Hans Blanck

Joint algal toxicity of 16 dissimilarly acting chemicals is predictable by the concept of independent action

For a predictive assessment of the aquatic toxicity of chemical mixtures, two competing concepts are available: concentration addition and independent action. Concentration addition is generally regarded as a reasonable expectation for the joint toxicity of similarly acting substances. In the opposite case of dissimilarly acting toxicants the choice of the most appropriate concept is a controversial issue. In tests with freshwater algae we therefore studied the extreme situation of multiple exposure to chemicals with strictly different specific mechanisms of action. Concentration response analyses were performed for 16 different biocides, and for mixtures containing all 16 substances in two different concentration ratios. Observed mixture toxicity was compared with predictions, calculated from the concentration response functions of individual toxicants by alternatively applying both concepts. The assumption of independent action yielded accurate predictions, irrespective of the mixture ratio or the effect level under consideration. Moreover, results even demonstrate that dissimilarly acting chemicals can show significant joint effects, predictable by independent action, when combined in concentrations below individual NOEC values, statistically estimated to elicit insignificant individual effects of only 1%. The alternative hypothesis of concentration addition resulted in overestimation of mixture toxicity, but differences between observed and predicted effect concentrations did not exceed a factor of 3.2. This finding complies with previous studies, which indicated near concentration-additive action of mixtures of dissimilarly acting substances. Nevertheless, with the scientific objective to predict multi-component mixture toxicity with the highest possible accuracy, concentration addition obviously is no universal solution. Independent action proves to be superior where components are well known to interact specifically with different molecular target sites, and provided that reliable statistical estimates of low toxic effects of individual mixture constituents can be given. With a regulatory perspective, however, fulfilment of both conditions may be regarded as an extraordinary situation, and hence concentration addition may be defendable as a pragmatic and precautionary default assumption.

Predicting the joint algal toxicity of multi-component s-triazine mixtures at low-effect concentrations of individual toxicants.

Herbicidal s-triazines are widespread contaminants of surface waters. They are highly toxic to algae and other primary producers in aquatic systems. This results from their specific interference with photosynthetic electron transport. Risk assessment for aquatic biota has to consider situations of simultaneous exposure to various of these toxicants. In tests with freshwater algae we predicted and determined the toxicity of multiple mixtures of 18 different s-triazines. The toxicity parameter was the inhibition of reproduction of Scenedesmus vacuolatus. Concentration-response analyses were performed for single toxicants and for mixtures containing all 18 s-triazines in two different concentration ratios. Experiments were designed to allow a valid statistical description of the entire concentration-response relationships, including the low concentration range down to EC1. Observed effects and effect concentrations of mixtures were compared to predictions of mixture toxicity. Predictions were calculated from the concentration-response functions of individual s-triazines by applying the concepts of concentration addition and independent action (response addition) alternatively. Predictions based on independent action tend to underestimate the overall toxicity of s-triazine mixtures. In contrast, the concept of concentration addition provides highly accurate predictions of s-triazine mixture toxicity, irrespective of the effect level under consideration and the concentration ratio of the mixture components. This also holds true when the mixture components are present in concentrations below their individual NOEC values. Concentrations statistically estimated to elicit non-significant effects of only 1% still contribute to the overall toxicity. When present in a multi-component mixture they can co-operate to give a severe joint effect. Applicability of the findings obtained with s-triazines to mixtures of other contaminants in aquatic systems and consequences for risk assessment procedures are discussed.

A Critical Review of Procedures and Approaches Used for Assessing Pollution-Induced Community Tolerance (PICT) in Biotic Communities

Pollution-induced community tolerance (PICT) is used for the detection of minor effects of toxicants in biotic communities. Organisms survive in toxic environments only if they are tolerant to the chemicals present in their habitat. In the selection phase, toxicants hinder the success of sensitive individuals and species and replace them by more tolerant ones. The resulting increase in community tolerance is quantified in the detection phase by short-term toxicity tests. In this way PICT can establish causal linkages between contaminants and effects. An increase in community tolerance compared to the baseline tolerance at reference sites suggests that the community has been adversely affected by toxicants. PICT has been used in aquatic and terrestrial environments with communities of periphyton, phytoplankton, bacteria, nematodes and insects. A variety of methods have been used for quantification of community tolerance including photosynthesis, sulfolipid synthesis, respiration, thymidine and leucine incorporation, survival, and substrate utilisation patterns. PICT has been observed for copper, zinc, nickel, mercury, cadmium, arsenate, monomethylarsonic acid, diuron, tributyl tin, 4,5,6-trichloroguaiacol, irgarol 1051, seanine 211, atrazine, and trinitrotoluene. It is necessary to validate PICT, at least by showing that it is related to the preexposure concentration of the toxicants and that it is coupled to a toxicant-induced succession (TIS) in the community. Care must also be taken to ascertain that PICT interpretation is not confounded by co-tolerance or bioavailability differences. Co-tolerance patterns, which are indicative of the specificity of PICT, have only been investigated for arsenate, diuron and a few metals. For the further improvement of PICT methodology special attention should be given to co-tolerance patterns and development of new integrating short-term tests for quantification of tolerance. It is also important to broaden the scope of organisms and toxicants used. Properly validated, PICT is a powerful tool for detection of community effects and its use in monitoring and site-specific risk assessment should be encouraged.

Toxic effects of the antifouling agent Irgarol 1051 on periphyton communities in coastal water microcosms

In the late 1980s, a number of countries restricted the use of tri-n-butyltin (TBT) as an active ingredient in antifouling paint for small boats, including pleasure craft. Irgarol 1051—2-(tert-butylamino)-4-(cyclopropylamino)-6-(methylthio)-1,3,5-triazine, belonging to the s-triazine group of herbicides—is now used in combination with copper in several antifouling products. Irgarol 1051 contamination of the marine environment was studied close to a marina on the west coast of Sweden. Highest concentrations (1.6 nm, 0.4 μg l−1) were observed during the peak of the boating season. To investigate the potential for toxic effects, marine periphyton communities were established on artificial substrata in 22-litre flowthrough aquaria and exposed to Irgarol 1051 during a 3-week period in April 1994. The algicide was continuously added at concentrations ranging from 0.06 to 260 nm. Irgarol 1051 was found to significantly (p < 0.05) inhibit periphyton photosynthetic activity at 3.2 nm in short-term (hour) tests. Long-term (weeks) exposure produced effects at even lower concentrations. A significant change in community structure was found at 1 nm, which produced a shift towards tolerant species. Photosynthetic activity and algal biomass (chlorophyll a content) was significantly lowered at concentrations of 1 and 4 nm, respectively, indicating only minor functional redundancy in the communities. The most sensitive long-term effects were detected at 0.25-1 nm (0.063–0.25 μg l−1) of Irgarol 1051, which is within the concentration range detected in the contaminated areas around the marina. It can be concluded that the present use of Irgarol 1051 is likely to damage microalgal communities in contaminated coastal waters.

Species-dependent variation in algal sensitivity to chemical compounds.

Nineteen miscellaneous chemical compounds were tested on thirteen freshwater algal species grown in 250-microliter liquid cultures on plastic microtitration plates. It was demonstrated that the species-dependent variation in algal sensitivity (EC100) may reach over three orders of magnitude, the degree of variation depending on the chemical tested. No generally sensitive or generally insensitive species could be identified. An effort was made to quantify the predictive value of algal test batteries, at a given confidence level with respect to chemicals. The predictive value of small test batteries was low. Although it did increase with size it took the presence of more than nine members in the test batteries to improve the value to 0.1. This means that a nine-membered test battery might underestimate the sensitivity of the most susceptible algae by a factor of 0.1. A predictive value of 0.01 requires a three-membered test battery when accounting for 95% of the chemicals and a five-membered battery at the 99% confidence level. Implications for toxicity test strategies are discussed and it is suggested that algal test batteries, using simple test techniques, replace single unialgal cultures in routine work.

Pollution-induced community tolerance (PICT) in marine periphyton in a gradient of tri-n-butyltin (TBT) contamination

Abstract Before the ban of tri- n -butyl tin (TBT) compounds as antifouling agents, yacht marinas were important sources of TBT contamination in coastal waters. The impact of TBT on marine periphyton communities were investigated around a small marina on the Swedish west-coast, using the PICT (pollution-induced community tolerance) methodology. The increase in community tolerance due to elimination of TBT-sensitive algae, is an indicator of damage to the algal community. Periphyton was sampled on artificial substrata in temporal and spatial gradients of TBT exposure during the boating season 1988, i.e. before the ban. The community tolerance was quantified as EC50-values for TBTCl, TBTO and the reference compound DCMU based on short-term inhibition of periphyton photosynthesis. Photosynthesis was measured as the incorporation of 14 CO 2 into acid-stable organic compounds. Ratios between EC50-values for the monomer TBTCl and the dimer TBTO suggests that the toxic effect is exerted by the same dissociation product, most likely the TBT cation. Water samples for chemical analyses were taken to characterise TBT exposure during the colonization period. Periphyton communities showed elevated tolerances to TBTCl and TBTO, but not to DCMU, both in the spatial and temporal gradient, provided that the TBT exposure exceeded 0.5 nM. This is the same effect threshold as estimated earlier in controlled microcosm experiments. We have demonstrated that the PICT methodology is sensitive, specific and robust enough to detect minor effects of TBT on marine periphyton in a complex field situation. The estimated NEC value is 0.5 nM TBT, above which damage to algal communities should be expected.

Long-term toxicity of zinc to bacteria and algae in periphyton communities from the river Göta Älv, based on a microcosm study

This study aims to clarify the relation between pollution-induced community tolerance (PICT), community structure and net production in periphyton communities exposed to environmentally realistic concentrations of zinc and to determine levels of no-effect (NEC). Therefore, periphyton communities from a relatively uncontaminated river (Got a A8 lv, Sweden) were exposed to zinc during 4 weeks in a flow-through aquaria system. PICT was estimated as the increase in EC50 for the short-term inhibition of photosynthesis and thymidine incorporation. Community structure was measured as species richness and Bray-Curtis similarity index, and biomass as dry weight and chlorophyll a. NEC values were estimated as the intercept between a regression line and a control base line. NEC values for biomass and biomass dependent variables were 0.12-0.42 mM which implies that there may be many rivers affected by zinc. However, the PICT response and marked changes in community species composition were found only at much higher concentrations (9.7 mM). We hypothesise that this discrepancy in effect concentrations between biomass-dependent variables and other structure-related variables (including PICT) is due to an interaction between zinc and phosphorus leading to nutrient depletion. An indirect toxic effect on biomass due to nutrient deficiency should not be detectable as an increased zinc tolerance.

Detection of pollution-induced community tolerance (PICT) in marine periphyton communities established under diuron exposure

In the present work an effort aiming at a partial validation of the pollution-induced community tolerance (PICT) methodology is reported. Periphyton communities were established on artificial substrata in a marine microcosm system for 3 to 4 weeks. The communities were exposed to concentrations of the herbicide diuron (DCMU, 3-(3.4-dichlorophenyl)-1.1-dimethyl urea) ranging from 1.6 nM to 1 μM. A short-term test on periphyton photosynthesis was used to quantify the tolerances of the different communities, and an increase in the tolerance (PICT) was detected at concentrations above 40 nM. The results are discussed in terms of a ‘comparison triad’ where comparisons are made between the short-term effects on photosynthesis of control communities, the PICT of diuron-exposed communities, and other long-term effects such as cholorophyll a content, specific pholosynthetic activity (P) and diatom species richness. The first effect of diuron on periphyton photosynthesis in short-term tests was recorded at about 3.2 nM. while effects after long-term exposure, reflected by the various long-term parameters, were detectable only at higher concentrations, about 20 to 40 nM.

Water quality objectives for mixtures of toxic chemicals: problems and perspectives

The need to develop water quality objectives not only for single substances but also for mixtures of chemicals seems evident. For that purpose, the conceptual basis could be the use of the two existing biometric models: concentration addition (CA) and independent action (IA), which is also called response addition. Both may allow calculation of the toxicity of mixtures of chemicals with similar modes of action (CA) or dissimilar modes of action (IA), respectively. The joint research project Prediction and Assessment of the Aquatic Toxicity of Mixtures of Chemicals (PREDICT) within the framework of the IVth Environment and Climate Programme of the European Commission, provided the opportunity to address (a) chemometric and QSAR criteria to classify substances as supposedly similarly or dissimilarly acting; (b) the predictive values of both models for the toxicity of mixtures at low, statistically nonsignificant effect concentrations of the individual components; and (c) the predictability of mixture toxicity at higher levels of biological complexity. In this article, the general outline, methodological approach, and some preliminary findings of PREDICT are presented. A procedure for classifying chemicals in relation to their structural and toxicological similarities has been developed. The predictive capabilities of CA and IA models have been demonstrated for single species and, to some extent, for multispecies testing. The role of very low effect concentrations in multiple mixtures has been evaluated. Problems and perspectives concerning the development of water quality objectives for mixtures are discussed.

Effects of sulfonylurea herbicides on non-target aquatic micro-organisms. Growth inhibition of micro-algae and short-term inhibition of adenine and thymidine incorporation in periphyton communities

Abstract To study the differential sensitivity to sulfonylurea herbicides of aquatic micro-organisms we used the micro-plate technique as previously described by Blanck and Bjornsater [Blanck, H., Bjornsater, B., 1989. The algal microtest battery—a manual for routine tests of growth inhibition. Report to the Swedish National Chemicals Inspectorate, no 3/89]. This technique enables growth-inhibition tests to be carried out for a large number of species of micro-algae, and growth inhibition data for the two sulfonylureas chlorsulfuron and metsulfuron methyl were obtained for 20 marine species and 20 fresh-water species, respectively. The species-dependent variation in algal sensitivity was found to be very large, with strains of cyanobacteria and dinoflagellates being the most sensitive. The most sensitive species had EC 50 values for the inhibition of growth in the nanomolar range while EC 50 was higher than 1000 μM for the most tolerant species. Field experiments were also carried out to examine the sensitivity of periphyton communities in an agricultural area in southern Sweden. Periphyton communities were established on an artificial substratum in a creek affected by the runoff of agricultural pesticides and a creek with a watershed with no use of pesticides. Short-term toxicity of the sulfonylurea tribenuron methyl was assayed as inhibition of adenine and thymine incorporation weekly for 8 weeks at all sites. At the site with high exposure to pesticides and fertilisers the lowest observed effect concentration (LOEC) values for inhibition of adenine incorporation were always higher than environmentally expected concentrations indicating a high tolerance of periphyton to tribenuron methyl at this site. The toxicity of tribenuron methyl at the reference site was very variable over time. Adenine incorporation in periphyton from the reference site was in three out of seven experiments significantly inhibited at 3 nM tribenuron methyl, but in the remaining four experiments, LOEC values were much higher. The apparent high background variability in the short-term toxicity of tribenuron methyl makes an interpretation of the observed differences between the reference site and the site with higher exposure to pesticides very difficult. It is never the less interesting to compare the growth inhibition test data with some of the field toxicity tests at the reference site, showing that sulfonylurea herbicides can adversely affect both algal growth and periphyton activity at nanomolar concentrations.