Ecotoxicological assessment of galaxolide and tonalide as contaminants of emerging concern in marine ecosystems

The polycyclic musk compounds Galaxolide (HHCB) and Tonalide (AHTN) are the most frequently consumed fragrance materials applied in many household and personal care products. These substances have been detected in all environmental compartments and, due to their inherent lipophilicity, they easily bioaccumulate in aquatic organism. The aim of this PhD thesis was to assess the risk of environmental concentrations of HHCB and AHTN in the marine environments. In order to provide a more complete assessment on marine biota, empirical approach based on data of ecotoxicological assays was explored based on environmental risk assessment (ERA) scheme. In this case, it was intended to employ organisms from different trophic levels like microalgae, echinoderms, bivalves, shrimps, and fish. This diversification in the species allows assessing how organisms with different biological complexity can be affected. In addition, different endpoints were used for each species, taking as basis the sensitivity and relevance of the endpoints. Another important aspect considered to select the endpoints was their level of biological organization, so that the responses measured were based on acute and chronic exposure, focusing on growth, survival, development, behaviour, and biomarkers at the biochemical and molecular levels. The results obtained in this thesis show that environmental concentrations of both compounds are detrimental to microalgae growth posing significant risk to Phaeodactylum tricornutum and Isochrysis galbana. It was also observed that environmental concentrations of HHCB and AHTN significantly altered the early life stages endpoints such as fertilization, larval development, and survival in tested species. Furthermore, this thesis has proven that in a scenario of heterogenous pollution where lethality is not expected to occur, HHCB and AHTN may trigger spatial avoidance, which might reduce the local biodiversity of ecosystems due to emigration to safer environments. Sublethal effects assessed in bivalves after chronic exposure to HHCB and AHTN showed that these substances are bioavailable to marine organisms and they have the potential to induce oxidative stress, genotoxicity, neurotoxicity and alter the health status of marine organisms. It was also observed that both compounds modulated endocrine disruption biomarkers in small fish. Despite the significant alterations in endocrine disruption biomarkers induced by both compounds, AHTN appeared to be a more potent inhibitor of endocrine activity in the marine environments. This PhD Thesis has demonstrated the adverse effects of environmental concentrations of HHCB and AHTN in marine ecosystems. Therefore, the data presented in this study should be integrated with other available data required for policy actions that will aid the conservation and management of the sea

Effects and Risk Assessment of the Polycyclic Musk Compounds Galaxolide (R) and Tonalide (R) on Marine Microalgae, Invertebrates, and Fish

The current research investigated the potential environmental risk of the polycyclic musk compounds, Galaxolide (R) (HHCB) and Tonalide (R) (AHTN), in the marine environments. These substances are lipophilic, bioaccumulated, and potentially biomagnified in aquatic organisms. To understand the toxicity of HHCB and AHTN, acute toxicity tests were performed by exposing marine microalgae (Phaeodactylum tricornutum, Tretraselmis chuii, and Isochrysis galbana), crustaceans (Artemia franciscana), echinoderms (Paracentrotus lividus), bivalves (Mytilus galloprovincialis), fish (Sparus aurata), and a candidate freshwater microalga (Raphidocelis subcapitata) to environmentally relevant concentrations (0.005-5 mu g/L) following standardized protocols (US EPA, Environment Canada and OECD). P. tricornutum and I. galbana were sensitive to both substances and for P. tricornutum exposed to HHCB and AHTN, the IC10 values (the inhibition concentration at which 10% microalgae growth inhibition was observed) were 0.127 and 0.002 mu g/L, respectively, while IC10 values calculated for I. galbana were 5.22 mu g/L (a little higher than the highest concentration) and 0.328 mu g/L, for HHCB and AHTN, respectively. Significant (p < 0.01) concentration dependent responses were measured in P. lividus and M. galloprovincialis larvae developments, as well as S. aurata mortality tested with HHCB. The effect of HHCB on P. lividus larvae development was the most sensitive endpoint recorded, producing an EC50 value (the effect concentration at which 50% effect was observed) of 4.063 mu g/L. Considering the risk quotients both substances seem to represent high environmental risk to P. tricornutum and M. galloprovincialis in marine environments

Evaluación ecotoxicológica de la galaxolida y la tonalida como contaminantes de interés emergente en ecosistemas marinos

Tesis doctoral presentada para optar al grado de Doctor por la Universidad de Cádiz, Programa de Doctorado en Gestión y Conservación del Mar.--The polycyclic musk compounds Galaxolide (HHCB) and Tonalide (AHTN) are the most frequently consumed fragrance materials applied in many household and personal care products. These substances have been detected in all environmental compartments and, due to their inherent lipophilicity, they easily bioaccumulate in aquatic organism. The aim of this PhD thesis was to assess the risk of environmental concentrations of HHCB and AHTN in the marine environments. In order to provide a more complete assessment on marine biota, empirical approach based on data of ecotoxicological assays was explored based on environmental risk assessment (ERA) scheme. In this case, it was intended to employ organisms from different trophic levels like microalgae, echinoderms, bivalves, shrimps, and fish. This diversification in the species allows assessing how organisms with different biological complexity can be affected. In addition, different endpoints were used for each species, taking as basis the sensitivity and relevance of the endpoints. Another important aspect considered to select the endpoints was their level of biological organization, so that the responses measured were based on acute and chronic exposure, focusing on growth, survival, development, behaviour, and biomarkers at the biochemical and molecular levels. The results obtained in this thesis show that environmental concentrations of both compounds are detrimental to microalgae growth posing significant risk to Phaeodactylum tricornutum and Isochrysis galbana. It was also observed that environmental concentrations of HHCB and AHTN significantly altered the early life stages endpoints such as fertilization, larval development, and survival in tested species. Furthermore, this thesis has proven that in a scenario of heterogenous pollution where lethality is not expected to occur, HHCB and AHTN may trigger spatial avoidance, which might reduce the local biodiversity of ecosystems due to emigration to safer environments. Sublethal effects assessed in bivalves after chronic exposure to HHCB and AHTN showed that these substances are bioavailable to marine organisms and they have the potential to induce oxidative stress, genotoxicity, neurotoxicity and alter the health status of marine organisms. It was also observed that both compounds modulated endocrine disruption biomarkers in small fish. Despite the significant alterations in endocrine disruption biomarkers induced by both compounds, AHTN appeared to be a more potent inhibitor of endocrine activity in the marine environments. This PhD Thesis has demonstrated the adverse effects of environmental concentrations of HHCB and AHTN in marine ecosystems. Therefore, the data presented in this study should be integrated with other available data required for policy actions that will aid the conservation and management of the sea

Effects and Risk Assessment of the Polycyclic Musk Compounds Galaxolide® and Tonalide® on Marine Microalgae, Invertebrates, and Fish

The current research investigated the potential environmental risk of the polycyclic musk compounds, Galaxolide® (HHCB) and Tonalide® (AHTN), in the marine environments. These substances are lipophilic, bioaccumulated, and potentially biomagnified in aquatic organisms. To understand the toxicity of HHCB and AHTN, acute toxicity tests were performed by exposing marine microalgae (Phaeodactylum tricornutum, Tretraselmis chuii, and Isochrysis galbana), crustaceans (Artemia franciscana), echinoderms (Paracentrotus lividus), bivalves (Mytilus galloprovincialis), fish (Sparus aurata), and a candidate freshwater microalga (Raphidocelis subcapitata) to environmentally relevant concentrations (0.005–5 µg/L) following standardized protocols (US EPA, Environment Canada and OECD). P. tricornutum and I. galbana were sensitive to both substances and for P. tricornutum exposed to HHCB and AHTN, the IC10 values (the inhibition concentration at which 10% microalgae growth inhibition was observed) were 0.127 and 0.002 µg/L, respectively, while IC10 values calculated for I. galbana were 5.22 µg/L (a little higher than the highest concentration) and 0.328 µg/L, for HHCB and AHTN, respectively. Significant (p &lt; 0.01) concentration dependent responses were measured in P. lividus and M. galloprovincialis larvae developments, as well as S. aurata mortality tested with HHCB. The effect of HHCB on P. lividus larvae development was the most sensitive endpoint recorded, producing an EC50 value (the effect concentration at which 50% effect was observed) of 4.063 µg/L. Considering the risk quotients both substances seem to represent high environmental risk to P. tricornutum and M. galloprovincialis in marine environments

Galaxolide and Tonalide Modulate Neuroendocrine Activity In Marine Species From Two Taxonomic Groups

Galaxolide (HHCB) and tonalide (AHTN) are polycyclic musk compounds (PMCs) used in household and personal care products that have been included on the list as emerging contaminants of environmental concern due to their ubiquity in aquatic and terrestrial environments. There still exists a dearth of information on the neurotoxicity and endocrine disrupting effects of these contaminants, especially for marine and estuarine species. Here, we assessed the neuroendocrine effects of HHCB and AHTN using adult clams, Ruditapes philippinarum, and yolk-sac larvae of sheepshead minnow, Cyprinodon variegatus. The clams were treated with concentrations (0.005–50 μg/L) of each compound for 21 days. Meanwhile, sheepshead minnow larvae were exposed to 0.5, 5 and 50 μg/L of HHCB and AHTN for 3 days. Enzyme activities related to neurotoxicity (acetylcholinesterase - AChE), neuroendocrine function (cyclooxygenase - COX), and energy reserves (total lipids - TL) were assessed in R. philippinarum. Gene expression levels of cyp19 and vtg1 were measured in C. variegatus using qPCR. Our results indicated induction of AChE and COX in the clams exposed to HHCB while AHTN exposure significantly inhibited AChE and COX. Gene expression of cyp19 and vtg1 in yolk-sac C. variegatus larvae exposed to 50 μg/L AHTN was significantly downregulated versus the control. The results of this study demonstrate that HHCB and AHTN might pose neurotoxic and endocrine disrupting effects in coastal ecosystems

Avoidance behaviour of the shrimp Palaemon varians regarding a contaminant gradient of galaxolide and tonalide in seawater

The musk fragrances galaxolide (HHCB) and tonalide (AHTN) are compounds of emerging concern that have been found in various environmental compartments. The present study addressed the ability of HHCB and AHTN to elicit the avoidance response in the estuarine shrimp Palaemon varians and to predict the population immediate decline (PID) of P. varians when exposed to HHCB and AHTN by integrating both avoidance (non-forced exposure) and lethality (forced exposure) responses. The avoidance response was tested in a non-forced multi-compartmented static system, in which the shrimps could move freely among the compartments with different concentrations. The shrimps (n = 3 shrimps per compartment/concentration; 18 shrimps per system) were exposed to a gradient (0, 0.005, 0.05, 0.5, 5 and 50 μg/L) of both substances and their positions were checked at every 20 min for a 3 h period. The results from 24-h forced exposure showed no dose-response relationship and the highest percentage mortality was 17% for HHCB at 0.005 and 0.5 μg/L. In the 3-h non-forced exposure to a gradient of HHCB and AHTN, significant concentration-dependent spatial avoidance was observed for both substances. The shrimps avoided the lowest concentration of HHCB and AHTN (0.005 μg/L) by 15% and 16%. The avoidance increased significantly (p < 0.005) to a 61% and 57%, respectively, for the highest concentration (50 μg/L). The population immediate decline was driven by the avoidance behaviour of the shrimps rather than mortality. These results indicated that the aversiveness of HHCB and AHTN might have serious consequences for habitat selection processes by organisms.This research was supported by the Spanish Ministry of Economy and Competitiveness (Ref. CTM2012-37591), FEDER Funds from European Union and also partially funded by the Spanish Ministry of Science, Innovation and Universities (MICINN) through the Explora project (#CGL2017-92160-EXP). C.V.M. Araújo is grateful to the MICINN for the Juan de la Cierva (IJCI-2014-19318) and Ramón y Cajal (RYC-2017-22324) contracts