Specific toxicity of azithromycin to the freshwater microalga Raphidocelis subcapitata.

Pharmaceuticals are produced to inflict a specific physiological response in organisms. However, as only partially metabolized after administration, these types of compounds can also originate harmful side effects to non-target organisms. Additionally, there is still a lack of knowledge on the toxicological effects of legacy pharmaceuticals such as the antibiotic azithromycin. This macrolide occurs at high concentrations in the aquatic environment and can constitute a threat to aquatic organisms that are at the basis of the aquatic food chain, namely microalgae. This study established a high-throughput methodology to study the toxicity of azithromycin to the freshwater microalga Raphidocelis subcapitata. Flow cytometry and pulse amplitude modulated (PAM) fluorometry were used as screening tools. General toxicity was shown by effects in growth rate, cell size, cell complexity, cell viability and cell cycle. More specific outcomes were indicated by the analysis of mitochondrial and cytoplasmatic membrane potentials, DNA content, formation of ROS and LPO, natural pigments content and photosystem II performance. The specific mode of action (MoA) of azithromycin to crucial components of microalgae cells was revealed. Azithromycin had a negative impact on the regulation of energy dissipation at the PSII centers, along with an insufficient protection by the regulatory mechanisms leading to photodamage. The blockage of photosynthetic electrons led to ROS formation and consequent oxidative damage, affecting membranes and DNA. Overall, the used methodology exhibited its high potential for detecting the toxic MoA of compounds in microalgae and should be considered for future risk assessment of pharmaceuticals.

Review: ecotoxicity of organic and organo-metallic antifouling co-biocides and implications for environmental hazard and risk assessments in aquatic ecosystems.

Hazard assessments of Irgarol 1051, diuron, 2-(thiocyanomethylthio)benzothiazole (TCMTB), dichloro-octylisothiazolin (DCOIT), chlorothalonil, dichlofluanid, thiram, zinc pyrithione, copper pyrithione, triphenylborane pyridine (TPBP), capsaicin, nonivamide, tralopyril and medetomidine were performed to establish robust environmental quality standards (EQS), based on predicted no effect concentrations (PNECs). Microalgae, zooplankton, fish and amphibians were the most sensitive ecological groups to all the antifoulants evaluated, especially in the early life stages. No differences were identified between freshwater and seawater species. The use of toxicity tests with non-standard species is encouraged because they increase the datasets, allowing EQS to be derived from probabilistic-based PNECs whilst reducing uncertainties. The global ban of tributyltin (TBT) has been heralded as a major environmental success; however, substitute antifoulants may also pose risks to aquatic ecosystems. Environmental risk assessments (ERAs) have driven decision-makings for regulating antifouling products, but in many countries there is still a lack of regulation of antifouling biocides which should be addressed.

A call for action: Improve reporting of research studies to increase the scientific basis for regulatory decision-making.

This is a call for action to scientific journals to introduce reporting requirements for toxicity and ecotoxicity studies. Such reporting requirements will support the use of peer-reviewed research studies in regulatory decision-making. Moreover, this could improve the reliability and reproducibility of published studies in general and make better use of the resources spent in research.

A tiered assessment strategy for more effective evaluation of bioaccumulation of chemicals in fish.

There is currently limited guidance available for regulators and risk assessors on how to use data from non-guideline methods when assessing the bioaccumulation potential of a chemical. Furthermore, bioaccumulation assessments can be more subjective than they need to be due to the lack of a guidance framework on how to use/include the range of information that may be available for a substance. Under some circumstances, in silico, in vitro and/or in vivo non-test guideline data may be sufficient to classify whether a substance is bioaccumulative without the need for further animal testing. Classifying the bioaccumulative potential of a substance is especially difficult when the bioconcentration factor (BCF) is close to the threshold for defining it as bioaccumulative/very bioaccumulative (B/vB), and a more structured process is required to reduce uncertainty in the BCF estimates. In these situations, in silico and in vitro data can, and should, be used to provide greater confidence in classifying these substances. To aid future evaluations of bioaccumulation data, a proposed tiered assessment strategy is presented incorporating all available data on the bioaccumulative properties of a substance. In addition, a revised scheme is recommended for improving the classification of the bioaccumulative potential of a substance.

Assessment of the direct effects of biogenic and petrogenic activated carbon on benthic organisms.

Activated carbon (AC) has long been associated with the capacity to effectively remove organic substances from aquatic and sediment matrices; however, its use in remediation purposes has drawn some concern due to possible impacts on benthic communities. Within the inner Oslofjord, the use of AC has been well documented for reducing the risks associated with dioxins or dioxin-like compounds from contaminated areas. However, benthic surveys performed on areas treated with AC have revealed that the abundance of organisms inhabiting these areas can be reduced significantly in the subsequent years following treatment. The reason for the reduction in the benthic communities is currently unknown, and therefore, an integrated approach to assess the effects of 2 different forms of AC (biogenic and petrogenic) on benthic organisms has been performed. A battery of 3 different benthic organisms with different feeding and life-cycle processes has been used encompassing sediment surface feeders, sediment ingestors, and sediment reworkers. Results of the tests indicated that although AC is not acutely toxic at concentrations up to 1000 mg/L, there may be physical effects of the substance on benthic dwelling organisms at environmentally relevant concentrations of AC at remediated sites.

OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing.

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV<30%) for most chemicals and laboratories. The reproducibility was lower (CV>30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes.

In vivo passive sampling of nonpolar contaminants in brown trout (Salmo trutta).

Equilibrium passive sampling through in vivo implantation can help circumvent complex extractions of biological tissues, provide more accurate information on chemical contaminant burden based on the fugacity of a chemical in an organism rather than conventional normalization to lipid content, and improve the assessment of contaminant bioaccumulation potential. Here, we explored the feasibility of in vivo implantation for the passive sampling of neutral hydrophobic contaminants through the insertion of a silicone tag into brown trout (Salmo trutta). Implanted fish from the upper reaches of the River Alna (Oslo, Norway) were relocated to a polluted section of the river for a 28 day caged exposure. "Whole fish" lipid-silicone distribution coefficients (Dlip-sil) were calculated for chlorinated compounds measured in whole fish and in silicone tags of 13 fish. Dlip-sil ranged from 13.6 to 40.0 g g(-1) for polychlorinated biphenyl congeners 28-156 (CB28 and CB156), respectively, and are in close agreement with literature in vitro lipid phase and tissue-based lipid-silicone partition coefficients. After dissection a further of eight fish, muscle and liver samples were analyzed separately. Muscle-based Dlip-sil values similar to the whole fish data were observed. However, lipid-normalized concentrations in the liver tended to be lower than in muscle for most compounds (by up to 50%). Values of whole fish Dlip-sil for brominated diphenyl ethers determined for three fish were in the range of 8.6-51 g g(-1) and in agreement with chlorinated substances. Finally, fugacity ratios calculated from equilibrium concentrations in fish-implanted and water-exposed silicone provided information on the bioaccumulation for chlorinated compounds as well as for some polycyclic aromatic hydrocarbons. Equilibrium passive sampling through in vivo implantation can allow the comparison of a chemical's activity or fugacity in biotic as well as abiotic environmental compartments and at different trophic levels up to humans.

A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment.

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance.

Ciona spp. and ascidians as bioindicator organisms for evaluating effects of endocrine disrupting chemicals: A discussion paper.

In context of testing, screening and monitoring of endocrine-disrupting (ED) type of environmental pollutants, tunicates could possibly represent a particularly interesting group of bioindicator organisms. These primitive chordates are already important model organisms within developmental and genomics research due to their central position in evolution and close relationship to vertebrates. The solitary ascidians, such as the genus Ciona spp. (vase tunicates), could possibly be extra feasible as ED bioindicators. They have a free-swimming, tadpole-like larval stage that develops extremely quickly (<20 h under favorable conditions), has a short life cycle (typically 2-3 months), are relatively easy to maintain in laboratory culture, have fully sequenced genomes, and transgenic embryos with 3D course data of the embryo ontogeny are available. In this article, we discuss possible roles of Ciona spp. (and other solitary ascidians) as ecotoxicological bioindicator organisms in general but perhaps especially for effect studies of contaminants with presumed endocrine disrupting modes of action.

Weight of evidence tools in the prediction of acute fish toxicity.

Acute fish toxicity (AFT) is a key endpoint in nearly all regulatory implementations of environmental hazard assessments of chemicals globally. Although it is an early tier assay, the AFT assay is complex and uses many juvenile fish each year for the registration and assessment of chemicals. Thus, it is imperative to seek animal alternative approaches to replace or reduce animal use for environmental hazard assessments. A Bayesian Network (BN) model has been developed that brings together a suite of lines of evidence (LoEs) to produce a probabilistic estimate of AFT without the testing of additional juvenile fish. Lines of evidence include chemical descriptors, mode of action (MoA) assignment, knowledge of algal and daphnid acute toxicity, and animal alternative assays such as fish embryo tests and in vitro fish assays (e.g., gill cytotoxicity). The effort also includes retrieval, assessment, and curation of quality acute fish toxicity data because these act as the baseline of comparison with model outputs. An ideal outcome of this effort would be to have global applicability, acceptance and uptake, relevance to predominant fish species used in chemical assessments, be expandable to allow incorporation of future knowledge, and data to be publicly available. The BN model can be conceived as having incorporated principles of tiered assessment and whose outcomes will be directed by the available evidence in combination with prior information. We demonstrate that, as additional evidence is included in the prediction of a given chemical's ecotoxicity profile, both the accuracy and the precision of the predicted AFT can increase. Ultimately an improved environmental hazard assessment will be achieved. Integr Environ Assess Manag 2023;19:1220-1234. © 2022 SETAC.

Unravelling reasons for variability in the OECD 306 marine biodegradation test.

The recommended test for assessing if a chemical can be biodegraded in the marine environment is performed according to the Organisation for Economic Cooperation and Development Marine biodegradation test guideline (OECD 306). However, this test is known to generate highly variable test results when comparing interlaboratory test results for the same compound. One reason can be the relatively low bacterial content compared to the inoculum used for OECD readily biodegradation tests (OECD 301). Some of the variability in data obtained from OECD 306 tests can also be due to the flexibility on how to store the seawater inoculum before starting a test. Another variable in the seawater inoculum is the source of seawater used by different laboratories, i.e., geographical location and anthropogenic activities at the source. In this study, the effect of aging seawater and the source of seawater (sample time and depth) were investigated to determine differences in the biodegradation of the reference compound aniline. Aging the seawater before starting the test is recommended in OECD 306 to reduce the background levels of organic carbon in the water. However, it also functions to acclimatize the bacterial community from the environmental source temperature to the test temperature (normally 20 °C). Herein, the microbial community was monitored using flowcytometer during the aging process. As expected, the microbial community changed over time. In one experiment, aging significantly improved the biodegradation of aniline, while in two experiments, there was no significant difference in biodegradation. Interestingly however, there was significant variability in the biodegradation of aniline between sampling seasons and depths, even when all experiments were performed in the same lab, by the same operator and seawater obtained from the same source. This highlights the need for a more robust and consistent microbial inoculum source to reduce variability in seawater biodegradation tests.

Limitations and uncertainties of acute fish toxicity assessments can be reduced using alternative methods.

Information about acute fish toxicity is routinely required in many jurisdictions for environmental risk assessment of chem­icals. This information is typically obtained using a 96-hour juvenile fish test for lethality according to OECD test guideline (TG) 203 or equivalent regional guidelines. However, TG 203 has never been validated using the criteria currently required for new test methods including alternative methods. Characterization of the practicality and validity of TG 203 is important to provide a benchmark for alternative methods. This contribution systematically summarizes the available knowledge on limitations and uncertainties of TG 203, based on methodological, statistical, and biological consider­ations. Uncertainties stem from the historic flexibility (e.g., use of a broad range of species) and constraints of the basic test design (e.g., no replication). Other sources of uncertainty arise from environmental safety extrapolation based on TG 203 data. Environmental extrapolation models, combined with data from alternative methods, including mechanistic indicators of toxicity, may provide at least the same level of environmental protection. Yet, most importantly, the 3R advan­tages of alternative methods allow a better standardization, characterization, and an improved basic study design. This can enhance data reliability and thus facilitate the comparison of chemical toxicity, as well as the environmental classifi­cations and prediction of no-effect concentrations of chemicals. Combined with the 3R gains and the potential for higher throughput, a reliable assessment of more chemicals can be achieved, leading to improved environmental protection.

Interlaboratory Validation of Toxicity Testing Using the Duckweed Lemna minor Root-Regrowth Test.

The common duckweed (Lemna minor), a freshwater monocot that floats on the surfaces of slow-moving streams and ponds, is commonly used in toxicity testing. The novel Lemna root- regrowth test is a toxicity test performed in replicate test vessels (24-well plates), each containing 3 mL test solution and a 2-3 frond colony. Prior to exposure, roots are excised from the plant, and newly developed roots are measured after 3 days of regrowth. Compared to the three internationally standardized methods, this bioassay is faster (72 h), simpler, more convenient (requiring only a 3-mL) and cheaper. The sensitivity of root regrowth to 3,5-dichlorophenol was statistically the same as using the conventional ISO test method. The results of interlaboratory comparison tests conducted by 10 international institutes showed 21.3% repeatability and 27.2% reproducibility for CuSO4 and 21.28% repeatability and 18.6% reproducibility for wastewater. These validity criteria are well within the generally accepted levels of <30% to 40%, confirming that this test method is acceptable as a standardized biological test and can be used as a regulatory tool. The Lemna root regrowth test complements the lengthier conventional protocols and is suitable for rapid screening of wastewater and priority substances spikes in natural waters.

Evaluation of a Bayesian Network for Strengthening the Weight of Evidence to Predict Acute Fish Toxicity from Fish Embryo Toxicity Data.

The use of fish embryo toxicity (FET) data for hazard assessments of chemicals, in place of acute fish toxicity (AFT) data, has long been the goal for many environmental scientists. The FET test was first proposed as a replacement to the standardized AFT test nearly 15 y ago, but as of now, it has still not been accepted as a standalone replacement by regulatory authorities such as the European Chemicals Agency (ECHA). However, the ECHA has indicated that FET data can be used in a weight of evidence (WoE) approach, if enough information is available to support the conclusions related to the hazard assessment. To determine how such a WoE approach could be applied in practice has been challenging. To provide a conclusive WoE for FET data, we have developed a Bayesian network (BN) to incorporate multiple lines of evidence to predict AFT. There are 4 different lines of evidence in this BN model: 1) physicochemical properties, 2) AFT data from chemicals in a similar class or category, 3) ecotoxicity data from other trophic levels of organisms (e.g., daphnids and algae), and 4) measured FET data. The BN model was constructed from data obtained from a curated database and conditional probabilities assigned for the outcomes of each line of evidence. To evaluate the model, 20 data-rich chemicals, containing a minimum of 3 AFT and FET test data points, were selected to ensure a suitable comparison could be performed. The results of the AFT predictions indicated that the BN model could accurately predict the toxicity interval for 80% of the chemicals evaluated. For the remaining chemicals (20%), either daphnids or algae were the most sensitive test species, and for those chemicals, the daphnid or algal hazard data would have driven the environmental classification. Integr Environ Assess Manag 2020;16:452-460. © 2020 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

An ecotoxicological assessment of mine tailings from three Norwegian mines.

The study assessed the environmental toxicity of three Norwegian mine tailings from Omya Hustadmarmor, Sydvaranger, and Sibelco, which are all released into a seawater recipient. Ecotoxicity assessments were performed on the overlying water extracted from the mine tailings, the transformation/dissolution waters obtained from the mine tailings, and whole sediment assessment using a suite of marine organisms including algae, Crustacea, and Mollusca. Overall, based on the toxicity evaluation of the transformation/dissolution data, Sibelco tailings resulted in the highest toxicity albeit at relatively high concentrations, followed by Sydvaranger and Hustadmarmor. Sibelco was the only mine where process chemicals were not used. In contrast, the Corophium sediment contact assay revealed a significantly higher toxicity exerted by Hustadmarmor tailings, which may indicate a physical impact of the fine tailings. The effects observed were discussed with respect to both the measured chemical concentrations of the tailings and the potential physical impact of the tailing particles on organism health.

Performance of Three-Dimensional Rainbow Trout (Oncorhynchus mykiss) Hepatocyte Spheroids for Evaluating Biotransformation of Pyrene.

The aquatic bioconcentration of a chemical is typically determined using conventional fish tests. To foster the approach of alternatives to animal testing, a combination of computational models and in vitro substrate depletion bioassays (e.g., primary hepatocytes) can be used. One recently developed in vitro assay is the three-dimensional (3D) hepatic spheroid model from rainbow trout (Oncorhynchus mykiss). The aim of the present study was to evaluate the metabolic competence of the 3D spheroids from rainbow trout when exposed to pyrene, using 2 different sampling procedures (SP1 and SP2). The results were compared with previously published intrinsic clearance (CL) results from S9 fractions and primary hepatocyte assays. Extraction of pyrene using SP1 suggested that the spheroids had depleted 33% of the pyrene within 4 h of exposure, reducing to 91% after 30 h. However, when applying SP2 a substantial amount (36%) of the pyrene was bound to the exposure vial within 2 h, decreasing after 6 h of exposure. Formation of hydroxypyrene-glucuronide (OH-PYR-Glu) was obtained throughout the study, displaying the metabolic competence of the 3D spheroids. The 2 sampling procedures yielded different CLin vitro , where pyrene depletion using SP2 was very similar to published studies using primary hepatocytes. The 3D spheroids demonstrated reproducibile, log-linear biotransformation of pyrene and displayed formation of OH-PYR-Glu, indicating their metabolic competence for 30 h or more. Environ Toxicol Chem 2019;38:1738-1747. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Characterization of multiple biomarker responses using flow cytometry to improve environmental hazard assessment with the green microalgae Raphidocelis subcapitata.

Microalgal toxicity tests using integrative endpoints as algal growth are regularly required to analyse the toxicity of potentially hazardous substances in the aquatic environment. However, these do not provide mechanistic information on the toxic mode of action by which contaminants may affect algae. Bottled waters can be used as a substitute for culturing media and should not impose any stress to the cultured organisms. However, certain chemical components can interfere with specific cell targets which are not revealed by general toxicity assays. The present study investigated the sensitivity of flow cytometry (FCM) to analyse sub-lethal effects of different bottled waters to the freshwater microalgae Raphidocelis subcapitata. Several endpoints were analysed including growth rate, natural pigments content, cell size, complexity, viability and cycle, Reactive Oxygen Species (ROS) formation, mitochondrial membrane potential and Lipid Peroxidation (LPO). Additionally, photosystem II (PSII) performance was analysed by PAM fluorometry, to provide further information on the absorption, distribution and use of energy in photosynthesis. Results indicated that the most sensitive endpoints were the oxidative stress related endpoints ROS formation and LPO, pigment content, morphological endpoints as cell size, complexity and cycle, with growth rate being one of the least sensitive. Although being essential macronutrients for algal growth, the chemical elements Ca, Na, Mg, and NH4 were identified as being primarily responsible for the observed toxicological effects to exposed algae. The applied methodology proved to be of high throughput, simultaneously assembling information on morphological, biochemical, and physiological status of algal cells. FCM also showed potential to reveal mechanistic information on the toxic mode of action of the bottled waters before any effects on algal growth was observed. The used approach demonstrated its potential for being integrated into future microalgal toxicity bioassays for testing chemicals to improve the hazard information obtained from currently approved internationally accepted test guidelines.

Repeatability and Reproducibility of the RTgill-W1 Cell Line Assay for Predicting Fish Acute Toxicity.

Predicting fish acute toxicity of chemicals in vitro is an attractive alternative method to the conventional approach using juvenile and adult fish. The rainbow trout (Oncorhynchus mykiss) cell line assay with RTgill-W1 cells has been designed for this purpose. It quantifies cell viability using fluorescent measurements for metabolic activity, cell- and lysosomal-membrane integrity on the same set of cells. Results from over 70 organic chemicals attest to the high predictive capacity of this test. We here report on the repeatability (intralaboratory variability) and reproducibility (interlaboratory variability) of the RTgill-W1 cell line assay in a round-robin study focusing on 6 test chemicals involving 6 laboratories from the industrial and academic sector. All participating laboratories were able to establish the assay according to preset quality criteria even though, apart from the lead laboratory, none had previously worked with the RTgill-W1 cell line. Concentration-response modeling, based on either nominal or geometric mean-derived measured concentrations, yielded effect concentrations (EC50) that spanned approximately 4 orders of magnitude over the chemical range, covering all fish acute toxicity categories. Coefficients of variation for intralaboratory and interlaboratory variability for the average of the 3 fluorescent cell viability measurements were 15.5% and 30.8%, respectively, which is comparable to other fish-derived, small-scale bioassays. This study therefore underlines the robustness of the RTgill-W1 cell line assay and its accurate performance when carried out by operators in different laboratory settings.

Defining the chronic impacts of atenolol on embryo-larval development and reproduction in the fathead minnow (Pimephales promelas).

Atenolol is a beta-adrenergic receptor antagonist ('beta-blocker') widely used for the treatment of angina, glaucoma, high blood pressure and other related conditions. Since atenolol is not appreciably metabolized in humans, the parent compound is the predominant excretory product, and has been detected in sewage effluent discharges and surface waters. Consequently, atenolol has been chosen as a reference pharmaceutical for a European Union-funded research consortium, known as ERAPharm (http://www.erapharm.org), which focused on the fate and effects of pharmaceuticals in the environment. Here, we present data generated within this project from studies assessing population-relevant effects in a freshwater fish species. Using fathead minnows (Pimephales promelas) as a standard OECD test species, embryo-larval development (early life stage or ELS) and short-term (21 d) adult reproduction studies were undertaken. In the ELS study, the 4d embryo NOEC(hatching) and LOEC(hatching) values were 10 and >10mg/L, respectively, and after 28 d, NOEC(growth) and LOEC(growth) values were 3.2 and 10mg/L, respectively (arithmetic mean measured atenolol concentrations were >90% of these nominal values). In the short-term reproduction study, NOEC(reproduction) and LOEC(reproduction) values were 10 and >10mg/L, respectively (mean measured concentrations were 77-96% of nominal values), while the most sensitive endpoint was an increase in male fish condition index, giving NOEC(condition index) and LOEC(condition index) values of 1.0 and 3.2mg/L, respectively. The corresponding measured plasma concentration of atenolol in these fish was 0.0518 mg/L. These data collectively suggest that atenolol has low chronic toxicity to fish under the conditions described, particularly considering the low environmental concentrations reported. These data also allowed the assessment of two theoretical approaches proposed as predictors of the environmental impact of human pharmaceuticals: the Huggett 'mammalian-fish leverage model'; and the acute:chronic ratio (ACR). The Huggett model gave a measured human: fish effect ratio (ER) of 19.3 for atenolol, which compared well with the predicted ER of 40.98. Moreover, for an ER of 19.3, the model suggests that chronic testing may be warranted, and from our resultant effects data, atenolol does not cause significant chronic effects in fathead minnow at environmentally realistic concentrations. The calculated ACR for atenolol is >31.25, which is far lower than that of 17 alpha-ethinylestradiol and other potent steroidal oestrogens, thus further supporting the observed low toxicity. The data produced for atenolol here fit well with both approaches, but also highlight the importance of generating 'real' experimental data with which to calibrate and validate such models.

Evaluation of the reproductive effects of tamoxifen citrate in partial and full life-cycle studies using fathead minnows (Pimephales promelas).

Laboratory studies were conducted to investigate potential adverse effects on development, growth, reproduction and biomarker responses (vitellogenin [VTG] and gonad histology) in fathead minnows (Pimephales promelas) exposed to tamoxifen citrate. Based on the results of a partial life cycle study (nominal [mean measured] concentrations ranged from 0.18 [0.11] to 18 [15.74] microg/L), a 284-d fish full life-cycle (FFLC) flow-through study was conducted using newly fertilized embryos (<24 h postfertilization) exposed to nominal (mean measured) concentrations of 14C-tamoxifen citrate that ranged from 0.01 (0.007) to 5.12 (4.08) microg/L. Triethylene glycol (2.0 microl/L) was used as a solvent carrier, with 17beta-estradiol (E2) as a positive control (nominal 0.1 microg/L). Among the biomarkers measured, significant effects on VTG and gonad histology were observed, although these results required care in their interpretation. Among important population-relevant endpoints, no effects on reproduction were observed at nominal concentrations < or = 5.12 microg/L. Effects on growth (length and weight) were observed in some treatments; however, some of these showed irregular concentration-response relationships, which made interpretation uncertain, or were deemed transient in nature (e.g., reduction in growth of F1 28-d posthatch larval fish at nominal concentrations of 0.08, 0.64, and 5.12 microg/L) and judged not to be biologically significant. Interpretation of results from fish chronic studies is challenging and frequently calls for scientific judgement about statistical and biological significance and what constitutes an adverse effect. Using the principles used in mammalian toxicology studies, data from partial and FFLC studies were evaluated from both statistical and biological perspectives in order to determine no-observed-adverse effect concentrations (expressed as (adverse)NOEC) for use in environmental risk assessment. Careful consideration of both biological and statistical outcomes from these studies suggested overall (adverse)NOEC concentration and lowest-observed-effect concentration ((adverse)LOEC) values for tamoxifen citrate of 5.12 microg/L and 5.6 microg/L, respectively.