Algal Growth Inhibition Test Research Articles

Systematization of a toxicity screening method based on a combination of chemical analysis and the delayed fluorescence algal growth inhibition test for use in emergency environmental surveys.

In recent years, heavy rainfall disasters linked to climate change have become more frequent, raising concerns about the release of chemicals stored in factories. Assessing chemical contamination during such emergencies therefore necessitates the development of a quick and easy method for evaluating hazardous contaminants in combination with toxicity testing. This study proposes a "toxicity screening" method that combines biological response testing and chemical analysis to systematically evaluate hazardous contaminants in emergency situations. The toxicity screening method evaluates the water quality in three steps, including water quality measurements and a delayed fluorescence (DF) assay, metal content measurements and a DF assay, and targeted screening analysis and a DF assay. The efficacy of this method was tested using industrial wastewater from 14 locations. Seven of the samples were non-toxic, while the other seven samples were toxic, displaying no observed effect concentration (NOEC) values ranging from 0.625 to 20%. Two toxic samples in the first phase possessed high total chlorine concentrations (0.4mg L-1) and conductivities (2200 mS m-1), indicating that the main sources of toxicity were residual chlorine and a high salt concentration. In the second phase, metal content analysis identified metals as the toxicity cause in four samples. In the third phase, the organic contaminants were analyzed, and tri-n-octyl phosphate (TNOP) was detected at a concentration of 0.00027mg L-1. The results of solid-phase extraction experiments and exposure tests with TNOP alone indicated that the contribution of TNOP to the toxicity was negligible and that chemicals not adsorbed on the solid-phase extraction cartridges were the cause of toxicity. The proposed method can therefore be considered effective for disaster-related water quality assessment, delivering results within 12days.

Effects of Non-steroidal Anti-inflammatory Drugs (Ibuprofen and Diclofenac) and Their Mixtures on the Growth of Three Green Algae

Ibuprofen (IBU) and diclofenac (DFN) are the most widely consumed non-steroidal anti-inflammatory (NSAIDs) worldwide. Since they are not completely metabolized in the human body, the unaltered active ingredients can reach aquatic ecosystems through domestic effluents. The aim of this study was to analyse the toxic effects of IBU and DFN, individually and in mixtures, on three freshwater green algae strains, the international standard strain Raphidocelis subcapitata and two native strains, Ankistrodesmus fusiformis and Tetradesmus obliquus. Bioassays were carried out according to the 72-h algal growth inhibition test. The concentration-response curve of the mixture was compared to predicted effects based on both the concentration addition (CA) and the independent action (IA) models. The two drugs tested individually were toxic to all three algal strains, with EC50 values <100 mg L-1. According to these values, the most sensitive strain was R. subcapitata (IBU = 20 mg L-1 and DFN = 8 mg L-1). The most resistant strain was T. obliquus (IBU = 74.1 mg L-1 and DFN = 92.7 mg L-1) and the A. fusiformis strain showed intermediate sensitivity (IBU = 26.7 mg L-1 and DFN = 14.6 mg L-1). The mixtures showed a synergistic effect on R. subcapitata, an additive effect on A. fusiformis, and an antagonistic effect on T. obliquus. Neither the CA nor the IA model was appropriate for predicting the toxicity of the mixtures. In conclusion, IBU and DFN showed toxic effects on the algae growth at the concentrations tested. Individual and mixed toxicity was different according to the species. These differences could be species-specific, showing the importance of including native strains in ecotoxicological studies to evaluate the potential effects of pollutants in regional aquatic environments.

Algal growth inhibition test with TEMPO-oxidized cellulose nanofibers

Ecotoxicity data on cellulose nanofibers (CNFs) are limited despite their wide potential applications prospects, such as structural and packaging materials, filters, coatings, foods, and cosmetics. In this study, toxicity tests of 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized CNFs (TEMPO-CNFs), which are one of the major CNF products commercially available in Japan, on the green alga Raphidocelis subcapitata were conducted. As nanomaterials are considered difficult-to-test substances, the Organisation for Economic Co-operation and Development has released a guidance document that provides considerations regarding ecotoxicity tests of nanomaterials. In the algal growth inhibition tests of TEMPO-CNFs, there were specific issues to be examined, including the effects of medium components on the characteristics of TEMPO-CNFs, CNF interference with algal density measurements, algal interference with CNF measurements, and the effects of ion concentration changes in the test medium by the addition of CNFs on algal growth. To examine these issues, we conducted preliminary studies and established a suitable test method for algal growth inhibition tests of TEMPO-CNFs. We confirmed that the components in the medium for algal growth inhibition tests had negligible effects on the characteristics (zeta-potential, viscosity, and morphology) and concentration stability of TEMPO-CNFs and that in vitro and in vivo fluorescence measurements were applicable for estimating the algal densities, without interference by TEMPO-CNFs. In contrast, we observed that the grown algae interfered with the CNF concentration measurements. Therefore, we established a method to correct the measured CNF concentrations by estimating the algal contribution. Furthermore, we found that the nutrient salt concentrations in the medium changed due to interactions with CNFs; however, this change did not affect algal growth. Based on the results of the preliminary studies, algal growth inhibition tests of TEMPO-CNFs were conducted using in vitro and in vivo fluorescence measurements, along with measurements of CNFs and ion concentrations in the test dispersions. The test results showed that no growth inhibition was observed on growth rate or yield even at the maximum CNF concentration of 100 mg/L, suggesting that the ecological effect of TEMPO-CNFs on algae was relatively low. The results of this study will be valuable for conducting ecotoxicity assessments on additional CNFs and comparable nanomaterials in future studies.

Assessing the genotoxic potential of wastewater effluents from three wastewater treatment plants in South Africa.

Wastewater treatment plants are mainly monitored for quality in terms of their biological oxygen demand and microbiological constituents as stipulated in the specific discharge permit. Wastewater influents and effluents were taken from three WWTPs in South Africa over the summer and winter seasons. Previous toxicity tests such as the Vibrio fischeri bioluminescence assay and the Selenastrum capricornutum algal growth inhibition test have shown that the effluents displayed acute toxicity. To further investigate the quality of the effluent, the genotoxic potential was determined using the SOS Chromosome and UMU Chromosome test. The SOS Chromotest demonstrated induction factor values of above 1.5 for influents during both seasons indicating that the influents were genotoxic (p < 0.05). Effluents discharged during winter and summer also had induction factors greater than 1.5 (p < 0.05). A range of induction factors was detected with the UMU-Chromotest for influents and effluents (1.98 ± 0.38 and 2.40 ± 0.51, respectively). Findings show point sources in the area can lead to influents and effluents that are potentially genotoxic. Designing a monitoring programme that encompasses testing of both the regulatory determinants with additional specialized tests can provide a more holistic view of wastewater quality and the efficiency of WWTP to reduce the discharge of hazards.

Investigation of toxic effects of BPA and BPA analogues (BPS and BPAF) on Spirulina sp., Desmodesmus subspicatus and Chlorella vulgaris

Bisphenols (BPs) are produced for many applications for used in industry. BPs have been found all part of aquatic environments such as sediment and surface water that is poses a risk to the aquatic ecosystem. Restricting the use of BPA, environmental concentrations of bisphenol S, and bisphenol AF begin to increase. The present study aims to indicate that toxicity BPA and BPA analogues (BPS and BPAF) by algal growth inhibition test for the green algae Chlorella vulgaris, Spirulina sp., Desmodesmus subspicatus. In this way, result of this study present the nominal effective concentrations of BPA analogues and the suitability of the species for use as a biomarker in ecotoxicology tests. IC50 values (growth rate inhibition by 50%, respectively) for three toxicants were determined separately. Results of this study showed the effects of these chemicals on photosynthesis (primer production). The result of algal growth inhibition test showed that BPAF (72h EC50 3.80 mg/L) was found to be more toxic than BPS (3d EC50 6.31 m L-1) for Spirulina sp. BPS (3d EC50 2.43 mg/L) showed the most toxic effect on the growth of C. vulgaris, followed by BPAF with 3d EC50 3.32 mg/L. BPS (3d EC50 0.88 mg/L) and BPAF (3d EC50 6.48 mg/L) were found to be toxic for D. subspicatus, respectively, from highest to lowest toxicity. These results indicate that bisphenol analogues are hazardous to primer production. Therefore, it is necessary to study their combined effects as well as to study how they act individually.

Assessing the ecological risk of representative wastewater based on a growth inhibition method with freshwater algae (Raphidocelis subcapitata)

Wastewater contains a vast number of pollutants that potentially threaten the ecosystem after being discharged into the environment. The present study aimed to develop a rapid and sensitive toxicity detection method for assessing the ecological risk of wastewater. Raphidocelis subcapitata was used as the test organism in the algal growth inhibition test, and the anti-interference capacity of R. subcapitata itself and three biomass measuring methods (spectrophotometer, electron particle counter and chlorophyll fluorescence) on sample color and sample particulate matter were examined. The results revealed that chlorophyll fluorometry was more suitable for determining biomass in the growth inhibition test. Furthermore, chlorophyll fluorometry was selected to evaluate the combined toxic effects and sensitivity of R. subcapitata to wastewater from different industries. Microalgae respond differently to the toxicity of wastewater from various sources. The LID values for chemical wastewater 2, urban sewage 1, pharmaceutical wastewater 1, electroplating effluent and pharmaceutical wastewater 2 were 4.0, 3.0, 2.7, 2.4 and 2.0, respectively. The present study investigated the toxicity response of R. subcapitata to wastewater from various industries by growth inhibition experiments to comprehend the possible ecological concerns of wastewater from diverse businesses. This study will provide a significant basis for the design of detection methods and a comprehensive evaluation of toxicity in surface water environments.Graphical

Bakırçay nehri yüzey sularının Scenedesmus dimorphus yeşil algi gelişimi üzerine etkileri hakkında bir ön çalışma

Bakırçay River, one of the important rivers of the Aegean region, has a length of 129 km and an approximate catchment area of 3160 km2. Bakırçay River, which carries agricultural drainage and polluted wastewater with high nitrogen and phosphorus content, is polluted due to domestic and agricultural resources. The water of the Bakırçay River is used for irrigation in agriculture and fishing from the dam lakes on the river for feeding purposes also negatively affects the health of the people in the vicinity. In the Bakırçay River, there is widespread pollution caused by mining areas as well as domestic, industrial and agricultural pollution. For this purpose, the “Algal Growth Inhibition Test, OECD 201” was applied to the water samples obtained from 10 stations on the Bakırçay River. Scenedesmus dimorphus (Turpin) Kützing, 1834 green algae culture, accepted as the primary trophic level representative for the " Algal Growth Inhibition Test, OECD 201" test, one of the short-term phytotoxicity test methods, was used in this test. Water samples were tested at five different dilutions (40, 60, 80, 100 %). To determine the effects on the growth of Scenedesmus dimorphus, the cells were counted by fluorimeter (Turner design) daily at the same time and the test duration was 72 hours. In conclusion, the highest level of toxicity was found at Stations 1, 9, 10,13,14, and 15. The reason for this was that there was a coal facility near Station 9 and that all branches of the Bakırçay River joined near Station15 and then drained into Çandarlı Bay.

Toxicity and Environmental Risk Assessment of Polycarbamate and Its Main Components to Marine Algae and Crustaceans.

Polycarbamate is commonly used as an antifoulant coating on fishing nets in Japan. Although its toxicity to freshwater organisms has been reported, its toxicity to marine organisms is currently unknown. We conducted algal growth inhibition and crustacean immobilization tests to assess the effects of polycarbamate on marine organisms. We also evaluated the acute toxicity of the main components of polycarbamate, namely, dimethyldithiocarbamate and ethylenebisdithiocarbamate, to algae, which are the most sensitive tested organisms to polycarbamate. The toxicities of dimethyldithiocarbamate and ethylenebisdithiocarbamate partially explain that of polycarbamate. To assess the primary risk, we derived the predicted no-effect concentration (PNEC) for polycarbamate in a probabilistic manner using species sensitivity distributions. The 72 h no observed effect concentration (NOEC) of polycarbamate to the alga Skeletonema marinoi-dohrnii complex was 0.45 μg/L. The toxicity of dimethyldithiocarbamate may have contributed up to 72% of the toxicity observed for polycarbamate. The fifth percentile of hazardous concentration (HC5) derived from the acute toxicity values was 0.48 μg/L. Comparison of previously reported environmental polycarbamate concentrations in Hiroshima Bay, Japan, to the PNEC estimated using the minimum NOEC and HC5 suggest that polycarbamate currently poses a high ecological risk. Therefore, reducing the risk by restricting polycarbamate use is necessary.

Removal of residues of psychoactive substances during wastewater treatment, their occurrence in receiving river waters and environmental risk assessment

Continuous consumption combined with incomplete removal during wastewater treatment means residues of psychoactive substances (licit drugs, medications of abuse and illicit drugs) are constantly introduced into the aquatic environment, where they have the potential to affect non-target organisms. In this study, 17 drug residues of psychoactive substances were determined in wastewater influent, effluent and in receiving rivers of six Slovene municipal wastewater treatment plants employing different treatment technologies. Variations in removal efficiencies (REs) during spring, summer and winter were explored, and ecotoxic effects were evaluated using in silico (Ecological Structure-Activity Relationships software–ECOSAR) and in vivo (algal growth inhibition test) methods. Drug residues were detected in influent and effluent in the ng/L to μg/L range. In receiving rivers, biomarkers were in the ng/L range, and there was good agreement between measured and predicted concentrations. On average, REs were highest for nicotine, 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THC-COOH), cocaine residues, and amphetamine (>90 %) and lowest for methadone residues (<30 %). REs were comparable between treatments involving activated sludge and membrane bioreactors, while the moving biofilm bed reactor (MBBR) removed cotinine, cocaine, and benzoylecgonine to a lesser extent. Accordingly, higher levels of nicotine and cocaine residues were detected in river water receiving MBBR discharge. Although there were seasonal variations in REs and levels of drug residues in receiving rivers, no general pattern could be observed. No significant inhibition of algal growth (Chlamydomonas reinhardtii) was observed for the tested compounds (1 mg/L) during 72 h and 240 h of exposure, although effects on aquatic plants were predicted in silico. In addition, environmental risk assessment revealed that levels of nicotine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), morphine, and 3,4-methylenedioxymethamphetamine (MDMA) pose a risk to aquatic organisms. Since nicotine and EDDP can have acute and chronic effects, the authors support regular monitoring of receiving surface waters, followed up by regulatory actions.

Integrated Exposure and Algal Ecotoxicological Assessments of Effluents from Secondary and Advanced-Tertiary Wastewater-Treatment Plants.

The great concern over the environmental impact of wastewaters has led to the designing of advanced treatment processes to upgrade conventional treatment plants and achieve a significant reduction of contaminants in receiving waters. In the present study we combined chemical and ecotoxicological analyses, aiming to evaluate the reduction of toxicity effects associated with the removal of micropollutants and to define the contribution of the detected compounds to the overall toxicity of the mixtures in a series of wastewater effluents collected from a secondary treatment (OUT 2) and from a tertiary activated carbon treatment (OUT 3) plant. The target compounds were selected after a screening procedure among pharmaceuticals, musk fragrances, and trace metals. The classical algal growth inhibition test was conducted on the original effluent samples and on different fractions obtained by solid-phase extraction (SPE) treatment. A good accordance was found between the removal of toxicity (30%-80%) and organic compounds (70%-80%) after the tertiary treatment, suggesting its high efficiency to improve the wastewater quality. The discrepancy between the contribution to the overall toxicity of the nonadsorbable compounds (i.e., inorganic or very polar organic compounds) as experimentally measured by the SPE bioassays (18%-76%) and calculated by the concentration addition approach (>97%) could be mitigated by including the bioavailability correction in metal-toxicity modeling of wastewater mixtures. For the organic compounds, the toxic equivalency method enabled us to quantify the portion of toxicity explained by the detected chemicals in both OUT 2 (82%-104%) and OUT 3 (5%-57%), validating the selection of the target molecules. The applied integrating approach could be implemented by the inclusion of both additional target chemicals and toxicity endpoints. Environ Toxicol Chem 2022;41:2404-2419. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Passive dosing: Assessing the toxicity of individual PAHs and recreated mixtures to the microalgae Raphidocelis subcapitata

Risk assessment of hydrophobic organic compounds (HOCs) is difficult because maintaining a well-defined exposure during aquatic toxicity testing is challenging due to the limited water solubility and various loss processes such as volatilization, biodegradation and sorption. Passive dosing techniques help to overcome these challenges by providing a well-controlled and solvent-free exposure. In this study, the algal growth inhibition test (DIN EN ISO 8692) was converted into a miniaturized passive dosing setting. For this purpose, biocompatible O-rings were used as substance reservoirs and loaded with polycyclic aromatic hydrocarbons (PAHs). The growth inhibition of the microalgae Raphidocelis subcapitata induced by single PAHs (log KOW 3.24-5.91) was investigated. In addition, recreated PAH mixtures were tested representing field compositions of the pore water North Sea sediments. Some of the single PAHs revealed strong growth inhibiting effects on the algal growth, while the recreated mixture compositions had slightly lower effect on the growth inhibition in the highest concentrations. Overall, the toxicity of the PAHs generally increased with the maximum chemical activities (amax) of the PAHs and the inhibition data could be fitted with one maximum chemical activity response curve. Therefore, the miniaturized passive dosing approach appears as a promising practical and economical method that can be used for toxicity testing of the different trophic levels to improve comprehensive risk assessment.

Testing particles using the algal growth inhibition test (OECD 201): the suitability of in vivo chlorophyll fluorescence measurements

BackgroundThe freshwater algae and cyanobacteria growth inhibition test (OECD test guideline 201) is frequently used to assess the ecotoxicity of chemicals or particles. A central issue is the measurement of algal growth by quantifying algal biomass over time. Chlorophyll fluorescence measurements are recommended for the testing of particles. The analysis of in vivo fluorescence is the simplest and fastest approach, but is only suitable if there is no interference with the materials. Therefore, in vitro fluorescence analysis is often preferred. We carried out a comprehensive comparison of chlorophyll fluorescence measurements in vitro and in vivo to evaluate the suitability of rapid in vivo testing for the determination of Raphidocelis subcapitata biomass in the presence of diverse particles.ResultsFor the in vitro measurement, we applied a method that separates particles from chlorophyll using locust bean gum. We tested inorganic and organic particles (including alloys and polymers), ion-releasing and non-releasing materials, and particle sizes in the nanometer to micrometer range with a variety of shapes (spherical, flaky and fibrous). Some of the materials were nontoxic, whereas others showed varying degrees of toxicity (ErC50 = 0.2–100 mg/L in both methods). There were only minor differences between the methods in ErC50 values and the percent inhibition at various test concentrations, but the confidence intervals for the ErC50 values in vivo were narrower and were covered by the range observed in vitro. The in vivo approach showed no limitations, whereas the validity criteria listed in OECD test guideline 201 were not always fulfilled by the in vitro measurements.ConclusionThe in vivo approach was a suitable and time-saving method for a wide range of particles, although we cannot completely exclude the possibility that some particles may interfere with fluorescence measurement. To avoid false assessments, pre-tests with simple measurements are therefore recommended.Graphical abstract

Separating toxicity and shading in algal growth inhibition tests of nanomaterials and colored substances

Nanoparticles and colored substances can inhibit algal growth by light shading and chemical toxicity. This study presents two complementary approaches to account for shading in algal growth inhibition tests of engineered nanomaterials (ENMs) and colored substances. The first approach distinguishes between shading effects and toxicity by varying the light path in parallel algal growth inhibition tests. This Multiple Path-Length (MPL) test was applied to TiO2 ENMs and the colored substances sodium picramate and Rhodamine B. A left shifting of concentration-response curves, with increasing light path lengths, indicated shading for Rhodamine B, sodium picramate and TiO2 ENMs. EC50-values obtained at the shortest light path length were generally found best suited to quantify the toxicity of ENMs and colored substances. The second approach addresses shading at the cellular level, where particles can attach to the cell surface and affect photo-pigment content and composition. Pigments associated with photosystem I and II were determined at varying light intensities and concentrations of TiO2 ENMs. The photo-pigments that increased in response to physical shading, decreased after TiO2 ENMs exposure. This indicates that toxicity rather than cellular shading dominated the response of algae exposed to TiO2 ENMs. Additional tests were conducted with the nanomaterials CeO2 and goethite to evaluate the applicability of this approach to other ENMs. On this basis, we recommend MPL testing for determining EC50-values that are not confounded by shading in the test solution, and the pigment-based approach for investigating shading on the cellular level.

Application of a Multidisciplinary Weight of Evidence Approach as a Tool for Monitoring the Ecological Risk of Dredging Activities

The use of multidisciplinary investigations for the evaluation of aquatic ecosystems status is recommended by the European Directives, but it is still a challenging practice. In this study, we apply a quantitative weight of evidence (WOE) approach (Sediqualsoft) for the integration of extensive data obtained from different typologies of investigations, obtained over a 4-year monitoring study of dredging activities in the harbor of Leghorn (Italy). During different phases of such operations, selected sites have been characterized in terms of levels of trace metals and polycyclic aromatic hydrocarbons in sediments, bioaccumulation of contaminants, and a wide battery of biomarkers in transplanted mussels, ecotoxicological effects of sediments through a battery of bioassays (algal growth inhibition, bioluminescence inhibition, and embryotoxicity tests), and the status of benthic communities. Each typology of data, line of evidence (LOE), has been initially elaborated through dedicated logical flowcharts and algorithms providing specific hazard indices, followed by their overall integration based on different weights assigned to each LOE. This approach allowed to summarize more than 10,000 results, reaching robust conclusions on environmental impact during various phases of dredging operations. This approach was confirmed as a useful tool for monitoring the risk, supporting a “site-oriented” decision making process by providing stakeholders simple interpretation of complex data.

Innovative electrochemical biosensor for toxicological investigations on algae and cyanobacteria

An electrochemical biosensor is presented that directly reflects the metabolic activity of prokaryotic and eukaryotic cells. This biosensor can be used measure the biological activity of bacteria, yeasts and mammalian cells. This makes the sensor interesting for various applications in industry and science. A possible application is bioprocess control, monitor activities from yeasts, bacteria and fungi to increase the yield. Other applications are starter culture quality studies in the food industry and cytoxicological evaluation with mammalian cells.Our latest investigations additionally indicate the applicability of the electrochemical biosensor to measure algae and cyanobacteria. In our investigations, we were able to show that it was also possible to detect photosynthetic organisms with the electrochemical measurement method, used for investigations on prokaryotic and eukaryotic organisms before. Therefore, this the present study demonstrates an alternative to using this electrochemical biosensor equipped with alga and cyanobacteria for toxicological investigations based on selected test chemicals. The results of this study show a good correlation with those from reference methods, such as the Algal Growth Inhibition Test and the Microtox Test. The advantages of the new electrochemical biosensor are easy handling and shorter measurement time by using different types of test organisms.The evaluation of the sensor signal is based on the current–time curves of a potentiostatic measurement produced by the detection of microbially reduced mediator molecules immobilized in a gel structure. The mediator molecules are reduced during the measurement process. The reduced mediator molecules produce a current signal, which rapidly provides information about the vigor and vitality of living bacteria, yeasts, fungi or cells.

Pesticide mixture toxicity to algae in agricultural streams – Field observations and laboratory studies with in situ samples and reconstituted water

Long-term pesticide water concentrations were investigated in four agricultural streams and their mixture toxicity on algae was assessed, based on realistic (i.e. observed) concentrations in laboratory tests using (i) natural weekly water samples and (ii) reconstituted pesticide-spiked water samples representing mixtures with predicted high mixture. This approach both covered the full complexity of natural water samples and the controlled approach of reconstituted water samples. Long-term monitoring data (time-integrated, weekly samples) revealed more than 11 pesticides (range 11.0 ± 0.25–24.0 ± 0.44) in 75% or more of the almost 1600 samples collected between 2002 and 2018. ∑TUalgae exceeded 0.1 for 29 observations (or 1.8%). Despite the multitude of pesticides in a sample, ∑TUalgae was frequently set by one or a few dominating pesticides that contribute to more than 90% of the mixture’s toxicity. Algal growth inhibition tests with in situ stream water showed a high frequency of inhibition, despite the low ∑TU for most of these samples (range 0.000014–0.3858). These “false positive” results were attributed to confounding effects of turbidity, the complexation of nutrients, and toxic effects of metals and/or other unknown contaminants. Algal inhibition tests with spiked reconstituted water showed significant inhibitory effects in the range of 1–10x the ∑TUalgae observed in worst-case field samples. Although these tests disregard the chemical complexity of natural water, they show that inhibitory effects of pesticides on algae may occur at the ∑TUalgae observed in monitoring. Furthermore, considering that the ∑TUalgae of stream water are based on weekly average concentrations and likely underestimate short-term peak concentrations of pesticides, these results strongly suggest that inhibitory effects on algae may occur in the agricultural streams of southern Sweden. We conjecture, however, that the rapid recovery of algae contributes to ameliorate these short-term effects and that pesticide contamination should be seen as one of many stressors in the streams that drain agricultural landscapes.

The effect of using light emitting diodes and fluorescent lamps as different light sources in growth inhibition tests of green alga, diatom, and cyanobacteria.

Aquatic organisms have been used to investigate the safety of chemicals worldwide. One such assessment is an algal growth inhibition test. Algal growth inhibition tests are commonly performed using a growth chamber with fluorescent lamps as the lighting source, as test guidelines require continuous uniform fluorescent illumination. However, fluorescent lamps contain mercury, which has been identified as hazardous to humans and other organisms. The Minamata Convention (adopted in 2013) requires reduction or prohibition of products containing mercury. On the other hand, light-emitting diodes do not contain mercury and provide a photosynthetically effective wavelength range of 400-700 nm which is an adequate light intensity for algal growth. Light-emitting diodes are thus preferable to fluorescent lamps as a potential light source in algal growth inhibition tests. In this study, we investigated if light-emitting diodes could be substituted for fluorescent lamps in growth inhibition studies with green alga (Pseudokirchneriella subcapitata), diatom (Navicula pelliculosa), and cyanobacteria (Anabaena flos-aquae). Algal growth inhibition tests were performed using five different chemicals known to have different modes of action and are assigned as reference substances in the test guidelines. The results of each algal test showed similar values between light-emitting diodes and fluorescent lamps in terms of conditions for the growth inhibition rate and percent inhibition in yield of each chemical. It was therefore concluded that using light-emitting diodes instead of fluorescent lamps as a lighting source had no effect on the algal growth inhibition test results.

Performance of Chlorella Vulgaris Exposed to Heavy Metal Mixtures: Linking Measured Endpoints and Mechanisms.

Microalgae growth inhibition assays are candidates for referent ecotoxicology as a fundamental part of the strategy to reduce the use of fish and other animal models in aquatic toxicology. In the present work, the performance of Chlorella vulgaris exposed to heavy metals following standardized growth and photosynthesis inhibition assays was assessed in two different scenarios: (1) dilutions of single heavy metals and (2) an artificial mixture of heavy metals at similar levels as those found in natural rivers. Chemical speciation of heavy metals was estimated with Visual MINTEQ software; free heavy metal ion concentrations were used as input data, together with microalgae growth and photosynthesis inhibition, to compare different effects and explain possible toxicity mechanisms. The final goal was to assess the suitability of the ecotoxicological test based on the growth and photosynthesis inhibition of microalgae cultures, supported by mathematic models for regulatory and decision-making purposes. The C. vulgaris algae growth inhibition test was more sensitive for As, Zn, and Pb exposure whereas the photosynthesis inhibition test was more sensitive for Cu and Ni exposure. The effects on growth and photosynthesis were not related. C. vulgaris evidenced the formation of mucilaginous aggregations at lower copper concentrations. We found that the toxicity of a given heavy metal is not only determined by its chemical speciation; other chemical compounds (as nutrient loads) and biological interactions play an important role in the final toxicity. Predictive mixture effect models tend to overestimate the effects of metal mixtures in C. vulgaris for both growth and photosynthesis inhibition tests. Growth and photosynthesis inhibition tests give complementary information, and both are a fast, cheap, and sensitive alternative to animal testing. More research is needed to solve the challenge of complex pollutant mixtures as they are present in natural environments, where microalgae-based assays can be suitable monitoring tools for pollution management and regulatory purposes.

Micro- and Nanoplastic Exposure Effects in Microalgae: A Meta-Analysis of Standard Growth Inhibition Tests

Background: Ecological impacts of micro- and nanoplastics particles (MNP) are among the most discussed environmental concerns. In algae, MNP are commonly hypothesized to reduce growth, which is a standard ecotoxicological endpoint. However, the reported test outcomes vary, with both growth inhibition and stimulation being observed. Due to this conflict of information, a data synthesis for MNP potential to cause growth inhibition in toxicity testing is needed. Methods: We performed a meta-analysis study to assess the effect of MNP exposure on algal growth. Twenty studies published between 2010 and 2020 and representing 16 algal species and five polymer materials administered as particles in size range 0.04 to 3000 µm were included in this meta-analysis. A random-effect model was used to estimate the effect size in three datasets: (1) Low concentration range (< 100 mg/L), (2) High concentration range (≥ 100 mg/L), and (3) Full range model (0.004 to 1100 mg/L), which encompassed all studies using the combination of experimental settings (test species, MNP concentration, polymer material, and particle size) yielding the highest effect size within a study. Results: The exposure to MNP was not significantly associated with growth inhibition in any of the models tested. However, a high heterogeneity between the studies was found in all three models. Neither MNP concentration nor polymer material contributed significantly to the heterogeneity, whereas polymer density had a significant moderating effect, with a higher risk of growth inhibition at lower densities. We also identified a publication bias, with small studies that reported significant inhibition being overrepresented in our dataset. Conclusions: The meta-analysis found limited evidence for MNP effect on microalgal growth in the standard algal growth inhibition test. The heterogeneity and varying methodological quality of studies limited the interpretation and the confidence in the findings. For hazard assessment, standardization and controlled exposure are needed as well as more sensitive endpoints that can inform us about the effect mechanisms. Finally, using particle-free controls in such tests cannot account for the presence of inert particulates in the test system, and, hence, does not allow to attribute observed effects to the test polymers.

Effect of humic acids on the toxicity of pollutants to Chlamydomonas reinhardtii: Investigation by a microscale algal growth inhibition test.

Dissolved humic substances (DHSs) are the major components of organic matter in the aquatic environment. DHSs are well known to considerably affect the speciation, solubility, and toxicity of a wide variety of pollutants in the aquatic environment. In this study, the effects of the toxicity of heavy metals and hydrophobic organic pollutants (HOPs) on Chlamydomonas reinhardtii in the presence of humic acid (HA) were examined by a microscale algal growth inhibition (μ-AGI) test based on spectrophotometric detection. To clarify the relationship between the chemical properties of HAs and the toxicity change of pollutants, eight HAs from different sources were prepared and used. HAs were responsible for mitigating the toxicity of Hg, Cu, pesticides (γ-HCH, 2,4-D, and DDT), and polycyclic aromatic hydrocarbons (PAHs) such as naphthalene (Nap), anthracene (Ant), and benzo[a]pyrene (BaP). In particular, an approximately 100-fold decrease in the toxicity of BaP was observed in the presence of 10 ppm HAs extracted from tropical peat. The results indicated that the carboxylic group content and the HA molecular weight are correlated to the changes in the heavy metal toxicity. For HOPs, the aromaticity and polarity of HAs are crucial for mitigating their toxicity. Furthermore, it was clearly shown that the lake water including a high concentration of DHSs collected from Central Kalimantan, Indonesia, reduced the toxicity of Hg and γ-HCH on Chlamydomonas reinhardtii. Graphical abstract.