Model Deviation Ratio Research Articles

Predicting the Toxicities of Ternary Mixtures of two Metals and Sodium Dodecyl Sulfate to Serratia marcescens (SerEW01) from Otamiri River Water

Background: Otamiri river server as a source of water for domestic activities, urban farming, recreation, aquatic foods in Owerri and environs. It also receives untreated domestic, industrial and agricultural waste water and run offs from the municipality. Seepages from solid wastes dumps at the river banks and sand mining activity going on in the river could also constitute environmental hazards
 Aims: This study aims at evaluating the interactive effects of the ternary mixtures of sodium dodecyl sulfate (SDS) and some divalent metals on preponderant bacterium (Serratia marcescens (SerEW01)) from the river.
 Study Design: Fixed ratio ray design was used for the study, with inhibition of dehydrogenase activity as end point.
 Place and Duration of Study: Owerri, Imo State, Nigeria, June – December, 2019.
 Methodology: The bacterium was earlier isolated as the preponderant bacterium isolate from the river water. Fixed ratio ternary mixtures (Equieffect concentration (EEC50) and arbitrary concentration (ABCR) ratios), SDS + Pb + Zn, SDS + Cd +Zn, SDS + Pb +Ni, SDS + Ni + Cd, SDS + Co + Pb and SDS + Co + Cd were designed to evaluate the combined toxicities of these toxicants. Toxicities predicted by concentration addition (CA) and independent action (IA) models were compared with the experimentally observed toxicities.
 Results: The EC50S observed ranged from 0.046 ± 0.003 mM (Zn) to 2.329 ± 0.092 mM (SDS). The EC50S of the toxicants were statistically different from each other (P<0.05). The order of increasing toxicities were SDS >Pb >Ni > Co > Cd(II) >Zn. Concentration-dependent toxicities with progressive inhibition of the dehydrogenase activity as the concentration increased were observed.. In all ternary mixtures, both the experimentally derived, CA and IA-predicted EC50S were statistically different from each other. Both models predicted lower toxicities compared to the experimental data. The Toxic Index and Model Deviation Ratio indicated synergistic interaction of SDS and metal ions against S. marcescens (SerEW01)
 Conclusion: This study could constitute base line information towards assessing the possible environmental hazards associated with co-contamination of the environment by SDS and divalent heavy metals, more so when both pollutants are common aquatic pollutants.

Dose–response analysis of toxicological and pharmacological mixtures with the model deviation ratio method: Problems and solutions

Risk assessment for mixtures of chemicals requires to investigate the magnitude of their potential adverse effects on living organisms. This is usually done by assessing how experimental toxicological mixture data depart from the model of Loewe additivity. Several recent scientific studies propose to perform this task using an ad hoc method known as model deviation ratio (MDR) method. Moreover, the first official European regulatory document for the study of combined exposures explicitly recommends the use of the MDR method (EFSA Scientific Committee et al. Guidance on harmonised methodologies for human health, animal health and ecological risk assessment of combined exposure to multiple chemicals. EFSA Journal, 2019).We show here that the MDR method is not rooted in statistical principles and can lead to erroneous claims. We show however that the distribution of the MDR can be evaluated by simulations and show how this allows us to devise and carry out a bona fide statistical test. The proposed method accounts for uncertainty in the estimation of ED/EC50 and does not require a minimum sample size. The computer code developped in this study is made available as an R package called MDR.

Investigating combined toxicity of binary mixtures in bees: Meta-analysis of laboratory tests, modelling, mechanistic basis and implications for risk assessment

Bees are exposed to a wide range of multiple chemicals “chemical mixtures” from anthropogenic (e.g. plant protection products or veterinary products) or natural origin (e.g. mycotoxins, plant toxins). Quantifying the relative impact of multiple chemicals on bee health compared with other environmental stressors (e.g. varroa, viruses, and nutrition) has been identified as a priority to support the development of holistic risk assessment methods. Here, extensive literature searches and data collection of available laboratory studies on combined toxicity data for binary mixtures of pesticides and non-chemical stressors has been performed for honey bees (Apis mellifera), wild bees (Bombus spp.) and solitary bee species (Osmia spp.). From 957 screened publications, 14 publications provided 218 binary mixture toxicity data mostly for acute mortality (lethal dose: LD50) after contact exposure (61%), with fewer studies reporting chronic oral toxicity (20%) and acute oral LC50 values (19%). From the data collection, available dose response data for 92 binary mixtures were modelled using a Toxic Unit (TU) approach and the MIXTOX modelling tool to test assumptions of combined toxicity i.e. concentration addition (CA), and interactions (i.e. synergism, antagonism). The magnitude of interactions was quantified as the Model Deviation Ratio (MDR). The CA model applied to 17% of cases while synergism and antagonism were observed for 72% (MDR > 1.25) and 11% (MDR < 0.83) respectively. Most synergistic effects (55%) were observed as interactions between sterol-biosynthesis-inhibiting (SBI) fungicides and insecticide/acaricide. The mechanisms behind such synergistic effects of binary mixtures in bees are known to involve direct cytochrome P450 (CYP) inhibition, resulting in an increase in internal dose and toxicity of the binary mixture. Moreover, bees are known to have the lowest number of CYP copies and other detoxification enzymes in the insect kingdom. In the light of these findings, occurrence of these binary mixtures in relevant crops (frequency and concentrations) would need to be investigated. Addressing this exposure dimension remains critical to characterise the likelihood and plausibility of such interactions to occur under field realistic conditions. Finally, data gaps and further work for the development of risk assessment methods to assess multiple stressors in bees including chemicals and non-chemical stressors in bees are discussed.

Sensitivity of larval and juvenile freshwater mussels (unionidae) to ammonia, chloride, copper, potassium, and selected binary chemical mixtures

In aquatic environments, organisms such as freshwater mussels are likely exposed to complex contaminant mixtures related to industrial, agricultural, and urban activities. With growing interest in understanding the risk that chemical mixtures pose to mussels, this investigation focused on the effects of various waterborne contaminants (ammonia, chloride, copper, and potassium) and selected binary mixtures of these chemicals following a fixed-ratio design to Villosa iris glochidia and juvenile Lampsilis fasciola. In individual exposures, 48-h EC50 values were determined for V. iris glochidia exposed to ammonia chloride (7.4 [95% confidence interval (CI) 6.6–8.2] mg N/L), ammonia sulfate (8.4 [7.6–9.1] mg N/L), copper sulfate (14.2 [12.9–15.4] μg Cu2+/L), potassium chloride (12.8 [11.9–13.7] mg K+/L), potassium sulfate (10.1 [8.9–11.2] mg K+/L), and sodium chloride (480.5 [435.5–525.5] mg Cl−/L). The 7-d LC50 values for juvenile L. fasciola were determined for potassium sulfate (45.0 [18.8–71.2] mg K+/L), and sodium chloride (1738.2 [1418.6–2057.8] mg Cl−/L). In Ontario these waterborne contaminants have been reported to co-occur, with concentrations exceeding the EC10 for both life stages at some locations. Data from binary mixture exposures for V. iris glochidia (chloride-ammonia, chloride-copper, and copper-ammonia) and juvenile L. fasciola (chloride-potassium) were analyzed using a regression-based, dose-response mixture analysis modeling framework. Results from the mixture analysis were used to determine if an additive model for mixture toxicity [concentration addition (CA) or independent action (IA)] best described the toxicity of each mixture and if deviation towards dose-ratio (DR) or dose-level (DL) synergism/antagonism (S/A) occurred. For all glochidia binary mixture exposures, CA was the best fit model with DL deviation reported for the chloride-copper mixture and DR deviation reported for the copper-ammonia mixture. Using the model deviation ratio (MDR), the observed toxicity in all three glochidia mixture exposures were adequately described by both CA (mean = 0.71) and IA (mean = 0.97) whereas the juvenile mixture exposure was only adequately described by CA (mean = 0.64; IA mean = 0.05).

Environmental Stress Increases Synergistic Effects of Pesticide Mixtures on Daphnia magna.

Some widely used pesticide mixtures produce more than additive effects according to conventional combined effect models. However, synergistic effects have been so far generally observed at unrealistically high pesticide concentrations. Here, we used Daphnia magna as a test organism and investigated how food limitation-a common ecological stressor-affects the mixture toxicity of a pyrethroid insecticide and azole fungicide. We also compared three models regarding the prediction of mixture effects, including concentration addition (CA), effect addition (EA), and stress addition model (SAM). We revealed that especially under low food, the strength of synergism between esfenvalerate and prochloraz increased with an increasing concentration of prochloraz independent of the null model. Under high food conditions and at concentrations of prochloraz ≥32 μg/L, we observed a marginal synergistic effect with a model deviation ratio (MDR) = 2.1 at 32 μg/L prochloraz and 2.2 at 100 μg/L prochloraz when using CA as the null model. In contrast, the combination of both pesticides and food stress caused synergistic effects shown by an MDR = 10.9 even at 1 μg/L of prochloraz that is frequently detected in the environment. The combined effects of pesticides and food stress could be predicted best with the SAM that showed the lowest mean deviation between effect observation and prediction (mean deviation SAM = 16 [SD = 28], EA = 1072 [2105], CA = 1345 [2644]). We conclude that common environmental stressors can strongly increase the synergistic effects of toxicants. This knowledge is especially relevant considering current efforts to include the additional risk of pesticide mixtures and environmental stressors into the environmental risk assessment of pesticides.

Acute Effects of Binary Mixtures of Imidacloprid and Tebuconazole on 4 Freshwater Invertebrates.

Receiving waters from agricultural areas can contain multiple pesticides such as the neonicotinoid imidacloprid and the fungicide tebuconazole, leading to the potential for aquatic life to be exposed to such mixtures. In the present study, the effects of tebuconazole were tested alone and in binary mixtures with imidacloprid on 4 aquatic invertebrates: Chironomus dilutus, Hyalella azteca, Lumbriculus variegatus, and Neocloeon triangulifer. Acute (96-h) median lethal concentrations (LC50s) were derived for individual compounds and used to design a binary mixture study to determine cumulative effects. The LC50s showed that imidacloprid was more potent than tebuconazole by 1 to 3 orders of magnitude for the 4 species. Lethality data from mixture experiments were analyzed using MIXTOX to determine deviations from independent action, followed by the model deviation ratio (MDR) technique to determine the biological significance and reproducibility of observed mixture effects. MIXTOX showed that the cumulative toxicities of imidacloprid-tebuconazole differed between the species: for C. dilutus there was no deviation from independent action; however, for H. azteca the mixture was antagonistic (specifically dose ratio-dependent), and for N. triangulifer it was synergistic. The MDR method showed that only observations with H. azteca significantly deviated from independent action. Because of the lack of evidence of a clear deviation from independent action and the much greater potency of imidacloprid, the weight of evidence indicates that the presence of tebuconazole is unlikely to appreciably increase the hazard from imidacloprid exposure to aquatic invertebrates. Environ Toxicol Chem 2019;00:1-17. © 2019 SETAC.

Genotoxicity of selected pharmaceuticals, their binary mixtures, and varying environmental conditions – study with human adenocarcinoma cancer HT29 cell line

Pharmaceutical residues are present in the environment in mixtures and their adverse effects may also result from interactions that occur between compounds. Studies presented in this work focus on genotoxicity of pharmaceuticals from different therapeutic groups in mixtures and in individual solutions impacted with different environmental conditions assessed using comet assay (alkaline approach). Binary mixtures of pharmaceuticals (in different concentration ratios) and in individual solutions impacted with pH change (range from 5.5 to 8.5) or addition of inorganic ions, were incubated with HT29 cells and after 24 h time period cells were tested for the presence of DNA damage. To estimate whether mixtures act more (synergistic) or less (antagonistic) efficiently Concentrations Addition (CA) and Independent Action (IA) approaches were applied followed by a calculation of the Model Deviation Ratio (MDR) to determine deviation from the predicted values. Addition of inorganic ions mainly reduced their genotoxicity. Diclofenac s. was the most susceptible to potassium, fluoride, and bromide ions. Change of the pH of pharmaceutical solutions had significant impact on genotoxicity of diclofenac s. and fluoxetine h. Among mixtures, more commonly observed interactions were synergistic ones, exactly twenty-five cases (ten pairs containing chloramphenicol or oxytetracycline h.) and ten cases of antagonism (four for pairs containing chloramphenicol or fluoxetine h.). The results obtained indicate that interactions between tested compounds occur frequently and can lead to DNA damage. This topic especially concerning in vitro tests using cells is still rare, however, it should not be neglected.

Investigation of the Synergistic Toxicity of Binary Mixtures of Pesticides and Pharmaceuticals on Aliivibrio fischeri in Major River Basins in South Korea.

This work introduces the potential synergistic toxicity of binary mixtures of pesticides and pharmaceuticals, which have been detected in substantial amounts in major river basins in South Korea. Different dose-response curve functions were employed in each experimental toxicity dataset for Aliivibrio fischeri. We tested the toxicity of 30 binary mixtures at two effect concentrations: high effect concentration [EC50] and low effect concentration (EC10) ranges. Thus, the toxicological interactions were evaluated at 60 effected concentration data points in total and based on model deviation ratios (MDRs) between predicted and observed toxicity values (e.g., three types of combined effects: synergistic (MDR > 2), additive (0.5 ≤ MDR ≤ 2), and antagonistic (MDR < 0.5)). From the 60 data points, MDRs could not be applied to 17 points, since their toxicities could not be measured. The result showed 48%-additive (n = 20), 40%-antagonistic (n = 17), and 12%-synergistic (n = 6) toxicity effects from 43 binaries (excluding the 17 combinations without MDRs). In this study, EC10 ratio mixtures at a low overall effect range showed a general tendency to have more synergistic effects than the EC50 ratio mixtures at a high effect range. We also found an inversion phenomenon, which detected three binaries of the combination of synergism at low concentrations and additive antagonism at high concentrations.

Bisphenols (A, S, and F) affect the basic hormonal activity determined for pharmaceuticals – Study of Saccharomyces cerevisiae

Pharmaceuticals and analogs of bisphenol A (BPA) are increasingly threatening environmental pollutants. In this study, mixtures of selected pharmaceuticals (diclofenac sodium salt, chloramphenicol, oxytetracycline hydrochloride, fluoxetine hydrochloride, estrone, ketoprofen, progesterone, gemfibrozil and androstenedione) were prepared with BPA and its two analogs (namely, bisphenols F and S) at such ratios to reflect environmentally detectable levels. Then, the mixture solutions were studied with a XenoScreen YES/YAS assay to determine the variations in the initial hormonal response of each pharmaceutical compound due to the presence of a bisphenol analog. The results obtained were modeled with the concentration addition (CA) and independent action (IA) approaches, the trueness of which was studied with model deviation ratios (MDR). The estrogenic agonistic activity of the drugs studied was most strongly affected by the presence of BPA in solution (twenty-one cases of synergy observed for CA models versus twelve cases of antagonism in the case of IA predictions). BPS shows a strong agonistic estrogenic impact on most of the drugs studied at medium and high concentration levels; androgenic agonistic activity was also impaired with elevated concentrations of BPS. Increasing the concentration of BPF in a reaction mixture also increased the number of YES + synergism incidences (for CA modeling). Estrone, progesterone and androstenedione were mostly affected by the highest BPF concentrations studied in the case of androgenic agonistic research performed.

Ecotoxicological and biochemical mixture effects of an herbicide and a metal at the marine primary producer diatom Thalassiosira weissflogii and the primary consumer copepod Acartia tonsa.

Mixture effects of chemicals and their potential synergistic interactions are of great concern to the public and regulatory authorities worldwide. Intensive agricultural activities are leading to discharges of chemical mixtures to nearby estuarine and marine waters with possible adverse effects on the aquatic communities and for the trophic food web interlinking these communities. Further information about the impacts of these stressors on aquatic organisms is needed. This study addresses ecotoxicological and biochemical effects of single and mixtures of the metal copper and the herbicide Primextra® Gold TZ on the marine diatom Thalassiosira weissflogii and on the estuarine calanoid copepod Acartia tonsa by determining growth rate and survival, respectively, and changes on fatty acid(FA) profiles in both species. Mixture effects on diatom species revealed that copper and Primextra® acted most likely additively with respect to the concentration addition (CA) and independent action (IA) models with model deviation ratios (MDR), 0.752 and 1.063, respectively. For the copepod species, copper and Primextra® were most likely non-interactive with respect to the CA model (MDR = 1.521) but acted most likely synergistically with respect to the IA model (MDR = 2.026). A significant decline in the absolute FA concentration was observed for copepod species after mixture exposure including a considerable decrease of essential FAs that cannot be synthesized de novo by these grazers. We concluded that the mixture effects are more hazardous for primary consumer than for primary producer species in terms of both abundance and biomass quality, suggesting a potential for harmful effects for higher trophic levels and thus a decrease in energy flow through the ecosystem.

Assessing ecotoxicity and the endocrine potential of selected phthalates, BADGE and BFDGE derivatives in relation to environmentally detectable levels

There is no doubt that the subject area of plastic materials (e.g., production of epoxy resins or polyesters) is inherently connected to issues concerning bisphenol A (BPA) and its analogues. Unfortunately, much less attention has been given to other compounds, which are also used for the production of these materials. Bisphenol A diglycidyl ether (BADGE) is a synthetic industrial compound obtained by a condensation reaction between epichlorohydrin (ECH) and BPA. Similarly, novolac glycidyl ether (BFDGE) is produced in the reaction between novolac and epichlorohydrin. Nevertheless, there is a lack of information on the combined effects of BADGE derivatives at environmentally relevant levels. In the current study, toxicity levels in Microtox® and XenoScreen YES/YAS assays were determined for several analogues alone, then the biological effects of compound pairs mixed in 33, 66 and 100% of each compounds' EC50 ratios were evaluated. The Microtox® test has been chosen as a relevant tool, and the results were referred to the Xenoscreen YES/YAS assay, which has been chosen for the fast determination of the endocrine potential of the compounds tested. The results obtained constitutes the basis for model studies, with Concentration Addition (CA) and Independent Action (IA), followed by Model Deviation Ratio (MDR) interpretation, to evaluate the possible interactions occurring between analytes when present in mixtures. The results indicate that the hydrochloric derivatives of BADGE and BFDGE are of the greatest toxicological and endocrine threat. Thus, their presence in mixtures under certain environmental conditions (including presence in the tissues of living organisms) should be strictly monitored and reported, especially in acidic environments. Strong evidence on the synergic behaviors of these analytes, which expressed high toxicity (EC50 2.69–117.49μg/mL), is demonstrated with Model Deviation Ratio (MDR).

Toxicity of Quaternary Ammonium Compounds (QACs) as single compounds and mixtures to aquatic non-target microorganisms: Experimental data and predictive models

The toxic effects of five Quaternary Ammonium Compounds (QACs) that are widely used as active ingredients in personal care products were assessed using the bioluminescent bacterium Aliivibrio fischeri (formerly Vibrio fischeri) (Microtox® test system). The experimental results showed a relevant toxicity for almost all of the single QACs, with IC50 values lower than 1mgL-1. Analysis of the mode of action through the application of the Quantitative Structure-Activity Relationship (QSAR) models indicated an a-specific reactivity for most of the QACs toward A. fischeri. Only hexadecyl trimethyl ammonium chloride (ATMAC-16) behaved as a polar-narcotic, with a low reactivity toward the bacterial cell membrane. The concentration response curves of the different binary and multicomponent mixtures of QACs were also evaluated with respect to the predictions from the Concentration Addition (CA) and Independent Action (IA) models. For almost all of the binary and multicomponent mixtures (7 out of 11 mixtures tested), an agreement between the experimental and predicted ICx was observed and confirmed via application of the Model Deviation Ratio (MDR). In four cases, some deviations from the expected behaviour were observed (potential antagonistic and synergistic interactions) at concentrations on the order of hundreds of µgL-1, which could be of environmental concern, especially in the case of synergistic effects. The analysis of aquatic ecotoxicity data and the few available values of the measured environmental concentrations (MECs) from the literature for wastewaters and receiving waterbodies suggest that a potential risk toward aquatic life cannot be excluded.

Assessing interactions of binary mixtures of Penicillium mycotoxins (PMs) by using a bovine macrophage cell line (BoMacs)

Penicillium mycotoxins (PMs) are toxic contaminants commonly found as mixtures in animal feed. Therefore, it is important to investigate potential joint toxicity of PM mixtures. In the present study, we assessed the joint effect of binary combinations of the following PMs: citrinin (CIT), ochratoxin A (OTA), patulin (PAT), mycophenolic acid (MPA) and penicillic acid (PA) using independent action (IA) and concentration addition (CA) concepts. Previously published toxicity data (i.e. IC25; PM concentration that inhibited bovine macrophage (BoMacs) proliferation by 25%) were initially analyzed, and both concepts agreed that OTA+PA demonstrated synergism (p<0.05), while PAT+PA showed antagonism (p<0.05). When a follow-up dilution study was carried out using binary combinations of PMs at three different dilution levels (i.e. IC25, 0.5∗IC25, 0.25∗IC25), only the mixture of CIT+OTA at 0.5∗IC25 was determined to have synergism by both IA and CA concepts with Model Deviation Ratios (MDRs; the ratio of predicted versus observed effect concentrations) of 1.4 and 1.7, respectively. The joint effect of OTA+MPA, OTA+PA and CIT+PAT complied with the IA concept, while CIT+PA, PAT+MPA and PAT+PA were better predicted with the CA over the IA concept. The present study suggests to test both IA and CA concepts using multiple doses when assessing risk of mycotoxin mixtures if the mode of action is unknown. In addition, the study showed that the tested PMs could be predicted by IA or CA within an approximate two-fold certainty, raising the possibility for a joint risk assessment of mycotoxins in food and feed.

Single- and mixture toxicity of three organic UV-filters, ethylhexyl methoxycinnamate, octocrylene, and avobenzone on Daphnia magna

In freshwater environments, aquatic organisms are generally exposed to mixtures of various chemical substances. In this study, we tested the toxicity of three organic UV-filters (ethylhexyl methoxycinnamate, octocrylene, and avobenzone) to Daphnia magna in order to evaluate the combined toxicity of these substances when in they occur in a mixture. The values of effective concentrations (ECx) for each UV-filter were calculated by concentration-response curves; concentration-combinations of three different UV-filters in a mixture were determined by the fraction of components based on EC25 values predicted by concentration addition (CA) model. The interaction between the UV-filters were also assessed by model deviation ratio (MDR) using observed and predicted toxicity values obtained from mixture-exposure tests and CA model. The results from this study indicated that observed ECxmix (e.g., EC10mix, EC25mix, or EC50mix) values obtained from mixture-exposure tests were higher than predicted ECxmix (e.g., EC10mix, EC25mix, or EC50mix) values calculated by CA model. MDR values were also less than a factor of 1.0 in a mixtures of three different UV-filters. Based on these results, we suggest for the first time a reduction of toxic effects in the mixtures of three UV-filters, caused by antagonistic action of the components. Our findings from this study will provide important information for hazard or risk assessment of organic UV-filters, when they existed together in the aquatic environment. To better understand the mixture toxicity and the interaction of components in a mixture, further studies for various combinations of mixture components are also required.

Toxicity of quaternary mixtures of phenolic compounds and formulated glyphosate to microbial community of river water

The toxicity of mixtures of formulated glyphosate, phenol, 4-chlorophenol and 2,4-dichlorophenol to river water microbial community was investigated using non specific dehydrogenase activity as endpoint. The microbial community was exposed to individual toxicant and quaternary mixture ratios at concentrations ranging from 50 mg L-1 to 3000 mg L-1 over 24 h. The ecological doses (EC50) as estimated from logistic or hormesis dose-effect models were 1317.946 ± 51.460 mg L-1, 1046.414 ± 65.534 mg L-1, 85.080 ± 4.468 mg L-1 and 60.897 ± 5.199 mg L-1 for formulated glyphosate, phenol, 4-chlorophenol and 2,4-dichlorophenol respectively. The toxicity and interaction of the mixtures were evaluated using concentration addition and toxic index models. The model deviation ratios (MDR) ranged from 0.553 ± 0.024 to 1.096 ± 0.021 while the toxic index (TI) varied between 0.912 ± 0.017 and 1.810 ± 0.078. However, the MDR and TI for all mixtures lie between 0.5 and 2.0. Thus, the joint action of the mixtures were considered additive.

Individual and joint toxicity of the herbicide S-metolachlor and a metabolite, deethylatrazine on aquatic crustaceans: Difference between ecological groups

We studied the individual and joint acute toxicity of S-metolachlor (SMOC) and deethylatrazine (DEA - a metabolite of atrazine) on different non-target freshwater crustaceans. We used animals from different ecological groups: two amphipods from surface running water (Gammarus pulex and Gammarus cf. orinos), an isopod from surface stagnant water (Asellus aquaticus) and an amphipod living in groundwater (Niphargus rhenorhodanensis). Organisms were exposed to different levels of SMOC and DEA, alone or in binary mixture. Temperature effect on SMOC toxicity was assessed by exposing G. pulex and N. rhenorhodanensis to SMOC at 11 °C and 15 °C. Studying mortality as the biological endpoint, N. rhenorhodanensis was more resistant than surface water species towards SMOC and DEA. Among surface water species, G. pulex was the most sensitive while Gammarus cf. orinos and A. aquaticus showed similar responses to both compounds. Temperature increase did not change SMOC toxicity but modify the shape and steepness of the dose-response curve. We used a Model Deviation Ratio (MDR) approach to evaluate the predictability of Concentration Addition (CA) and Independent Action (IA) models to mixture toxicity. Results indicated either an additive or an antagonistic or a synergistic interaction depending on the concentrations combination and the test species. Our finding conclusively show the suitability of CA and IA in predicting mixture toxicities but results should be interpreted with caution according to ecological group of exposed species in risk assessment procedures.

Modeling of pharmaceuticals mixtures toxicity with deviation ratio and best-fit functions models

The present study deals with assessment of ecotoxicological parameters of 9 drugs (diclofenac (sodium salt), oxytetracycline hydrochloride, fluoxetine hydrochloride, chloramphenicol, ketoprofen, progesterone, estrone, androstenedione and gemfibrozil), present in the environmental compartments at specific concentration levels, and their mutual combinations by couples against Microtox® and XenoScreen YES/YAS® bioassays. As the quantitative assessment of ecotoxicity of drug mixtures is an complex and sophisticated topic in the present study we have used two major approaches to gain specific information on the mutual impact of two separate drugs present in a mixture. The first approach is well documented in many toxicological studies and follows the procedure for assessing three types of models, namely concentration addition (CA), independent action (IA) and simple interaction (SI) by calculation of a model deviation ratio (MDR) for each one of the experiments carried out. The second approach used was based on the assumption that the mutual impact in each mixture of two drugs could be described by a best-fit model function with calculation of weight (regression coefficient or other model parameter) for each of the participants in the mixture or by correlation analysis. It was shown that the sign and the absolute value of the weight or the correlation coefficient could be a reliable measure for the impact of either drug A on drug B or, vice versa, of B on A. Results of studies justify the statement, that both of the approaches show similar assessment of the mode of mutual interaction of the drugs studied. It was found that most of the drug mixtures exhibit independent action and quite few of the mixtures show synergic or dependent action.

Mixture Toxicity of Imidacloprid and Cyfluthrin to Two Non-target Species, the Fathead Minnow Pimephales promelas and the Amphipod Hyalella azteca

Two species, the fathead minnow Pimephales promelas and the amphipod Hyalella azteca, were tested to examine acute toxicity to two insecticides, cyfluthrin and imidacloprid individually and as a mixture. Cyfluthrin was acutely toxic to P. promelas and H. azteca with EC50 values and 95 % confidence intervals of 0.31 µg L(-1) (0.26-0.35 µg L(-1)) and 0.0015 µg L(-1) (0.0011-0.0018 µg L(-1)), respectively. Imidacloprid was not acutely toxic to P. promelas at water concentrations ranging from 1 to 5000 µg L(-1), whereas it was toxic to H. azteca with a EC50 value of 33.5 µg L(-1) (23.3-47.4 µg L(-1)). For the P. promelas mixture test, imidacloprid was added at a single concentration to a geometric series of cyfluthrin concentrations bracketing the EC50 value. A synergistic ratio (SR) of 1.9 was found for P. promelas, which was calculated using the cyfluthrin-only exposure and mixture-exposure data. Because cyfluthrin and imidacloprid were toxic to H. azteca, the mixture test was designed based on an equipotent toxic unit method. Results from the mixture test indicated a model deviation ratio (MDR) of 1.7 or 2.7 depending on the model. Mixture test results from the simultaneous exposure to cyfluthrin and imidacloprid with both species indicated a greater than expected toxic response because the SR or MDR values were >1. Because these two insecticides are commonly used together in the same product formulations, nontarget species could be more affected due to their greater-than-additive toxicity observed in the current study.

Combined effects of pharmaceuticals, personal care products, biocides and organic contaminants on the growth of Skeletonema pseudocostatum

Organisms in the environment are exposed to a number of pollutants from different compound groups. In addition to the classic pollutants like the polychlorinated biphenyls, polyaromatic hydrocarbons (PAHs), alkylphenols, biocides, etc. other compound groups of concern are constantly emerging. Pharmaceuticals and personal care products (PPCPs) can be expected to co-occur with other organic contaminants like biocides, PAHs and alkylphenols in areas affected by wastewater, industrial effluents and intensive recreational activity. In this study, representatives from these four different compound groups were tested individually and in mixtures in a growth inhibition assay with the marine algae Skeletonema pseudocostatum (formerly Skeletonema costatum) to determine whether the combined effects could be predicted by models for additive effects; the concentration addition (CA) and independent action (IA) prediction model. The eleven tested compounds reduced the growth of S. pseudocostatum in the microplate test in a concentration-dependent manner. The order of toxicity of these chemicals were irgarol>fluoxetine>diuron>benzo(a)pyrene>thioguanine>triclosan>propranolol>benzophenone 3>cetrimonium bromide>4-tert-octylphenol>endosulfan. Several binary mixtures and a mixture of eight compounds from the four different compound groups were tested. All tested mixtures were additive as model deviation ratios, the deviation between experimental and predicted effect concentrations, were within a factor of 2 from one or both prediction models (e.g. CA and IA). Interestingly, a concentration dependent shift from IA to CA, potentially due to activation of similar toxicity pathways at higher concentrations, was observed for the mixture of eight compounds. The combined effects of the multi-compound mixture were clearly additive and it should therefore be expected that PPCPs, biocides, PAHs and alkylphenols will collectively contribute to the risk in areas contaminated by such complex mixtures.

Transgenic (cyp19a1b-GFP) zebrafish embryos as a tool for assessing combined effects of oestrogenic chemicals

Endocrine disrupting chemicals and especially oestrogen receptor (ER) agonists have been extensively studied over the years due to their potential effects on sexual development and reproduction in vertebrates, notably fish. As ER agonists can exist as complex mixtures in the aquatic environment, evaluating the impact of combined exposure on oestrogenic effects has become increasingly important. Use of predictive models such as concentration addition (CA) and independent action (IA) has allowed assessment of combined estrogenic effects of complex multi-compound mixtures of ER agonists in various fish in vitro and in vivo experimental models. The present work makes use of a transgenic zebrafish strain, tg(cyp19a1b-GFP), which expresses the green fluorescent protein (GFP) under the control of the cyp19a1b (brain aromatase or aromatase B) gene to determine the oestrogenic potency of ER agonists alone or in mixtures. In these studies, tg(cyp19a1b-GFP) zebrafish embryos were exposed for four days (from one to five days post fertilization) to five different oestrogenic chemicals; 17α-ethinylestradiol (EE2), 17β-estradiol (E2), estrone (E1), bisphenol A (BPA) and 4-tert-octylphenol (OP), and three mixtures of up to four of these compounds. The mixture of BPA, OP and E2 was also tested with primary cultures of rainbow trout hepatocytes by analysing the ER-mediated induction of the oestrogenic biomarker vitellogenin in order to compare the performance of the two methods for assessing oestrogenic effects of complex mixtures. The three tested mixtures were predominantly acting in an additive manner on the expression of GFP. Additivity was indicated by the overlap of the 95% confidence interval of the concentration response curves for the observed data with the CA and IA prediction models, and model deviation ratios within a factor of two for a majority of the mixture concentrations. However, minor deviations determined as more than additive effects for the mixture of EE2, E1 and E2 and less than additive effects for the mixture of BPA, OP, EE2 and E1 were observed at the higher mixture concentrations tested. The successful prediction of additivity by CA and IA in tg(cyp19a1b-GFP) zebrafish embryos and deviations at high mixture concentrations seemed to correspond well to results obtained in the rainbow trout hepatocyte assay. The present results clearly show the usefulness of combining predictive modelling and use of in vitro bioassays for rapid screening of oestrogenic effects of complex mixtures and environmental samples.