Toxicity testing with Vibrio Fischeri: A comparison between the long term (24 h) and the short term (30 min) bioassay

The acute bioluminescence inhibition assay using the marine bacterium Vihrio fischeri as the test organism is a widely used short term toxicity test. This paper compares the standard 30 minutes test with a 24 h assay using the same organism and the same test parameter. For that purpose concentration-response relationships were determined for a set of selected substances, reflecting different modes and mechanisms of action.

The results indicate a severe blind spot of the acute biotest: The toxicity of certain specifically acting chemicals is drastically underestimated here, while the chronic biotest gives a more reliable estimate of the toxicity of these substances.     

Ecotoxicological Characterisation and Classification of Existing Chemicals

GOAL, SCOPE AND BACKGROUND: In 1998, the International Council of Chemical Associations (ICCA) launched a global initiative to investigate more than 1,000 HPV chemicals (High Production Volume, > or = 1,000 t/a) within the refocused OECD HPV Chemicals Programme. Up to the OECD SIDS Initial Assessment Meeting in April 2004 (SIAM 18) 147 ICCA dossiers (ca. 230 CAS-No) have been assessed based on a harmonised data set. The environmental profile and an ecotoxicological characterisation of these chemicals are presented here. Data for acute aquatic toxicity were correlated among each other, as well as data for fish (LC50, LD50) and rodents (LD50). The data for acute aquatic toxicity are compared with other existing chemicals. METHODS: Data of the ICCA HPV chemicals from the OECD SIAM 11-18 are presented for: log Kow (as an indicator for bioaccumulation potential), biodegradation, acute aquatic toxicity and availability of long-term toxicity data. Correlation analysis was performed with log transformed data and a linear regression model was fitted to the data, if a significant correlation was found. Acute toxicity for fish and acute oral toxicity for rodents were correlated on a molar basis. Acute aquatic toxicity of the chemicals is compared with data from BUA reports 1-234 and a random EINECS sample (Knacker et al. 1995). RESULTS AND DISCUSSION: According to the dossier information, 71 of the 147 ICCA chemicals are not 'readily biodegradable', 21 have a log Kow > or = 3, and 44 are 'toxic' (LC/EC50 < or = 10 mg/L) or 'very toxic' (LC/EC50 < or = 1 mg/L) to aquatic organisms. For 77, only the base set (acute fish, Daphnia and algae) is available, for the rest at least one long-term test (fish or Daphnia) is available and three tests for a mere 14 others. Based on the data presented, the SIAM gives recommendations for Environment and Human Health. 22 chemicals have been identified as a 'candidate for further work' for Environment and 16 for Human Health. The highest correlation coefficient was obtained correlating fish and Daphnia (r2 = 0.79). LC50 (fish) is significantly correlated with LD50 (rodent), but data are widely scattered. The correlation is not improved after transforming LC50 (fish) to LD50 (fish), using BCF QSAR. Based on acute aquatic toxicity, 25.1% of the chemicals from the BUA reports 1-234 are classified as 'very toxic' (LC/EC50 < or = 1 mg/L). This proportion is 2.5-fold higher than the ICCA HPV chemicals and 1.4-fold higher than the random EINECS sample. CONCLUSIONS: Correlation coefficients for aquatic toxicity data are rather uniform (0.57-0.79) compared with literature data, but also the best correlation was observed between fish and Daphnia. Because the scatter around the regression lines is still considerable, simple predictions of ecotoxicity between species are not possible. Correlation of LC50 (fish) and LD50 (rodent) indicates that toxicity is different. Surprisingly, the correlation of fish and rodent toxicity is not improved by transforming LC50 values to internal LD50s. The selection of ICCA chemicals by market significance (production volume) leads to a classification of toxicity, which is more comparable to a random sample of EINECS chemicals than to German BUA chemicals. The latter were chosen for concern (for Environment or Human Health). RECOMMENDATIONS AND OUTLOOK: Of 147 dossiers assessed between SIAM 11-18, ca. 75% were sponsored by the three following countries: Germany (42), USA (37) and Japan (33). The current output is about 50 dossiers per year (70-100 CAS-No), but a trend for an increase of output is noticeable. Industry, national authorities, and OECD work on a further development to speed up the output. The number of chemicals with 'low priority for further work' and the work recommended for the 'candidates' (mainly exposure assessment) indicate that the data presented were adequate for an initial hazard assessment according to OECD requirements. From the ICCA HPV list (n = 880, state of 1999) 44% of the chemicals have data available to cover all SIDS endpoints for Environment and only 33% for Human Health (Allanou et al. 1999). This indicates the importance of the Initiative to provide information on existing chemicals. The authors agree with the expectation "...that the scientific information provided by this global initiative will be considered as an internationally accepted and harmonised basis for further steps of chemicals management." (ICCA 2002 b).     

Toxicity testing with luminescent bacteria – Characterization of an automated method for the combined assessment of acute and chronic effects

The luminescent bacteria test according to EN ISO 11348 is frequently applied in (eco) toxicity testing and is applicable for a huge variety of environmental and industrial samples. A big disadvantage of this method is the very short exposure time, which is expressed in a low sensitivity in regard to substances with a delayed effect. Chronic effects, i.e. interference with cell growth, cannot be assessed with this conventional standard method. The goal of this research was to develop an automated testing system for long term toxicity towards the luminescent bacteria Vibrio fischeri by implementing microtitration-based instrumentation. The optimized method, hereinafter referred to as “kinetic luminescent bacteria test”, can be described as a miniaturized combination of the conventional short-term luminescence inhibition test according to EN ISO 11348 and the Photobacterium phosphoreum growth inhibition test (DIN 38412-37). The validation procedure included the evaluation of six reference compounds (3,4-Dichloroaniline, 3,5-Dichlorophenol, Chloramphenicol, Streptomycin sulfate, Potassium dichromate, Zinc sulfate heptahydrate) and three different endpoints that are acute luminescence inhibition (acute LI) after 30 min, chronic luminescence inhibition (chronic LI) after 24 h and growth inhibition (GI) after 14 h. The optimized method allows the assessment of acute and chronic effects within one test, by what a misinterpretation of the toxicity of substances with delayed bacterial toxicity can be prevented, without abandoning most of the advantages of the conventional short-term test. Therefore, the kinetic luminescent bacteria test is exceptional as an initial screening test for environmental samples or substances with unknown (eco) toxicological characteristics.     

Monitoring the toxicity of phenolic chemicals to activated sludge using a novel optical scanning respirometer.

This paper reports the development of optical scanning respirometer for determining the toxicity of chemicals to activated sludge. The respirometer is used to measure the dissolved oxygen concentration by monitoring the luminescence intensity of ruthenium dye immobilized in a polymer film in contact with the wastewater sample. The intensity is related to the extent of oxygen quenching of luminescence. The toxicity of chemicals can be evaluated by measuring the inhibition on respiration rate of microorganism using the scanning respirometer. The IC50 values (concentration of a chemical that exhibits 50% respiration inhibition) of various phenolic chemicals in activated sludge were determined. The performance of this method is compared with other toxicity methods. The experimental results indicate that the reproducibility and sensitivity of this respirometer are reasonably good.     

Relative sensitivity of one freshwater and two marine acute toxicity tests as determined by testing 30 offshore E & P chemicals

Acute toxicity of 30 offshore E & P (Exploration and Production) chemicals was measured using the three standard test organisms Daphnia magna (freshwater cladoceran), Acartia tonsa (marine copepod) and Skeletonema costatum (marine diatom alga). Test chemicals included 20 water-soluble and 10 (partially) non-soluble products. For 22 out of the 30 chemicals, the difference in sensitivity between the three tests varied within one order of magnitude. A very good correlation was found between the two marine tests (r = 0.96, P < 0.01, n = 30), and a correlation coefficient of r = 0.78 (P < 0.01, n = 30) was found between D. magna and both A. tonsa and S. costatum, individually. When the comparison of D. magna and A. tonsa sensitivity was based only on the water-soluble chemicals, a significantly higher correlation was obtained (r = 0.84, n = 20), indicating that the sample preparation method used for the (partially) non-soluble chemicals (the water accommodated fraction (WAF) method) induces additional variation between tests performed with different test media. (Partially) non-soluble chemicals are characterised by phase separation or precipitation at the concentrations used for testing. In a WAF-based test, each test concentration/exposure level is prepared separately, and following mixing and separation, only the water phase is used for testing. Toxicity is related to the amount of substance originally added to the mixing vessels. For 25 of the 30 chemicals, D. magna was found to be less sensitive than the marine copepod by a factor >2. The generally higher sensitivity of the marine toxicity tests compared to the Daphnia test emphasise the importance of using marine data for environmental hazard classification as well as for environmental risk assessment purposes.     

Protocol for early detection and evaluation of inhibitory wastewater using combined aerobic respirometric and anaerobic batch techniques.

Faced with the task of treating significant volumes of complex industrial wastewaters, the biological components of municipal wastewater treatment plants are operating under the risk of toxic or inhibitory contaminants from the industrial effluents that may be detrimental to their operation. This might lead to undesirable effluent toxicity and/or result in permit violations. Therefore, there is a need for upset early warning systems that can protect full-scale plants from toxic or inhibitory constituents in the incoming wastewaters. This study focused on the development of a protocol for rapid detection of potentially toxic inhibitory or toxic wastewaters using combined aerobic respirometric and anaerobic batch techniques. Aerobic respirometers equipped with automated data acquisition systems were used as potential early warning devices. The inhibition effect on carbon and nitrogen oxidation was assessed. The degree of inhibition was evaluated as the concentration causing 50% reduction in microbial activity, which was estimated by an inhibition model. Anaerobic toxicity assays were also conducted to evaluate the inhibitory effects of the toxic compounds to anaerobic inocula obtained from a master culture reactor fed with ethanol. The developed protocol for early detection of toxicity was validated using wastewater samples from a biotechnology industry and a food processing industry, and pure chemicals such as furfural and phenol. Varying degrees of sensitivity were observed in the study when different groups of microorganisms, wastewater samples, and chemicals were tested. The comparison of aerobic and anaerobic inhibition suggested the importance of using both aerobic and anaerobic cultures to maximize the necessary sensitivity of the protocol.     

Effect of sediment turbidity and color on light output measurement for Microtox Basic Solid-Phase Test

In this work, sediment samples collected from several Spanish harbours were tested with two toxicity procedures, designed for solid samples: the Microtox Basic Solid-Phase Test (BSPT) and a modified procedure of the previous test protocol (mBSPT). According to the BSPT procedure, after initial light readings, pure bacteria were exposed to sediment suspension dilutions and light production was directly measured on suspended sediments without any further manipulation. As measurements are likely to be affected by sediment turbidity and color, a variation in initial light measurement has been here suggested, in order to consider the sample effect at all time readings during the test. Firstly, when sediment suspensions at different concentrations were added to bacteria suspension, immediately the initial light output drastically decayed by more than 50% in signal difference, resulting in a false inhibition, as effect of sample turbidity/color. This effect was more evident at high EC50 values, when slightly or not toxic samples were assessed. Secondly, the comparison of the EC50 obtained with both procedures, demonstrated that the mBSPT produced higher EC50 values (less toxic) than those obtained with the standard procedure. The mBSPT procedure resulted rapid and effective and it could be applied simultaneously with BSPT, in order to better evaluate the toxicity.     

Development of an optimal bacterial medium based on the growth inhibition assay with Vibrio fischeri.

Chronic toxicity level of chemicals to bacteria can depend on composition and concentration of medium ingredients. This was demonstrated by means of a growth rate inhibition test with the marine bacterium V. fischeri. In a minimal medium (following the validity criterion of achieving a least cell multiplication rate) containing only yeast extract as organic nutrient component, V. fischeri was to Cu2+ 11.9 times, to Hg2+ 3 times and to Zn2+ 2.8 times more sensitive than in the complete defined medium. Obviously yeast extract did not interfere greatly in the complexation mechanisms. The detection limits of the toxicity of Cd2+, 3,5-dichorophenol and nitrobenzene were not influenced by the substrate.     

Acute toxicity of chlorophenols to earthworms using a simple paper contact method and comparison with toxicities to fresh water organisms

An acute toxicity test of chlorophenols on earthworms (Eisenia fetida) was performed using a simple paper contact method proposed by OECD testing guideline no. 207, that were applied as an earthworm toxicity test. The median lethal concentration, EC50, had significant correlation with logP(ow) (1-octanol/water partition coefficient) of the chemicals. The toxicity of chlorophenols on E. fetida was compared with toxicities for other species: an algae (Selenostrum capricornutum), a crustacean (Daphnia magna), and a fish (Oryzias latipes). It was found that the toxicity of chlorophenols was almost same for E. fetida and for fresh water organisms. These results suggest the possibility of drawing correlations between the effects of pollutants on living things in different environments, fresh water and soil.     

The OECD validation program of the H295R steroidogenesis assay: Phase 3. Final inter-laboratory validation study

Background, goals, and scope

In response to increasing concerns regarding the potential of chemicals to interact with the endocrine system of humans and wildlife, various national and international programs have been initiated with the aim to develop new guidelines for the screening and testing of these chemicals in vertebrates. Here, we report on the validation of an in vitro assay, the H295R steroidogenesis assay, to detect chemicals with the potential to inhibit or induce the production of the sex steroid hormones testosterone (T) and 17??-estradiol (E2) in preparation for the development of an Organization for Economic Cooperation and Development (OECD) test guideline.

Methods

A previously optimized and pre-validated protocol was used to assess the potential of 28 chemicals of diverse structures and properties to validate the H295R steroidogenesis assay. These chemicals are comprised of known endocrine-active chemicals and ??negative?? chemicals that were not expected to have effects on the targeted endpoints, as well as a number of test chemicals with unknown modes of action at the level of the steroidogenic pathway. A total of seven laboratories from seven countries participated in this effort. In addition to effects on hormone production, confounding factors, such as cell viability and possible direct interference of test substances with antibody-based hormone detection assays, were assessed. Prior to and during the conduct of exposure experiments, each laboratory had to demonstrate that they were able to conduct the assay within the margin of predefined performance criteria.

Results

With a few exceptions, all laboratories met the key quality performance parameters, and only 2% and 7% of all experiments for T and E2, respectively, were excluded due to exceedance of these parameters. Of the 28 chemicals analyzed, 13 and 14 tested affected production of T and E2, respectively, while 11 and 8 did not result in significant effects on T and E2 production, respectively. Four and six chemicals produced ambiguous results for effects on T and E2 production, respectively. However, four of these cases each for T and E2 were associated with only one laboratory after a personnel change occurred. Significant interference of test chemicals with some of the antibody-based hormone detection systems occurred for four chemicals. Only one of these chemicals, however, significantly affected the ability of the detection system to categorize the chemical as affecting E2 or T production.

Discussion and conclusions

With one exception, the H295R steroidogenesis assay protocol successfully identified the majority of chemicals with known and unknown modes of interaction as inducers or inhibitors of T and E2 production. Thus it can be considered a reliable screen for chemicals that can alter the production of sex steroid hormones. One of the remaining limitations associated with the H295R steroidogenesis assay protocol is the relatively small basal production of E2 and its effect on quantifying the decreased production of this hormone with regard to the identification of weak inhibitors. An initial comparison of the data produced in this study with those from in vivo studies from the literature demonstrated the potential of the H295R steroidogenesis assay to identify chemicals affecting hormone homeostasis in whole organisms. Particularly promising was the lack of any false negatives during the validation and the very low number of false positives (1 out of 28 chemicals for each T and E2).

Perspectives

Based on the results obtained during this validation study and the accordingly revised test protocols, an OECD draft test guideline was developed and submitted to the OECD working group of the national coordinators of the test guidelines program (WNT) for comments in December 2009.     

Comparative acute toxicity of organic pollutants and reference values for crustaceans. I. Branchiopoda, Copepoda and Ostracoda

The acute toxicity of 468 organic pollutants to planktonic crustaceans (Branchiopoda, Copepoda and Ostracoda) from pre-existing data was compared by means of statistical analysis and relative tolerance indices (Trel). A surrogate species commonly used in toxicity bioassays (Daphnia magna) showed toxicity levels--within one order of magnitude--similar to all other Cladocera species, at least for 82% of the chemicals studied. All neurotoxic insecticides except neonicotinoids, PCBs, organometallic compounds and PAHs are the most toxic substances to these organisms. Sensitivity levels among taxa were compared for individual chemicals as well as groups of chemicals with similar characteristics. Whilst there are marked differences in sensitivity among taxa and particular groups of chemicals, no consistent trends were found for freshwater and saltwater species in relation to the latter groups. No correlation between LC50 and size of these organisms was found other than by chance, making extrapolations based on allometric equations impossible.     

Application of the criteria for classification of existing chemicals as dangerous for the environment

The criteria for classification and labelling of substances as “dangerous for the environment” agreed upon within the European Union (EU) were applied to two sets of existing chemicals. One set (sample A) consisted of 41 randomly selected compounds listed in the European Inventory of Existing Chemical Substances (EINECS). The other set (sample B) comprised 115 substances listed in Annex I of Directive 67/548/EEC which were classified by the EU Working Group on Classification and Labelling of Existing Chemicals. The aquatic toxicity (fish mortality,Daphnia immobilisation, algal growth inhibition), ready biodegradability and n-octanol/water partition coefficient were measured for sample A by one and the same laboratory. For sample B, the available ecotoxicological data originated from many different sources and therefore was rather heterogeneous. In both samples, algal toxicity was the most sensitive effect parameter for most substances. Furthermore, it was found that, classification based on a single aquatic test result differs in many cases from classification based on a complete data set, although a correlation exists between the biological end-points of the aquatic toxicity test systems.     

Physicochemical substance properties as indicators for unreliable exposure in microplate-based bioassays

In the last years many efforts were made to transform standardized algal test protocols into low-cost microplate assays. While advantages were pointed out frequently, limitations are not systematically addressed, thus hindering a widespread utilisation. In this study a group of organic substances with a wide distribution of volatility (log K_AW from −6.53 to −2.13) and lipophilicity (log K_OW from 1.26 to 4.92) was investigated with respect to the influence of these physicochemical properties on their algal toxicity in different assays. Therefore the EC₅₀ values were determined with a microplate assay based on ISO 8692 protocol and the results were compared with those of an established algal growth inhibition test conducted in air tight glass vessels. Using the ratio of the EC₅₀ values, a clear connection between biological response and volatility as well as lipophilicity of test substances could be detected. Chemicals with a log K_OW higher than 3 or a Henry coefficient log K_AW higher than −4 were identified as less effective in the microplate assay than in the comparative assay. The loss in nominal concentration due to physicochemical properties could be shown to contribute to this using HPLC analysis. Consequently, when using microplate assay’s one should be aware that lipophilic and volatile chemicals might be underestimated in their toxicity, which could be indicated from evaluating related physicochemical properties modelled from structural information prior to an experimental investigation.     

Bioluminescent yeast assays for detecting estrogenic and androgenic activity in different matrices

In this paper we describe the construction and use of a set of bioluminescent yeast strains for the detection of compounds that can affect androgen or estrogen receptor mediated hormonal signalling. The set includes Saccharomyces cerevisiae strains expressing human androgen receptor (AR), estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta), along with firefly luciferase controlled by a respective hormone responsive promoter. A constitutively luminescent strain was included in the set for determining the cytotoxicity of the sample. Yeast cells were incubated with pure chemicals or complex samples for 2.5 h, after which the signal could be detected from the cell-sample mixture after simply adding the D-luciferin substrate. The assays could be completed in one day and they required no cell lysis or centrifugation steps, which makes them suitable for high-throughput analysis of samples. Due to a short incubation time the assays are directly applicable to different sample matrices, requiring no pretreatment of the samples. The assays were used to assess the hormonal activity in moisturizing lotions as an example of a complex sample matrix known to contain endocrine disrupting chemicals. Six out of eight tested moisturisers showed high estrogenic activity, whereas no androgenic activity was observed in the samples.     

Toxicity identification evaluation of leachates from municipal solid waste landfills: a multispecies approach

The toxicity of leachates from two municipal solid waste (MSW) landfills in Southern Italy was characterized using a toxicity identification evaluation procedure. The chemical and physical fractionation techniques were: pH adjustment, pH adjustment/filtration, pH adjustment/C(18) solid phase extraction, graduated pH and EDTA chelation. All the samples exhibited acute toxicity towards the bacterium Vibrio fischeri, the freshwater rotifer Brachionus calyciflorus and the freshwater crustaceans Thamnocephalus platyurus and Daphnia magna. Statistical techniques were used to determine the discriminatory power and the toxicity detection capacity of the different assays and to choose a minimal battery of tests for the toxicity identification of leachates. Toxicity was closely associated with pH, generally increasing at higher pH levels and decliming at lower ones. Furthermore, results showed that toxicants could be characterized as cations, basic chemicals, suspended solids and apolar compounds.     

An invertebrate embryo test with the apple snail Marisa cornuarietis to assess effects of potential developmental and endocrine disruptors

A novel invertebrate embryo test with the apple snail, Marisa cornuarietis, comprising a test protocol for the following developmental endpoints is described: formation of eyes and tentacles, heart rate, hatching, weight after hatching. To evaluate effects on embryonic development, the snails were treated in a first step with 250 or 500 microg/l cadmium. Sublethal effects in terms of a significant delay in hatching could be found in the 250 microg/l treated animals, whereas 500 microg/l Cd were lethal for the snail embryos. To test endocrine disrupting chemicals with this protocol, experiments with bisphenol A (50 microg/l, 100 microg/l) and 17alpha-ethinylestradiol (10 microg/l) were performed. In both treatments an increase of weight after hatching was observed as well as a significant decline in the heart rate of the embryos. As shown here, the sensitivity of M. cornuarietis embryos test is equal or even higher than other test species like zebrafish embryos and, therefore, this test can be regarded as an alternative or supplement for ecotoxicological studies.     

Development of a three-stage system for wastewater toxicity monitoring: a design and feasibility study.

A three-stage system was developed to automate a batchwise toxicity testing protocol designed for assessing wastewater toxicity to activated sludge. The three-stage system used the luminescent bacterium Shkl. The three stages were cell storage, cell activation, and continuous toxicity testing. Shkl cells were stored in a bioreactor at 4 degrees C when the system was not in use and activated in another bioreactor for use in toxicity tests conducted in a continuous manner. The system could quickly be switched between the "off" and "on" modes, and operation of the system was easy. The stability of the system, in terms of cell density and bioluminescence in the storage and activation bioreactors, and the response of the activated cells to a metal and an organic toxicant were studied. The feasibility of the system design was demonstrated by simulating zinc toxicity episodes in synthetic wastewater. The needs for further modifications and improvements of the system were discussed.     

化学物质对发光菌的联合毒性评价方法

毒性单位法(TU)的理论基础来源于剂量加和模型(DA),目前仅在二元联合毒性评价中广泛应用。为了确定TU模型适合评价的混合物类型,实验选取5种剂量效应曲线类型不同的物质,采用微板光度计测试了一元、二元混合物对发光菌青海弧菌-Q67(Vibrio-qinghaiensis sp.-Q67)的急性毒性。根据物质的剂量效应曲线形状将物质分为A、B、C 3类,利用毒性单位法(TU)和联合作用定义法分别对AA类、AB类、AC类、BC类混合物进行分析。结果表明,TU法仅适合于由剂量效应曲线接近直线的物质组成的混合物进行联合毒性的评价。以效应为基准、TU模型为框架建立了TU’模型,该模型可以满足对任何类型已知成分的混合物或者未知成分的实际水样之间的多元联合作用的评价。     

Discrimination of excess toxicity from narcotic effect: Comparison of toxicity of class-based organic chemicals to Daphnia magna and Tetrahymena pyriformis

The discrimination of excess toxicity from narcotic effect plays a crucial role in the study of modes of toxic action for organic compounds. In this paper, the toxicity data of 758 chemicals to Daphnia magna and 993 chemicals to Tetrahymena pyriformis were used to investigate the excess toxicity. The result showed that mode of toxic action of chemicals is species dependent. The toxic ratio (TR) calculated from baseline model over the experimentally determined values showed that some classes (e.g. alkanes, alcohols, ethers, aldehydes, esters and benzenes) shared same modes of toxic action to both D. magna and T. pyriformis. However, some classes may share different modes of toxic action to T. pyriformis and D. magna (e.g. anilines and their derivatives). For the interspecies comparison, same reference threshold need to be used between species toxicity. The excess toxicity indicates that toxicity enhancement is driven by reactive or specific toxicity. However, not all the reactive compounds exhibit excess toxicity. In theory, the TR threshold should not be related with the experimental uncertainty. The experimental uncertainty only brings the difficulty for discriminating the toxic category of chemicals. The real threshold of excess toxicity which is used to identify baseline from reactive chemicals should be based on the critical concentration difference inside body, rather than critical concentration outside body (i.e. EC₅₀ or IGC₅₀). The experimental bioconcentration factors can be greatly different from predicted bioconcentration factors, resulting in different toxic ratios and leading to mis-classification of toxic category and outliers.     

Adaptation of the Daphnia sp. acute toxicity test: miniaturization and prolongation for the testing of nanomaterials

Manufacturing of nanomaterials (NMs) is often complex and expensive, and their environmental risks are poorly understood or even unknown. An economization of testing NMs is therefore desirable, which can be achieved by miniaturizing test systems. However, the downsizing of test vessels and volumes can enlarge the surface/volume ratio (SVR) which in turn can affect the bioavailable concentration of adsorbing substances like NMs. The present study focused on the miniaturization of the acute toxicity test with Daphnia magna. The adaptations were verified with three reference substances, the non-adsorbing potassium dichromate (K₂Cr₂O₇) and as potentially highly-adsorbing substances silver nanoparticles (AgNPs) and silver nitrate (AgNO₃). The miniaturized test was conducted in 24-well microtiter plates (MT) and simultaneously compared to the OECD standard test (ST). Furthermore, the test duration was prolonged from 48 to 96 h since NMs tend to show effects only after extended exposure. The toxicity of K₂Cr₂O₇ and AgNPs continued to increase within the prolonged test span. The test comparisons with K₂Cr₂O₇ did not reveal any significant differences between ST and MT. AgNO₃ toxicity was significantly decreased in MT compared to ST due to the enlarged SVR. The toxicity of AgNPs in MT after 24 h was equal to ST. Contrary to our expectations an exposure longer than 24 h resulted in an increase of AgNP toxicity in MT, possibly due to enhanced dissolution of silver. Microtiter plates are appropriate alternative test vessels for the Daphnia sp. acute toxicity test; thus, its miniaturization is feasible. The enlarged SVR has to be taken into account since it can affect the toxicity of potentially adsorbing substances. Furthermore, the standard test duration of 48 h might underestimate the toxicity of many substances, especially of NMs.