability of the methods for the evaluation of marine sediments and dredged material. Based on the rating of the individual bioassays, a tiered testing is suggested in a hierarchical approach representing a variety in taxa, biological processes and exposure routes, thereby covering the cellular, species, population and community level with a wide discriminatory and sensitivity range. The toxicological significance and complexity increases with the tiers: (1) screening and detection of impacts, (2) characterization of toxic effects, (3) verification of in situ alterations.     

Evaluation of time-to-effects as a basis for quantifying the toxicity of contaminated sediments

Due to uncertainties as to appropriate procedures and dilution materials, most sediment tests are conducted only with undiluted, whole samples. Hence, it is not possible to use conventional concentration-response approaches to quantify toxicity of samples that elicit a 100% effect (e.g., mortality) at a preset test interval (typically 10 d). An alternative approach to quantifying the relative toxicity of test sediments is to determine time-to-effects. The objective of this study was to assess the utility of a time-to-effects approach for quantifying toxicity of freshwater sediments to the invertebrates Hyalella azteca and Chironomus tentans. Survival of both species and growth of C. tentans was determined using five sediments (four test samples and a control sediment) by destructively sampling replicate test chambers over the course of a "standard" 10-d assay. Studies with the control sediment and a non-toxic test sample indicated excellent recovery of test animals, even early in the test (e.g., <24 h) when individuals of both species are relatively small. Reasonable, typically monotonic, time-to-death relationships were observed for both H. azteca and C. tentans exposed to three comparatively toxic test sediments, all of which caused significant mortality by 10 d. Use of the time-to-effects approach allowed expression of toxicity of the three samples relative to one another, as well as documentation of decreases in toxicity of one of the sediments with increased storage time. These studies demonstrate the feasibility of use of time-to-effects as a basis for quantifying the relative toxicity of contaminated sediments.     

A review on the application of microbial toxicity tests for deriving sediment quality guidelines

The results of microbial toxicity tests are needed for the risk assessment of polluted sediments. In comparison with animals the anaerobic microorganisms are more tolerant to natural sediment conditions whereas they are more sensitive for a number of specific pollutants. Microbial toxicity tests from a literature search were classified in seven categories. Category A, B and C use polluted sediments and are applied for sediment monitoring. In category D, a pure chemical is added and the organisms and the test conditions were derived from sediment. Therefore this category can be used for setting sediment quality guidelines which protect sediment functions for the toxic effects of chemicals. In category E, organisms from a polluted site are separated from the sediment and are tested with pure chemicals. Organisms from a more polluted site can be more tolerant to a local pollutant. This is called pollution-induced community tolerance and can be used as evidence for the occurrence of toxic effects in a specific sediment. In category F pure chemicals are tested with a pure culture of microorganisms under sediment conditions. The results of category F tests can be combined with single species tests with animals and plants to obtain sediment quality guidelines sufficient for species protection. This can be compared with the sediment quality guidelines which protect sediment functions. When one of these quality guidelines is exceeded for a compound at a specific location a category E test can be used to determine whether the compound shows toxic effects in that sediment.