Proposal of a testing strategy and assessment criteria for the ecotoxicological assessment of soil or soil materials

Background and Scope  Information on a potential contamination of soils or soil materials are derived by chemical analysis which takes place specifically for a given substance. For a comprehensive assessment, information on the bioavailable and mobile contaminant fraction, including all metabolites, is desirable. During the last years several research projects were initiated in Germany, to supplement the chemical analyses and to elaborate a suitable testing strategy. The main goal of this contribution is to elucidate the results of these research projects and to summarize the test strategy, which is recommended based on these results. Results and Conclusion  Ecotoxicological tests, which are standardized for the assessment of chemicals, were regarded as a suitable starting basis for a cost effective, pragmatic approach. Aquatic tests (testing of aqueous soil extracts) focus on the retention function of soils and terrestrial tests (testing of soil) on the habitat function. Suitable reference systems for the terrestrial tests and assessment criteria for both test types (terrestrial and aquatic) were elaborated. On the basis of a round robin test and a laboratory comparison test, a minimal test battery was established. This minimal test battery can be supplemented by further tests if more or specific information is required. Outlook  The recommendations should encourage the discussion regarding the application of biological methods for the assessment of soil quality. Such an assessment is or at least can be required by soil protection laws which have been adopted in some European countries within the last years.     

Toxicity of hexyl to<Emphasis Type="Italic">F. candida and E. crypticus</Emphasis>

Aim, Scope and Background  Hexyl is along with TNT a possible pollutant of ammunition contaminated soils. To assess the ecological risk of a pollutant biotests are obligatory to gain information of the effects on the habitat function of the contaminated soil. The effect of Hexyl was investigated using the standardised biotests for the springtailFolsomia Candida and the enchytraeidEnchytraeus crypticus. Method  The tests with Hexyl were performed in spiked standard soil (Lufa 2.2). The toxic endpoints were mortality (short-term test, exposure time 7 days) and reproduction rate (long-term test, exposure time 28 days). The effect on the reproduction was further investigated by transferring animals from contaminated soil material onto uncontaminated substrate. Then the number of offspring was determined for the collembola and the cocoon- and hatching rates for the enchytraeid. Results. The mortality of the collembola was not influenced by Hexyl, but the number of offspring was significantly reduced. On the bases of these data the influence of the contaminant on the fertility of the adults itself was investigated. Therefore a reproduction test was performed with adult springtails transferred from contaminated onto uncontaminated soil material. It was shown that the fertility of the adults was not affected by Hexyl indicating a strong lethal effect on the juveniles. In case of the enchytraeid the mortality as well as the reproduction was affected. The surviving enchytraeids turned yellow and rigid in the mortality tests. They were also reduced in size compared to individuals in the uncontaminated control soil material. Thus the cocoon and hatching rates of worms transferred from contaminated soil material onto agar-agar was further investigated. Indeed, the reproduction (both cocoon production and juvenile hatching) was affected by the contaminant. However, the effect was not as strong as might have been suggested by their change in colour and size. A rapid recovery of the worms was observed, too. Conclusion  Hexyl is toxic for the two terrestrial invertebrates, although in comparison to other explosives tested it is less toxic than TNT, but more toxic than the other explosives such as Hexogen (RDX) and Octogen (HMX) or TAT, the end product of the microbial reduction of TNT. So far, all these substances have only been tested with the luminescent bacteriaVibrio fischeri, for which Hexyl and TNT were classified as very toxic to aquatic organisms. With earthworms (Eisenia fetida andEisenia andrei) only TNT, Hexogen and Octogen have been investigated. TNT was also the most toxic of these substances. Outook  Hexyl is one of the main pollutants of ammunition-contaminated sites and has to be considered as a toxic compound. Therefore an assessment of its full ecological impact is necessary and should include tests with animals from different trophic levels as well as biotests with plants and microorganisms.     

Biotest— and chemistry-based hazard assessment of soils,sediments and solid wastes

Background and Objectives  The current environmental legislation regulating pollution issues is based on total levels of pollutants. This approach is not taking into account the bioavailability of pollutants (that is especially important for an analysis of soils and sediments as heavy metals and hydrophobic organic toxicants tend to sorb to solid matrix) and effects of toxicants in mixtures. Thus, toxicity-based criteria should be added to the currently existing chemical ones for the meaningful evaluation of the environmental hazard. The aim of the current study was 1) to compare the ecotoxicity and chemistry-based environmental evaluations for 27 solid-phase environmental samples (soils, sediments, solid wastes); 2) to suggest the battery of biotests for the screening of water-extracted toxicity. Methods  14 soils, 9 sediments and 4 oil-shale industry solid waste samples from Estonia and Lithuania were analyzed for the concentration of total PAHs, heavy metals, oil products and water-extracted phenols. The pollution level was evaluated by comparison of measured concentrations with Estonian permitted limit values in residential (PLV_r) and industrial (PLV_i) areas for each key pollutant A battery of 8 aquatic toxicity tests was applied for the analysis of aqueous extracts (L/S=3) of samples: tests with microalgaeSelenastrum capricornutum, macroalgaeNitellopsis obtusa, protozoaTetrahymena thermophila, crustaceansDaphnia magna andThamnocephalus platyurus, rotifersBrachionus calyciflorus and photobacteriaVibrio fiscberi. Particle-bound bioavailable toxicity was evaluated using a kinetic photobacterial assay withVibrio fiscberi (Solid-Phase Flash-Assay). The toxicity data were evaluated by MaxTox index (highest toxic signal of the battery). Results  Chemical evaluation and toxicological evaluation pointed to the same direction in half of the cases (13/27): 5 samples (including 2 presumably clean control soils) proved harmless and 8 hazardous to environment in case of both evaluations. However, there was a disagreement between chemical and toxicological evaluations for the rest of the samples (14/27). In two mismatching cases (soils from the territory of former gasoline stations) the level of oil products exceeded the PLV_r, but no toxicity was detected, most probably due to the low bioavailability of aged pollutants. It must be taken as a warning that a majority of mismatching cases (12/14) of the samples proved to be toxic or even very toxic despite the fact that the measured hazardous key pollutant levels were below the PLVr. Within these 12 samples were 2 soils from municipal dumping sites, 2 soils from the territory of military airport, 6 sediments from Curonian lagoon (Lithuania) and 2 oil-shale industry solid wastes (Estonia). Conclusions  The results of this study show the necessity of biotesting in environmental risk assessment to avoid the falsenegative results that may result in harmful effects for the ecosystems and also to human health. The following, reduced test battery was proposed for the ecotoxicological hazard assessment of water-extractable toxicity of solid-phase samples:Tetrahymena thermophila growth inhibition assay,Daphnia magna mortality assay andSelenastrum capricornutum growth inhibition assay. Also, the Microtox test (Vibrio fiscberi luminescence inhibition assay) could be valuable for screening purposes. For the evaluation of particle-bound, bioavailable toxicity of soil suspensions, Solid-Phase Flash-Assay (test organismVibrio fiscberi) is suggested. Compared to the results obtained with the initial battery of 8 tests, the reduced battery detected the toxicity in 85% of the cases.     

Optimisation of the Solid-Contact Test with Arthrobacter globiformis (7 pp)

Background, Aim and Scope   A number of biotests are available for the characterisation of solid matters such as soil or sediment. Among these, bacterial biotests using single test species often analyse the toxicity of water-soluble contaminants in aquatic extracts, but there is also a need for a fast and inexpensive bacterial solid-contact test. In this study, a solid contact test with added bacteria (Arthrobacter globiformis) was optimised (through miniaturization) for the development of a test kit with conserved bacteria. As in other tests, the results can be influenced by natural soil factors, often masking anthropogenic impacts. For this reason, a further goal of this study was the investigation of the influence of natural soil characteristics on the result of the solid contact test. The project is part of the joint research project 'Optimization of ecotoxicological test methods for routine use' (abbreviated as ERNTE-Forschungsvorhaben: 0330305). Materials and Methods: This method is based on an existing German standard (DIN 38412 L 48) using Arthrobacter globiformi for testing whole soils and sediments. The test principle is the measurement of the dehydrogenase activity of the test organism A. globiformis after an incubation time of two hours with the solid material. To attain the miniaturization in microplates, dye measurement was changed from spectrophotometrical determination of the substrate resazurine to the fluorimetric measurement of the product resorufin. A second step towards optimisation was the use of freeze-dried bacteria. Seven selected uncontaminated soils were tested in order to determine the influence of natural soil characteristics on the results of the solid contact test with A. globiformis. Freshly spiked and polluted field soils were analysed in order to obtain information about the sensitivity of the test. Results: It is possible to perform the contact test in microplates. The fluorimetric dye measurement can be carried out in the presence of the solid material, so the work-intensive step of centrifugation and filtration is no longer necessary. The measurement in the optimised contact test is based on the kinetics of the enzyme reaction. The investigation showed that conserved bacteria have the same activity and sensitivity as cultivated bacteria. Discussion: The study of the uncontaminated soils demonstrated the influence of various soil characteristics on the results of the solid contact test. This information is the basis for the selection of the control and reference soils and is crucial for setting the threshold value in toxicity testing. The investigation of freshly spiked and contaminated soils showed a different sensitivity dependent on the kind of the contamination. Conclusions: The solid contact test was successfully optimised using microplates, whereas now less than six hours are necessary for the analysis. The optimised test is rapid and sensitive, requiring small samples and no stock culture of the bacteria A. globiformis if using freeze-dried bacteria. In this study, the effect of natural soil factors such as pH-value was shown. This information is used to define the threshold value for toxicity. Therefore, the optimised contact test can be used for an efficient assessment of soil or soil substrates. Further studies will clear up if this optimisation is also valid for aquatic sediments and waste. Recommendations and Perspectives: Due to its short analysis time, the test is suitable for screening different kinds of solid matter and can be used for on-site analysis. - The optimised contact test with freeze-dried bacteria as part of a battery of tests is appropriate for the assessment of contaminated soils, sediments and waste.     

Earthworm avoidance test for soil assessments

For ecotoxicological assessments of contaminated or remediated soils pointing to the habitat function of soils for biocenoses, standardized tests with earthworms (acute test, reproduction test) are available among others. Tests used for routine applications should be sensitive and indicate impacts on test organisms after short test periods. The usually applied earthworm tests do not satisfactorily fulfil these criteria. Therefore, in the present work, a behavioural test with earthworms (test criterion: avoidance) was investigated in detail using uncontaminated, artificially contaminated and originally contaminated soils. It was demonstrated that the avoidance behaviour is primarily determined by pollutants, and not by chemical-physical soil properties. The sensitivity of the presented test reaches the sensitivity of established tests. For waste sites, a considerably higher sensitivity was determined. An avoidance behaviour of at least 80% of the worms leaving the soil to be assessed is proposed as a criterion for toxicity.     

Bioassays for the ecotoxicological and genotoxicological assessment of contaminated soils (results of a round robin test)

For the assessment of contaminated or remediated soils, in addition to chemical analyses, ecotoxicological tests are performed which focus on the retention function of soils (determined by tests with aqueous soil extracts) and on the habitat function (determined by tests with soil). While numerous standardized test guidelines exist to investigate effects by chemical substances, respective test procedures for soil assessments are lacking. To close this gap, a round robin test was performed to support standardization. Four contaminated soils were tested using a total of fifteen test systems including ecotoxicological and genotoxicological tests with soil extracts and soil. The results obtained from the tests with soil extracts are presented in this article.     

Bioassays for the ecotoxicological and genotoxicological assessment of contaminated soils (Results of a round-robin test)

For the assessment of contaminated or remediated soils, aside from chemical analyses, ecotoxicological tests are performed which focus on the retention function of soils (determined by tests with aqueous soil extracts) and on the habitat function (determined by tests with soil). Whereas numerous tests exist as standardized guidelines for identifying the effect of chemicals, this is not the case for the assessment of soil quality. A round-robin test was performed to monitor the comparability of the results from ecotoxicological test methods on soils and to facilitate the standardization of corresponding test methods. Four contaminated soils were tested using a total number of fifteen test systems, including ecotoxicological and genotoxicological tests with soil extracts and soil. In the second part of this publication series, the results obtained from the tests with soil microorganisms and soil fauna are presented.     

Behavioural endpoints in earthworm ecotoxicology

Background, Aims and Scope  Endpoints in earthworm ecotoxicology scheduled in guidelines are mortality and reproduction rates. However, not only the direct influence of pollutants on population parameters but also changes in behaviour such as substrate avoidance can have an important impact on soil ecosystems. In practice two different avoidance response tests are applied in earthworm ecotoxicology: (i) a six-chamber test system and (ii) a two-chamber test system. Both avoidance response-test systems were compared to establish their respective advantages and disadvantages in order to advance the standardisation of behavioural tests. The earthworm avoidance-response tests were applied in addition to the standard acute and chronic earthworm toxicity tests (ISO 11268) in order to compare the sensitivity of the test endpoints. Methods  Test substrates were contaminated with crude oil and 2,4,6-Trinitrotoluene (TNT), respectively. The test species wasEisenia fetida. The earthworms were exposed to the contaminated substrates and their mortality (14 d), reproduction rates (number of cocoons after 28 d, juvenile hatching after 56 d), and substrate preference (48 h) determined. Results and Discussion  Whereas 1000 mg/kg TPH (Total Petroleum Hydrocarbons) did not show any lethal effects, 100% mortality occurred in soil with comparable TNT concentration. The acute tests consistently produced the highest effect concentrations whereas reproduction and substrate avoidance were the more sensitive test parameters. Both behavioural test systems, when compared, showed similar substrate avoidance after an incubation time of 48 h. The six-chamber test system provides the potential to test six different substrates/concentrations at one time. It was observed, however, that earthworms did not migrate among all test chambers within a test unit in order to select the most appropriate substrate. Orientation was observed only between directly neighbouring test compartments, which complicates the interpretation of the test results. Conclusion  Substrate avoidance and reproduction variables were clearly more sensitive test endpoints than mortality. Therefore avoidance-response tests proved to be useful test methods in detecting effects of sublethal concentrations of pollutants on earthworms. The test duration of the avoidance tests is much shorter compared to the standard acute and chronic earthworm toxicity tests, which makes them a quick screening tool for identifying potential soil toxicity. Both avoidance-response test systems showed comparable results regarding the test sensitivity. Nonetheless, the incomplete substrate use in the six-chamber avoidance test due to the reduced migration possibilities (orientation only to neighbouring chambers) might reduce the distinctness of test results as it allows only reliable information on the most avoided and therefore most toxic substrate but not on 1 a clear dose-response pattern. Thus, to gain valid results, the number of replicates and the arrangement of the different substrates must be adopted. The two-chamber test system is less time-consuming due to easy handling and test results can be quantified more easily. Recommendations and Outlook  In consequence of the better validity of test results, lower expenses for test containers and less time for handling, the use of the two-chamber system is preferred over the six-chamber test system to assess the toxicity of polluted soil. Because of the ecosystem consequences of behavioural effects and the fact that avoidance response tests can reveal the toxic potential of pollutants in low concentrations, such tests should be included into ecotoxicological test protocols.     

Experiences with Novel Approaches in Earthworm Testing Alternatives (7 pp)

Goal, Scope and Background   The earthworm avoidance test is a sensitive screening test. Currently, two test designs, a two-chamber system and a six-chamber system, are under standardization. In the scope of the present study, the two test systems are compared. To assess the results, two procedures are applied, which are based on a threshold value and a statistical method. Moreover, the sensitivity of the avoidance test is compared with the sensitivity of the reproduction test. Methods   The avoidance behaviour of E. fetida towards five chemicals (Cd, Cu, PCP, TBT, TNT) was tested in a sandy and a loamy soil. The ecotoxicological test was performed according to the draft guideline ISO/DIS 17512-1. The results were compared with the number of offspring determined in the reproduction test carried out according to ISO 11268-2. Results and Discussion   The results demonstrate that the avoidance behaviour towards organic chemicals and heavy metals is a suitable screening method showing first tendencies of a chemical's effects on the habitat function of soils. Effects caused by chemical substances become visible at low concentrations and within short test periods. The sensitivity of the reproduction test and the avoidance test is principally comparable; in some cases, the avoidance test showed more sensitive reactions. The dose-response-relationships were more pronounced in the two-chamber test than in the six-chamber-system. Recommendation and Outlook   The two-chamber-system proved to be more feasible than the six-chamber-system. As the sensitivity of the avoidance test and the reproduction test is comparable, the avoidance test can be considered as a suitable screening test as well. A possible field of application may be the selection of soil samples for which the reproduction assay seems necessary. To assess contaminated or remediated soils, the earthworm reproduction test is recommended as an indicator for the habitat function of the soils. To reduce costs, the labour intensive reproduction test could be largely replaced by the avoidance test in performing the earth-worm reproduction test only for relevant samples. Before the Draft International Standard of the guideline will be adopted as an ISO Guideline, there will be sufficient time left to gain further international experience required to finalize the method.     

综合化学法和生物标记物法评价土壤中豆磺隆的生物有效性

Bioavailability is a critical factor for assessing the environmental risk of organic pollutants in soil. In this study, extractions with 3 different solvents, including 2 aqueous solutions, calcium chloride (CaCl₂) and a phosphate buffer solution (PBS), and a mixture of aqueous solution and organic solvent, a PBS-methanol (8:2, volume/volume) mixture (PBS-M), were performed to assess the bioavailability of chlorimuron-ethyl in soil in comparison to a battery of toxicity tests in wheat seedlings. The results indicated that the peroxidase (POD) activity in wheat leaves after 7 d of exposure was one of the sensitive biomarkers of chlorimuron-ethyl in soil. The extractability of chlorimuron-ethyl by all the 3 solvents decreased with exposure time, and the rate of decrease of the PBS-M extraction between 1 and 7 d of exposure was substantially higher than those of the aqueous solution extractions. Chlorimuron-ethyl gradually changed from a water-soluble form into a soil organic matter (SOM)-bound form in the soil. The PBS extraction correlated best with the POD activity in the leaves after 7 d of exposure.     

A fuzzy logic-classification of sediments based on data from in vitro biotests

Background, aim, and scope  Ecotoxicological risk assessment of sediments is usually based on a multitude of data obtained from tests with different endpoints. In the present study, a fuzzy logic-based model was developed in order to reduce the complexity of these data sets and to classify sediments on the basis of results from a battery of in vitro biotests. Materials and methods  The membership functions were adapted to fit the specific sensitivity and variability of each biotest. For this end, data sets were categorized into three toxicity levels using the box plot and empirical methods. The variability of each biotest was determined to calculate the range of the gradual membership. In addition, the biotests selected were ranked according to the biological organisation level in order to consider the ecological relevance of the endpoints measured by selected over- or underestimation of the toxicity levels. In the next step of the fuzzy logic model, a rule-base was implemented using if...and...then decisions to arrive at a system of five quality classes. Results  The results of the classification of sediments from the Rhine and Danube Rivers showed the highest correlation between the biotest results and the fuzzy logic alternative based on the empirical method (i.e. the classification of the data sets into toxicity levels). Discussion  Many different classification systems based on biological test systems are depending on respective data sets; therefore, they are difficult to compare with other locations. Furthermore, they don‘t consider the inherent variability of biotests and the ecological relevance of these test systems as well. In order to create a comprehensive risk assessment for sediments, mathematical models should be used which take uncertainties of biotest systems into account, since they are of particular importance for a reliable assessment. In the present investigation, the variability and ecological relevance of biotests were incorporated into a classification system based on fuzzy logic. Furthermore, since data from different sites and investigations were used to create membership functions of the fuzzy logic, this classification system has the potential to be independent of locations. Conclusions  In conclusion, the present fuzzy logic classification model provides an opportunity to integrate expert knowledge as well as acute and mechanism-specific effects for the classification of sediments for an ecotoxicological risk assessment. Recommendations and perspectives  In order to achieve a more comprehensive classification, further investigation is needed to incorporate results of chemical analyses and in situ parameters. Furthermore, more discussions are necessary with respect to the relative weight attributed to different ecological and chemical parameters in order to obtain a more precise assessment of sediments.     

Sediment Toxicity Assessment: Rationale for effect classes (5 pp)

-  Dedicated to Prof. Dr. Ulrich Förstner on his 65th birthdayBackground, Aims and Scope   The main challenge in the application of biotests for hazard assessment of sediments is a transparent interpretation, especially if combinations of biotests are applied and an integrated assessment of all results is wanted. For decision making, assigning results of biotesting into different classes that indicate increasing hazards can be a useful tool. In this paper we suggest an approach on how to set up a site-independent classification system for sediment toxicity tests.Methods   About 250 sediment samples were collected from two rivers and evaluated. The bioassays applied are used in a standardized procedure for all samples over years. The test battery include toxicological endpoints like nematode growth and reproduction, algae growth, bacteria activity, and luminescence inhibition.Results and Discussion   The classification system described emphasizes the following steps: the assessment of test-specific response spans for each applied biotest and the subsequent interpretation of inhibition values in terms of toxicity, estimation of accuracy and uncertainty of different test systems, and the use of cluster- and k-means analyses in order to identify occurring pattern within a large biotest data base, followed by a ranking. The outcome of the procedure were 5 effect classes with an increasing potential of hazard. Conclusion   The effect classes represent the result of a logical procedure of deduction. The procedure is transparent giving an increased degree of certainty. The site independent ranking of 5 classes could be used for sediment monitoring and facilitate fast interpretation. Recommendation and Perspective   A large database comprising differently contaminated sediments is the precondition for setting up effect classes, because it is essential to know the range of effects and the variability of the test methods. The proposed procedure could then provide the basic rules for designing an expert system.     

Validation of Microplate Bioassays for the Assessment of Contaminated and Remediated Sites

Background, Goal and Scope  Bioassays are frequently used to investigate the water extractable ecotoxicological and genotoxicological potential of contaminated soil samples. A laboratory intercomparison study was performed for validation of miniaturised biological test systems for the assessment of contaminated and remediated sites. The successful performance of this study resulted in an optimisation of microplate assays with respect to the testing of chemicals and environmental samples. Methods  For this purpose, miniaturised bioassays were chosen, which, because of their stage of development, are suitable for routine application in the characterisation of the water extractable ecotoxicological and genotoxicological potential of soils. These ecotoxicological and genotoxicological assays were performed with contaminated soil samples by three institutions at the same time. Results and Discussion  The toxicological assessment of the contaminated and remediated soil samples using LID-values, as a rule, was highly uniform. Some minor deviations could, for the most part, be explained by the heterogeneity of the soil samples and, to a lesser extent, by methodical aspects. The difference in sensitivity towards contaminants of the two bacteria Vibrio fischeri and Pseudomonas putida was pointed out. In the algae test with Desmodesmus subspicatus, the influence of the highest sample concentrations on the growth controls became obvious. It was recommended to modify the experimental setup of the microtitration plate, i.e. to place growth controls located next to both the lowest and the highest dilution steps of the sample. The Ames-test did in some cases provide new information on the genotoxicity of the samples, but is not considered useful in a test battery for the evaluation of the genotoxic potential because of its great expense in time and work. Conclusions  The investigations in this laboratory-intercomparison study for the assessment of the water extractable toxic potential of soil samples show that different bioassays are needed, which, in contrast to chemical-analytical methods, can detect the complete effects of all present pollutants in contaminated and remediated soils and solid substrates path-specifically. Recommendations and Outlook  If the recommended modifications for the performance of the bacterial and algae growth inhibition assays on microplates are taken into consideration, these tests can substitute the tests performed on a macro scale. The usefulness of the umu-test and the NM2009-test for the investigation of the genotoxic potential has been proven. Although the tests performed on microplates require much lower sample amounts, it is recommended that sample amounts be eluted in accordance with current guidelines to ensure representativity of the sample. Further work should focus on toxicity identification studies in the future by combining chemical and toxicological analyses.     

Application of a new sediment contact test with <Emphasis Type="Italic">Myriophyllum aquaticum</Emphasis> and of the aquatic <Emphasis Type="Italic">Lemna</Emphasis> test to assess the sediment quality of Lake Skadar

Goal, Scope and Background  Situated in the transboundary belt between Montenegro and Albania, Lake Skadar is the largest freshwater reservoir in Southeastern Europe. Because of the wide range of endemic, rare or endangered plant and animal species it supports, Lake Skadar and its extensive adjacent wetlands are internationally recognised as a site of significance and importance (Ramsar site). Within the last 10 to 20 years, Lake Skadar was exposed to intensive pollution. For the assessment of the ecotoxic load of the sediments sampled in Lake Skadar, a triad approach was recently applied. Overall, a complex spectrum of ecotoxic loads was elucidated. The aim of the present study was to use plant-based bioassays for assessing the sediment quality of Lake Skadar in order to facilitate and complement the triad test battery. The newly developed sediment contact test with Myriophyllum aquaticum and the aquatic growth inhibition test with Lemna minor were applied to native sediments and pore water, respectively, allowing the investigation of different toxicity-effects caused by particle-bound pollutants as well as pollutants in the interstitial water. This investigation is the first application of the novel sediment contact test with Myriophyllum aquaticum to lake sediments. Methods  Sediment samples were taken from nine selected sites at Lake Skadar and investigated by the sediment contact assay with Myriophyllum aquaticum. The pore water was extracted from these sediment samples to be analysed in the aquatic growth inhibition test with Lemna minor. The results of the sediment contact tests were compared with each other and with those of the aquatic growth inhibition test. Results and Discussion  Both applied macrophyte biotests revealed distinct changes in the growth behaviour of the two macrophytes subsequent to the exposure to the investigated natural sediments of Lake Skadar. The Myriophyllum sediment contact test revealed significant toxicity in the sediment samples from Radus and Kamenik, whereas the aquatic Lemna test showed inhibition effects for the samples from Sterbeq, Plavnica and Kamice. Data obtained with the newly developed Danio rerio contact test and the Arthrobacter globiformis contact test confirmed the Myriophyllum results. Analyses of the heavy metal content in the sediments revealed low or moderate contamination levels. Correlation analyses between the content of heavy metals in the sediments and growth inhibition of Myriophyllum aquaticum showed a significant correlation between Cr concentrations and growth inhibition. Comparable findings are available for a German river system. In contrast, no significant correlation between inhibition rates and concentration of metals could be observed with Lemna minor. Conclusions  It was shown that the newly developed sediment contact test with Myriophyllum aquaticum is applicable to lake sediments. In both the sediment contact test with Myriophyllum aquaticum on whole sediments and the aquatic growth inhibition test with Lemna minor on pore water, plant growth was influenced by the natural sediments and its components. Therefore, both test systems were found to be suitable for the detection of phytotoxic effects upon exposure to sediments. Myriophyllum aquaticum as test organism of the contact test grows directly in the sediment without an additional water-layer. Thus, it is able to detect toxicity caused by particle-bound phytotoxic substances as well as pore water-related contamination, while the floating Lemna minor can only detect effects emanating from pore water. Significant differences of the results were observed between these two test systems and, accordingly, the two different exposure scenarios. Hence, none of the tests can replace the other one and, as a consequence, both should be included into a test battery for the assessment of sediment toxicity. Recommendations and Perspectives  Both plant assays were shown to be reliable tools for the evaluation of the eco-toxicological risk potentials of pore water and solid-phase sediment. They should become a complement to the standardised test battery generally used for comprehensive hazard assessment. ESS-Submission Editor: Dr. Ulrike Kammann (ulrike.kammann@ifo.bfa-fisch.de)     

Duration of the standard earthworm avoidance test: Are 48 h necessary?

Ecotoxicological tests are often recommended for the environmental risk assessment (ERA) of contaminated soils. In comparison to chemical residue analysis that focuses on individual contaminants, ecotoxicological tests are able to integrate the effects of the overall contamination, including interactions between individual contaminants, as well as between the contaminants and the various soil properties. However, their use is limited by the fact that the most sensitive tests are chronic tests such as the earthworm reproduction test which lasts 56 days. In order to promote the use of ecotoxicological tests for the routine assessment of contaminated test soils, the usage of short-term earthworm avoidance tests were investigated in the German project ERNTE. According to the International Organization for Standardization (ISO) guideline no. 17512-1, such a test has a duration of just 48 h which, although less than chronic tests, is still quite long in comparison to modern methods of chemical analysis whereby results are often available within 24 h (“on-site analysis”). Therefore, we assessed the effects of shortening the duration of the earthworm avoidance test from 48 to 24 h on the resulting ecotoxicological data. Eight uncontaminated natural soils and 22 contaminated natural soils containing a wide range of chemicals were tested concurrently for 24 and 48 h. Additionally, seven of the uncontaminated natural soils (one was excluded due to its low pH) plus Organization for Economic Co-operation and Development (OECD) artificial soil or LUFA (Landwirtschaftliche Untersuchungs- und Forschungsanstalt, Speyer, Germany) St. 2.2 soil were spiked with two model chemicals (zinc nitrate-tetrahydrate and tributyltin-oxide (TBT-O)) and tested accordingly. It was also investigated whether the results would differ when using either standard LUFA St. 2.2 or OECD artificial soil as a control when contaminated natural soils were tested. Statistical analysis of the data indicates that a decrease in test duration in general did not result in a different assessment of the test soils. In view of the fact that an ERA of contaminated soils is increasingly starting with an on-site analysis (i.e. data are available within 24 h) it is recommended to change the existing ISO guideline, i.e. to decrease the test duration from 48 to 24 h. In doing so, the use of a multi-concentration design increases the robustness of the test results. Both OECD and LUFA St. 2.2 soils are equally suitable as controls.     

Biochemical parameters in Tubifex tubifex as an integral part of complex sediment toxicity assessment

Background, aim, and scope

Restoration of lakes and reservoirs with extensive cyanobacterial water bloom often requires evaluation of the sediment quality. Next to the chemical analysis of known pollutants, sediment bioassays should be employed to assess toxicity of the present contaminants and to make predictions of associated risk. Brno reservoir in the Czech Republic is a typical example of water bodies with long-term problems concerning cyanobacterial water blooms. Comprehensive assessment of reservoir sediment quality was conducted since successful reservoir restoration might require sediment removal. An important part of this survey focused on an examination of the utility of Tubifex tubifex and its sublethal biochemical markers for the assessment of direct sediment toxicity.

Materials and methods

This complex study included chemical analysis of contaminants (heavy metals, organic pollutants), ecotoxicity testing of sediment elutriates (tests with Daphnia magna, Pseudomonas putida, Sinapis alba, Scenedesmus subspicatus), and other parameters. We have tested in more detail the applicability of T. tubifex as a test organism for direct evaluation of contact sediment toxicity. Survival tests after 14 days of exposure were complemented by an assessment of parameters serving as biomarkers for sublethal effects [such as total glutathione content (GSH), activities of the enzymes glutathione transferase (GST), glutathione peroxidase (GPx), and glutathione reductase (GR)]. The data matrix was subjected to multivariate analysis to interpret relationships between different parameters and possible differences among locations.

Results

The multivariate statistical techniques helped to clearly identify the more contaminated upstream sites and separate them from the less contaminated and reference samples. The data document closer relationships of the detected sediment contamination with results of direct sediment exposure in the T. tubifex test regarding mortality but namely regarding the sublethal endpoints rather than the results obtained with other test organisms exposed to sediment elutriates. Aside from the reduction in T. tubifex survival, the sediments with organic pollution caused an increase in glutathione content and increased activities of glutathione S-transferase and glutathione peroxidase in the exposed T. tubifex worms.

Discussion

Results of our study confirm the suitability of T. tubifex for toxicity testing of raw waters and sediments. This longer-lasting direct contact test has proven more sensitive and appropriate to reflect a lower level of pollution than do the elutriate tests. Sensitive biochemical changes in T. tubifex, including an elevation in GSH levels and GST activities, reflect a general stimulation of detoxification metabolisms in the presence of xenobiotics. The results also suggest an important role of glutathione and related enzymes in detoxification processes and possible involvement of oxidative stress in toxicity mechanisms in benthic sediment-dwelling worms such as T. tubifex.

Conclusions

The complex assessment has identified the more contaminated samples with locally increased concentration of organic pollutants and significant ecotoxicity. The direct sediment contact test with T. tubifex and especially the biochemical parameters corresponded better to the lower level of pollution than the other tests with sediment elutriates. Despite its greater time and cost demands, the direct sediment contact test can provide a more realistic picture of exposure.

Recommendations and perspectives

Sediment bioassays should always be included as an integral part of the sediment quality assessment. The direct contact tests also take into account the more hydrophobic pollutants that are not easily available for the water elution but can still be accessible to the organisms. The T. tubifex test is a suitable option for contact sediment toxicity tests also because these animals show measurable sublethal biochemical changes that can be associated with this exposure.     

曹家路流域面源污染的特征及规律

通过连续的野外试验,对北京市重要水源地密云水库水源区曹家路流域的土壤理化特征、面源污染产汇污机制、污染物迁移转化特征等进行了初步分析和研究。研究内容包括:不同土壤类型的理化特征及养分含量随深度的变化规律;一场短时暴雨水文情形下,各面源污染特征指标的变化规律及产汇污机制;不同土地覆盖条件下,流域内各子河段面源污染的变化特征。最后,针对该研究区域面源污染特征,提出了污染物削减及控制的相关对策。     

Terrestrial Ecotoxicity of Eight Chemicals in a Systematic Approach (7 pp)

Background and Objective   Terrestrial ecotoxicity data are required for many research purposes. The data are derived either from the literature or elaborated by own investigations. As the terrestrial toxicity tests are usually time-consuming and labour intensive, the experiments are performed with a limited number of test organisms and soils. In the context of a project sponsored by CEFIC-LRI (European Chemical Industry Council – Long-Range Research Initiative), EC50-values were systematically elaborated for eight chemicals with a wide range of logKow-values (CdCl2, Trinitrotoluene, 3,4-dichloroaniline, 2,4-dichlorophenol, Tributyltinchloride, Pentachlorophenol, Benzo(a)pyrene, p,p-dichloro-2,2-diphenyl-1,1,1-trichloroethane). The substances were selected covering a broad range of physico-chemical and ecotoxicological properties. As toxicity endpoints, microbial activities, plant germination and growth as well as reproduction of earthworms and collembola were determined. As such systematic investigations are rarely performed and for some substances no data existed, the data pool is made available to the scientific community.Methods   All toxicity tests were conducted on three different soil types (sandy soil, silty soil, loamy soil), according to ISO and OECD guidelines Results, Discussion, Conclusion and Outlook   The different toxicities of the chemicals, the influence of soil properties on bioavailability as well as different sensitivities of test organisms and test parameters are reflected by the EC50-values. The results showed that the EC50-values calculated on the basis of nominal concentrations can significantly vary from EC50-values derived from analytical concentrations for some substances. To avoid false conclusions, this has to be considered especially when concentrations determined in the field are compared with toxicity data obtained from the literature or calculated on the basis of nominal concentrations. Moreover, the results indicate that terrestrial ecotoxicological tests should be accompanied by chemical analyses, to be sure to derive sound EC50-values. As the tests were performed according to international guidelines, the comprehensive data pool relevant for the terrestrial ecotoxicity of eight chemicals is considered to be of great value for further research and discussions dealing with the habitat function of soils.     

Effects of boric acid on various microbes,plants, and soil invertebrates

Purpose  

Terrestrial ecotoxicology includes the investigation of the impact of chemicals on soils and soil-inhabiting organisms, whereby ecotoxicological effects are mainly assessed using single-species laboratory tests. As part of laboratory testing, reference substances, with known toxicity, are regularly used to confirm the sensitivity of the cultivated test organisms over time. Furthermore, reference substances are also applied in ring test evaluations for the validation of new test methods. The present work evaluates boric acid for its suitability as a potential reference substance for tests with higher plants and soil organisms (e.g., invertebrates and microbial communities).     

Influence of Soil Organic Carbon on Acute and Chronic Toxicity of Plant Protection Products to Poecilus cupreus (Coleoptera, Carabidae) Larvae (4 pp)

Background, Aims and Scope   Variability of results from terrestrial ecotoxicological tests with arthropods demonstrate the importance of understanding the impact of soil composition has on the bioavailability of pollutants. Beside other soil parameters, the organic C content is known to be relevant to bioavailability. The aim of the present paper was to detect lethal and sublethal effects of plant protection products on soil-dwelling larvae of the carabid beetle Poecilus cupreus under different concentrations of soil organic C. The study was based on a familiar laboratory test method. Methods   Larvae of the laboratory-reared carabid beetle P. cupreus were exposed to dimethoate in a standardised soil (Lufa 2.1), modified to four different concentrations of organic C between 0.5 to 2.8% by admixing peat. Dimethoate was applied to the soil surface at rates ranging between 40 and 250 g a.i./ha. Some larvae were exposed to an application of water to act as a control. One larva was released per test tube, with at least 20 larvae per treatment group. Effects on mortality and developmental time of the larvae and on the hatching weight of adult beetles were recorded. Dimethoate was selected as the test item because some dose-response data for different soils were already available and because the reproducibility of test results with dimethoate had already been shown in a previous ring test. Results and Discussion   With increasing concentration of organic C in the soil substrate, mortality decreased at all tested rates of dimethoate. Consequently, LC50 values increased from 39 to 107 g a.i./ha with increasing organic C content from 0.5 to 2.8%. The results suggest higher bioavailability of dimethoate with decreasing content of organic C. The replication and magnitude of sublethal effects (hatching weight and developmental time) was insufficient to detect a clear interaction between dimethoate and organic C. Conclusion   The results of the experiment reveal that the concentration of organic C in the soil substrate is influential on the effects and, by association, the toxicity of dimethoate to larvae of P. cupreus. For the interpretation of terrestrial ectotoxicological tests with arthropods, the composition of the substrate has to be taken into account. Recommendations and Outlook   For reliable and reproducible test results, a well-defined and thoroughly controlled composition of the test soil is highly recommended. Test results should be used for risk assessments only after having taken the soil composition, especially the organic C content, into account.