Multilevel assessment of chlorothalonil sediment toxicity to Latin American estuarine biota: Effects on biomarkers, reproduction and survival in different benthic organisms.

Chlorothalonil is an organochlorine compound that has long been used in agriculture. In recent years, this compound has been used as an antifouling booster biocide and its presence has been reported in marine coastal environments, especially in navigational areas. Although sediment can be a sink for chlorothalonil due to high affinity to fine particulate matter, toxicity studies with non-target marine and/or estuarine organisms is focused on waterborne exposure only. This study aimed to determine sediment-borne ecotoxicological effects of chlorothalonil on different benthic organisms of the Latin American biota using a integrative multilevel approach. Marine/estuarine organisms were exposed to sediments spiked with chlorothalonil (from 0 to 10.0 µg g-1) and effects at sub-individual (biochemical biomarkers in Anomalocardia flexuosa), individual (lethal effects to Tiburonella viscana and Artemia salina) and subpopulation levels (Nitokra sp. reproduction) were assessed. Increasing chlorothalonil concentrations in sediment caused increasing ecotoxicological effects in different levels of biological organisation, from biochemical to subpopulation levels. The highest exposure concentrations showed increased biomarkers of effects (lipid peroxidation and DNA damage in gills and/or digestive gland of A. flexuosa), lower fecundity and lower survival of the test organisms. GPx activity associated with LPO levels in the digestive gland suggested a response to the oxidant challenge provided by the biocide. At the lowest concentration (0.001 µg g-1), chlorothalonil detoxification mechanisms and defense against its oxidising action, involving GSH and glutathione-dependent enzymes (GST and GPx) were induced. At intermediate concentrations, there was a tendency of decreasing GSH levels, probably due to conjugation with chlorothalonil, which also affected the activities of the glutathione-dependent enzymes. At the highest tested concentration (10.0 µg g-1), chlorothalonil may have restimulated GSH synthesis in the gills of A. flexuosa, although the prooxidant activity has induced effects. This study contributes to assessing the environmental risk of chlorothalonil in sediment for non-target marine and estuarine organisms.

A preliminary study on multi-level biomarkers response of the tropical oyster Crassostrea brasiliana to exposure to the antifouling biocide DCOIT.

This study investigated the sublethal effects of environmentally relevant concentrations of DCOIT on the neotropical oyster Crassostrea brasiliana. Gills and digestive glands of animals exposed to increasing concentrations of DCOIT were analyzed for biochemical, cellular, and histopathological responses. Exposure to DCOIT (0.2 to 151 µg L-1) for 120 h triggered oxidative stress in both tissues (through the modulation of GPX, GST, GSH and GR), which led to damage of membrane lipids (increase of LPO and reduction of the NRRT). DCOIT increased histopathological pathologies in gills, such as necrosis, lymphocyte infiltration and epithelial desquamation. This study showed that short term exposure to environmental concentrations of DCOIT causes negative effects on C. brasiliana at biochemical, physiological, and histological levels. Therefore, the use of DCOIT as a booster biocide in antifouling paints should be further assessed, as it may cause environmental hazards to marine organisms.

Temporal evolution of imposex and butyltin contamination in Gemophos viverratus from São Vicente (Cabo Verde) - a countercurrent trend on the world scenario.

Organotin (OT) based Antifouling Systems (AFS) were globally banned by the AFS Convention since 2008, but the Republic of Cabo Verde did not ratify this Convention, nor did it develop a national legislation to control OT-AFS. Gemophos viverratus imposex and butyltin tissue contamination were assessed around the São Vicente Island (Cabo Verde) in 2019 and compared with the data available from 2012. The vas deferens sequence index (VDSI), the relative penis length (RPL), the percentage of females with imposex (%I) and the percentage of sterilized females (%S) in 2019 ranged from 0 to 4.0, 0-84.4%, 0-100% and 0-5.1%, respectively, whilst TBT reached maximum values of ≈30 ng TBT-Sn g-1 dw in the whelk tissues. These values are very similar to those registered in 2012, which indicates that TBT pollution has not decreased over the years, in contrast to the declining trend observed worldwide.

Long-term monitoring of Nucella lapillus imposex in Ria de Aveiro (Portugal): When will a full recovery happen?

Nucella lapillus imposex has been monitored in Ria de Aveiro (Portugal) in order to understand the evolution of organotin (OT) pollution after the EU ban on OT-based antifouling systems (OT-AFS). A rapid decrease of imposex occurred, but a countercurrent tendency upsurged recently (2018-2019) with a slight increase of imposex at some sites, instead of a continuous decline towards a complete recovery. In 2018 the vas deferens sequence index ranged between 0.6 and 1.0 across stations and the percentage of females with imposex varied from 63 to 100%. In 2019 these parameters varied between 0.4 and 1.0 and 37-100%, while the concentration of butyltins (TBT, DBT, MBT) in the dogwhelk tissues were < 1 ng Sn g-1 dw. Although N. lapillus imposex levels are low they are indicative of OT pollution. The full recovery of N. lapillus imposex in the future is uncertain and a cause for concern.

Legacy and emerging antifouling biocide residues in a tropical estuarine system (Espirito Santo state, SE, Brazil).

The contamination by antifouling biocide residues (booster biocides - diuron, Irgarol, chlorothalonil, dichlofluanid and DCOIT; butyltin compounds-BTs (TBT, DBT and MBT); and antifouling paint particles-APPs) was appraised in sediments of Vitoria Estuarine System (VES). Even at its historical lower (ΣBTs ≤113 ng Sn g-1 dry wt), the current environmental levels of BTs in areas with a predominance of boatyards still pose a risk to the local biota and human population. DCOIT, among booster biocides, was the most frequently detected, especially in boatyards (≤40 ng g-1 dry wt) and Vitoria Port (64 ng g-1 dry wt), while APPs were also detected mainly in sediments of boatyards (≤5,969 µg g-1 dry wt). Since levels of diuron and DCOIT in APPs were as high as 1,670,000 and 899,000 ng g-1 dry wt, respectively, they are acting as secondary sources of these antifouling biocides. Therefore, VES is threatened by antifouling biocide residues due to the multiple diffuse sources of contamination, showing the need for more efforts on public policies (including temporal trend monitoring studies).

Ecological risk assessment of booster biocides in sediments of the Brazilian coastal areas.

Although booster biocides (Irgarol, diuron, chlorothalonil, dichlofluanid, and DCOIT) have been detected in sediments along the Brazilian coastal areas, the risk associated to their occurrence and levels is still unknown. Thus, the ecological risk of booster biocides to sediment-dwelling organisms from the Brazilian coast was assessed using a risk characterization approach through the Risk Quotient (Measured environmental concentration (MEC)/Predicted no effect concentrations (PNECs)). Sedimentary PNECs for Irgarol, diuron, chlorothalonil and DCOIT were derived based on published ecotoxicological data from both freshwater and marine studies, while a NORMAN methodology was used to derived it for dichlofluanid. Results showed that DCOIT, diuron, Irgarol, chlorothalonil, and dichlofluanid can pose high risk on 47%, 35%, 15%, 1% and 1%, respectively, of the 113 Brazilian sites appraised. Considering the trend of expansion of navigation/maritime activities, DCOIT may worsen its impact over the coastal areas of Brazil, especially ports, but also ship/boatyards, marinas, and maritime traffic zones. The present study is an important contribution to support advance on policy formulation concerning booster biocides worldwide, particularly considering the lack of regulation on the use of antifouling biocides in Brazil.

Are antifouling residues a matter of concern in the largest South American port?

In the present study, levels of booster biocides (diuron, Irgarol, chlorothalonil, dichlofluanid and DCOIT), butyltin compounds (TBT, DBT and MBT) and antifouling paint particles (APPs) were assessed in sediments of areas under the influence of the largest Latin American port, marinas, boat traffic and ship/boat maintenance facilities located within Santos-São Vicente Estuarine System (SSES). Contamination profile was directly related to local maritime activities, where sediments from the main navigation channel (MNC) presented low levels of antifouling residues while adjacent areas (AA), characterized by the presence of boats and boatyards, showed higher contamination considering all analyzed residues. Moreover, areas under the influence of fishing boats/yards presented relevant levels of butyltins (ΣBTs > 300 ng g-1) and APPs (>100 µg g-1), while marinas dominated by recreational boats showed higher booster biocides occurrence. Sites located nearby shipyards in the MNC and boatyards in the AA presented expressive amounts of APPs (>200 µg g-1). These APPs represent an important long-term source of biocides to the SSES. Thus, the profile of maritime activities in association to local oceanographic conditions drive the spatial distribution of antifouling residues within SESS, which in some case presented levels above sediment guidelines for TBT, DCOIT and diuron.

Effects of chlorothalonil on the antioxidant defense system of mussels Perna perna.

Chlorothalonil is an effective fungicide used in agriculture and formulations of antifouling paints, which use and possible toxicity has been generating great concern. Thus, the present study investigated the effects of chlorothalonil on the antioxidant defense system (ADS) of the mussel Perna perna. The ADS was evaluated in gills and digestive gland after 24 h and 96 h of exposure to environmental relevant levels of chlorothalonil (0.1 and 10 µg/L). The activity of the enzymes superoxide dismutase (SOD), catalase (CAT), glutamate cysteine-ligase (GCL) and glutathione S-transferase (GST), levels of non-enzymatic defenses, represented by glutathione (GSH), and lipoperoxidation (LPO) and protein carbonyls (PCO) were evaluated. Results indicated that exposure to chlorothalonil is affecting the ADS in both tissues. While the activity of SOD increased and GST and GSH were not altered in gills, they decreased in digestive gland after 24 h of exposure to 10 µg/L of chlorothalonil. The contrasting results indicate that gills and digestive gland presented different patterns of responses after exposure to chlorothalonil. Moreover, a tissue-specific response to chlorothalonil was observed. Gills could be acting as the first line of defense, presenting higher enzymatic levels with minor effects on the parameters analyzed. On the other hand, digestive gland, with lower levels of antioxidant defenses, was the most affect organ by chlorothalonil. It also should be highlighted that the fungicide reduced the glutathione metabolism in the digestive gland, which can lead to an imbalance of the redox state within the cells of animals.

Antifouling paint particles: Sources, occurrence, composition and dynamics.

Sources, occurrence, composition and dynamics of antifouling paint particles (APPs) were assessed in Patos Lagoon estuary (PLE), Southern Brazil. Ten areas including boatyards, a marina and artisanal fishing harbors were identified in the estuarine system as potential sources of APPs. The APPs generated in these areas were highly heterogeneous considering the size, shape and composition. Based on an estimate of antifouling paint usage and amount of boats in each studied area, artisanal fishing harbors could be the main source of particles to PLE. However, relatively high amounts of APPs, which ranged from 130 to 40,300 µg g-1, were detected in sediments collected in front of boatyards and a marina. The uneven distribution of APPs levels among the sediment samples were probably due to the presence of diffuse sources (fishing harbors) associated to "hotspots" (boatyards and marina) along the study area. Additionally, data of settling experiment indicate that size, shape and density of APPs, combined to local hydrodynamics, appears to contribute to the mobility of these residues within the estuary. In the main channel of PLE, smaller particles tend to be transported to adjacent coastal zone while particles tend to be deposited in the sediment surface of sheltered areas. Since different trace metals, and booster biocides were detected in APPs that were not correctly disposed, these particles can be considered as an important source of contamination to aquatic environments. The present data suggest that APPs represent an environmental problem for aquatic systems in Brazil, since the country lacks legislation in addition to inefficient control mechanisms. An improvement in boat maintenance processes are urgently needed to avoid this continuous release of APPs into the aquatic systems.

Induction of oxidative stress by chlorothalonil in the estuarine polychaete Laeonereis acuta.

Chlorothalonil is an active biocide applied in antifouling paints, and also used as fungicide in agricultural activities with the purpose to protect plants from foliar and seed diseases. Thus, the aim of this study was to evaluate the effects of chlorothalonil exposure on biochemical biomarkers of oxidative metabolism as well as on cholinesterases in the estuarine polychaete Laeonereis acuta. Animals were exposed for 24 and 96 h to the following nominal concentrations of chlorothalonil: 0.1, 10.0 and 100.0 µg/L. The antioxidant capacity against peroxyl radicals (ACAP) and the activity of the enzymes catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), glutamate cysteine ligase (GCL), acetylcholinesterase (AChE) and propionylcholinesterase (PChE) were evaluated in whole-body tissue. In addition, the levels of reduced glutathione (GSH), lipid peroxidation (LPO), glycogen and lactate levels were also analyzed. A reduction in ACAP levels was observed in animals exposed to the higher chlorothalonil concentration, concomitantly with an induction of GST activity as well as diminution in GSH content in these animals. This disturbance in the redox state of animal tissues leads to an oxidative stress situation, resulting in an induction in LPO levels. It was also demonstrated that chlorothalonil exposure causes alteration in AChE activity, possibly related to damage to membrane lipids. These results demonstrated that chlorothalonil possesses harmful effects to estuarine animals and its use as antifouling biocide has to be carefully reconsidered in risk analysis studies.

Antifouling booster biocide extraction from marine sediments: a fast and simple method based on vortex-assisted matrix solid-phase extraction.

This paper reports the development of an analytical method employing vortex-assisted matrix solid-phase dispersion (MSPD) for the extraction of diuron, Irgarol 1051, TCMTB (2-thiocyanomethylthiobenzothiazole), DCOIT (4,5-dichloro-2-n-octyl-3-(2H)-isothiazolin-3-one), and dichlofluanid from sediment samples. Separation and determination were performed by liquid chromatography tandem-mass spectrometry. Important MSPD parameters, such as sample mass, mass of C18, and type and volume of extraction solvent, were investigated by response surface methodology. Quantitative recoveries were obtained with 2.0 g of sediment sample, 0.25 g of C18 as the solid support, and 10 mL of methanol as the extraction solvent. The MSPD method was suitable for the extraction and determination of antifouling biocides in sediment samples, with recoveries between 61 and 103% and a relative standard deviation lower than 19%. Limits of quantification between 0.5 and 5 ng g-1 were obtained. Vortex-assisted MPSD was shown to be fast and easy to use, with the advantages of low cost and reduced solvent consumption compared to the commonly employed techniques for the extraction of booster biocides from sediment samples. Finally, the developed method was applied to real samples. Results revealed that the developed extraction method is effective and simple, thus allowing the determination of biocides in sediment samples.

Are antifouling paint particles a continuous source of toxic chemicals to the marine environment?

Antifouling paint particles (APPs) are generated during periodical maintenance of boat hulls. Chemical composition and toxicity (either chronic or acute) of APPs found in the sediment was evaluated using the epibenthic copepod Nitokra sp. The APPs analyzed showed the presence of high levels of metals such as Cu (234,247±268µgg-1), Zn (112,404±845µgg-1) and the booster biocide DCOIT (0.13µgg-1). Even at low concentrations (as from 5mgg-1 of APPs by mass of sediment) a significantly decrease in the fecundity was observed in laboratory tests. When the sediment was disturbed in elutriate test, a LC50 of 0.14% for APPs was found. This study was the first assessment of toxicity associated with the presence of APPs in sediment to benthic organisms, and it calls attention to the need of improving regulations in boatyards and marina areas.