Evaluation of precopulatory pairing behaviour and male fertility in a marine amphipod exposed to plastic additives.

Plastics contain a mixture of chemical additives that can leach into the environment and potentially cause harmful effects on reproduction and the endocrine system. Two of these chemicals, N-butyl benzenesulfonamide (NBBS) and triphenyl phosphate (TPHP), are among the top 30 organic chemicals detected in surface and groundwater and are currently placed on international watchlist for evaluation. Although bans have been placed on legacy pollutants such as diethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP), their persistence remains a concern. This study aimed to examine the impact of plastic additives, including NBBS, TPHP, DBP, and DEHP, on the reproductive behaviour and male fertility of the marine amphipod Echinogammarus marinus. Twenty precopulatory pairs of E. marinus were exposed to varying concentrations of the four test chemicals to assess their pairing behaviour. A high-throughput methodology was developed and optimised to record the contact time and re-pair time within 15 min and additional point observations for 96 h. The study found that low levels of NBBS, TPHP, and DEHP prolonged the contact and re-pairing time of amphipods and the proportion of pairs reduced drastically with re-pairing success ranging from 75% to 100% in the control group and 0%-85% in the exposed groups at 96 h. Sperm count declined by 40% and 60% in the 50 µg/l and 500 µg/l DBP groups, respectively, whereas TPHP resulted in significantly lower sperms in 50 µg/l exposed group. Animals exposed to NBBS and DEHP showed high interindividual variability in all exposed groups. Overall, this study provides evidence that plastic additives can disrupt the reproductive mechanisms and sperm counts of amphipods at environmentally relevant concentrations. Our research also demonstrated the usefulness of the precopulatory pairing mechanism as a sensitive endpoint in ecotoxicity assessments to proactively mitigate population-level effects in the aquatic environment.

The natural anthraquinone dye emodin: Eco/genotoxicological characterization for aquatic organisms.

Emodin is an anthraquinone secondary metabolite produced by several species of plants and fungi. Emodin is known for its pharmacological versatility, and, in the textile industry, for its good dyeing properties. However, its use in the textile industry can result in the formation and disposal of large volumes of wastewater. Emodin mutagenicity has been shown in bacteria and in human cells, but little is known about its possible toxic, genotoxic, or mutagenic effects in aquatic organisms. We have evaluated the eco/genotoxicity of emodin to aquatic organisms. Emodin was toxic to Daphnia similis (EC50 = 130 µg L-1) and zebrafish embryos (LC50 = 25 µg L-1). No toxicity was observed for Raphidocelis subcapitata, Ceriodaphnia dubia, or Parhyale hawaiensis. Additional biochemistry/molecular studies are needed to elucidate the toxic/mutagenic pathways of emodin in aquatic organisms. The PNEC value for emodin was 0.025 µg L-1. In addition to mutagenicity in the Salmonella/microsome assay, emodin was mutagenic in the micronucleus assay in the amphipod P. hawaiensis. Among the anthraquinone dyes tested to date, natural or synthetic, emodin was the most toxic to aquatic species.

The effects of plastic additives on swimming activity and startle response in marine amphipod Echinogammarus marinus.

Plastic additives are widely used in plastic production and are found in the environment owing to their widespread applications. Among these additives, N-butyl benzenesulfonamide (NBBS) and triphenyl phosphate (TPHP) are under international watchlist for evaluation, with limited studies on amphipods. Di-ethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP) are banned in some countries and categorised as substances of very high concern. This study aimed to investigate the effects of NBBS, TPHP, DEHP and DBP on the swimming activity of a coastal intertidal marine amphipod, Echinogammarus marinus. Furthermore, this study is the first to quantify startle response in E. marinus in response to light stimuli. Amphipods were exposed to 0, 0.5, 5, 50 and 500 µg/l concentrations of all test compounds. Swimming activity and startle responses were assessed by video tracking and analysis using an 8-min alternating dark and light protocol after exposure on days 7 and 14. We observed an overall compound and light effect on the swimming activity of E. marinus. A significant decrease in swimming distance was found in 500 µg/l NBBS and TPHP. We observed that the startle response in E. marinus had a latency period of >2 s and animals were assessed at 1 s and the sum of the first 5 s. There was a clear startle response in E. marinus during dark to light transition, evident with increased swimming distance. NBBS exposure significantly increased startle response at environmental concentrations, while significant effects were only seen in 500 µg/l TPHP at 5 s. We found no significant effects of DEHP and DBP on swimming behaviour at the concentrations assessed. The findings of this study affirm the necessity for a continuous review of plastic additives to combat adverse behavioural effects that may be transferable to the population levels.

Toxicity and mutagenicity studies of 6PPD-quinone in a marine invertebrate species and bacteria.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-quinone), an oxidation product of the tire additive, 6PPD, has been associated with high mortality of salmonids (0.1 µg/L). The objective of this study was to determine the acute toxicity using neonates and mutagenicity (micronuclei in hemolymph of exposed adults) of 6PPD-quinone in the marine amphipod Parhyale hawaiensis. Also, we studied its mutagenicity in the Salmonella/microsome assay using five strains of Salmonella with and without metabolic system (rat liver S9, 5%). 6PPD-quinone did not present acute toxicity to P. hawaiensis from 31.25 to 500 µg/L. Micronuclei frequency increased after 96 h-exposure to 6PPD-quinone (250 and 500 µg/L) when compared to the negative control. 6PPD-quinone also showed a weak mutagenic effect for TA100 only in the presence of S9. We conclude that 6PPD-quinone is mutagenic to P. hawaiensis and weakly mutagenic to bacteria. Our work provides information for future risk assessment of the presence of 6PPD-quinone in the aquatic environment.

Genotoxic effects of silver nanoparticles on a tropical marine amphipod via feeding exposure.

Silver nanoparticles (AgNP) are widely used in several applications including as antifouling agents; therefore, they can end up in estuarine and marine environments. These nanoparticles tend to aggregate and to deposit in the sediment, where many organisms feed and reproduce. Parhyale hawaiensis is an epibenthic amphipod globally distributed in tropical zones, and has been considered a potential model for ecotoxicology. The aim of this study was to evaluate genotoxic effects of AgNP and Ag salt via feeding, as P. hawaiensis lives in the sediment where nanoparticles tend to accumulate. Organisms were cultivated in the laboratory, and adults were exposed to food containing both AgNP and Ag salt. We collected hemolymph after different times of exposure, and analysed the hemocytes for nuclear abnormalities (including micronuclei) and DNA damage using the standard alkaline comet assay. Conditions of both assays were developed/optimized to allow their successful application in marine invertebrates. Increased frequencies of micronuclei, nuclear buds and total abnormalities were detected in relation to concentration and time in organisms exposed to AgNP and Ag salt in comparison to the controls. No DNA damage was detected when the alkaline comet assay was applied. After 5 days of exposure, we observed higher micronuclei frequencies in Ag salt treatment in comparison with AgNP. After 13 days, micronuclei frequencies were similar for both silver forms. We believe that the Ag, in its ion form, is causing the mutagenic effect; therefore, more time would be needed for the release of the ion from AgNP, explaining the delayed mutagenic effect.

The amphipod Parhyale hawaiensis as a promising model in ecotoxicology.

Near-shore marine/estuarine environments play an important role in the functioning of the marine ecosystem and are extremely vulnerable to the presence of chemical pollution. The ability to investigate the effects of pollution is limited by a lack of model organisms for which sufficient ecotoxicological information is available, and this is particularly true for tropical regions. The circumtropical marine amphipod Parhyale hawaiensis has become an important model organism in various disciplines, and here we summarize the scientific literature regarding the emergence of this model within ecotoxicology. P. hawaiensis is easily cultured in the laboratory and standardized ecotoxicity protocols have been developed and refined (e.g., miniaturized), and effects of toxicants on acute toxicity (Cd, Cu, Zn, Ag, ammonia, dyes, pesticides, environmental samples), genotoxicity as comet assay/micronuclei, and gene expression (Ag ion and Ag nanoparticles) and regeneration (pesticides) have been published. Methods for determination of internal concentrations of metals (Cu and Ag) and organic substances (synthetic dye) in hemolymph were successfully developed providing sources for the establishment of toxicokinetics models in aquatic amphipods. Protocols to evaluate reproduction and growth, for testing immune responses and DNA damage in germ cells are under way. The sensitivity of P. hawaiensis, measured as 50% lethal concentration (LC50), is in the same range as other amphipods. The combination of feasibility to culture P. hawaiensis in laboratory, the recent protocols for ecotoxicity evaluation and the rapidly expanding knowledge on its biology make it especially attractive as a model organism and promising tool for risk assessment evaluations in tropical environments.

Unusual male size vs sperm count relationships in a coastal marine amphipod indicate reproductive impairment by unknown toxicants.

Sperm quantity/quality are significant reproductive endpoints with clear links to population level dynamics. Amphipods are important model organisms in environmental toxicology. Despite this, field monitoring of male fertility in invertebrates has rarely been used in monitoring programs. The aim of this study was to compare sperm quality/quantity in an amphipod collected at six UK locations with differing water quality. Due to low sperm counts and an observed lack of relationship between sperm count and weight in amphipods collected from a nationally protected conservation area (Langstone Harbour, England), we also compared datasets from this site over a decade to determine the temporal significance of this finding. One collection to evaluate a female reproductive endpoint was also performed at this site. Interestingly, this harbour consistently presented some of the lowest sperm counts comparable to highly industrial sites and low eggs number from females. Amphipods collected from all the sites, except from Langstone Harbour, presented strong positive correlations between sperm count and weight. Given Langstone Harbour has several international and national protected statutes primarily for marine life and birds, our results indicate that E. marinus, one important food component for wading birds, might be impacted by unknown reproductive stressors. These unknown stressors maybe related to agricultural runoff, leachate from historical landfills and effluent from storm water overflows. This study highlights the importance of exploring new reproductive endpoints such as sperm quantity/quality in marine monitoring programs.