Microorganisms are ubiquitous and present in animal microbiomes, particulates, and colonizable surfaces of test systems. From an ecotoxicological perspective, they are metabolically active biological compartments that respond to test conditions, including test substances. In exposure experiments, microorganisms can both alleviate toxicity via, for example, biotransformation, and reinforce the adverse effects via, for example, disrupted microbiome-host interactions. Acknowledging these interactions is essential for a mechanistic understanding of results in effect studies and developing assays towards more ecologically relevant hazard assessment. Therefore, there is increasing attention toward “microbiome aware ecotoxicology” in recent years, focusing mostly on test organism microbiomes. 

I studied how microorganisms present in systems designed for acute and chronic toxicity assays with Daphnia magna affect the test outcome. The experimental studies showed that bacteria introduced in the system intentionally (as a part of the experimental design; Papers I, II, and III) or unintentionally (with the microbiome of the test animals; Paper IV) responded to the test substances and mediated the exposure for the target species. In these studies, we employed the emerging contaminants ciprofloxacin (an antibiotic drug; Paper I) and various fossil-based polymers (microplastic; Papers II, III, and IV), representing a microbiome disrupting and a biofilm promoting type of substance respectively. 

In Paper I, we hypothesized that exposure to antibiotics would primarily target the daphnid microbiome with downstream effects on the host fitness. To test this hypothesis, we chronically exposed daphnids to ciprofloxacin, which resulted in decreased microbiome diversity. However, contrary to our hypothesis, there were significant stimulatory effects on the host fitness and antioxidant production due to the direct pro-oxidative ciprofloxacin effects on the host. Although the microbiome was not directly involved in the growth-related responses to the ciprofloxacin exposure, the microbiome’s alterations suggest that exposure to any antimicrobials, which – unlike ciprofloxacin – do not stimulate antioxidant production, would result in gut dysbiosis with possible adverse effects on the host. 

Further, we hypothesized that in assays with particulate test materials, such as microplastic, bacterial biofilms increase particle aggregation, affecting exposure levels. This hypothesis was tested using D. magna exposed to a mixture of kaolin clay and polystyrene with and without biofilm (Paper II). We found that biofilm significantly decreased the adverse effects exerted by particulates directly, most likely, by providing nutrition for the daphnids, and indirectly, by inducing particle aggregation. In Paper III, we compared biofilm communities established on the plastic (polyethylene, polypropylene, and polystyrene) vs. non-plastic (cellulose and glass) substrates. The biofilm communities on the plastic were significantly different from those on the non-plastic materials;  hence, microplastic contribution to the suspended solids in the exposure can drive the biofilm community composition in the system. Finally, in Paper IV, we found that in a closed system designed to evaluate microplastic effects on D. magna, bacteria originated from the daphnid microbiome colonize particulates and affect their aggregation and animal survival. Together, these findings suggest that chemical exposure (Paper I), the microbiome of the test animal (Paper IV), the composition of the suspended solids (SS) (Papers II and IV), and their surface properties (Paper III) contribute to the diversity and abundance of the biofilm in the test system, which can affect the test outcome. Thus, the microbiome reacts to and interacts with contaminants within a test system, which calls for the appreciation of these interactions when interpreting the results as well as new developments toward standardization of the bacterial component in (eco)toxicity assays with eukaryotic test species.

Increasing concerns arise regarding the environmental risk of microplastics (MP) and their interactions with biota, prompting numerous effect studies. However, current ecotoxicology methods fall short when it comes to assessing MP impacts. One intriguing aspect of MP is their propensity to aggregate and interact with various substances in their environment, including bacteria. The unresolved methodological challenges in MP ecotoxicology revolve around aggregation and biofilm formation. Consequently, to establish suitable protocols for MP hazard assessment, it is essential to comprehend how particle behaviour and microbial colonisation affect the responses of test species to MP. 

This thesis aimed to improve our understanding of MP behaviour in ecotoxicological assays, focusing on the interplay between aggregation, biofilms, and effects on test organisms. First, a metaanalysis of findings from 20 published experimental studies addressing MP effects on algal growth was conducted to identify material and particle characteristics associated with growth inhibition (Paper I). The results revealed poor experimental control over particle aggregation, sedimentation, and algal biofilm formation resulting in conflicting findings across studies, even for the same polymers and particle sizes, and insufficient evidence for growth inhibition due to the MP exposure. 

These challenges were addressed by applying experimental designs with clay particles typical for natural environments, bacteria and measuring aggregation as a particle size distribution (PSD) in assays with Daphnia magna as a model test organism in incubation systems preventing sedimentation (Papers II-IV). In Paper II, natural biofilms were grown on MP and clay particles that were used in the experiments at high suspended solid concentrations (10-1000 mg/L) and daphnid mortality as the endpoint. We found that MP directly stimulated aggregation in the particle mixture and indirectly daphnid survival because larger aggregates were associated with lower mortality. Moreover, biofilm had positive effects on aggregate size and daphnid survival. 

In Paper III, environmentally relevant levels of suspended solids (0.1-10 mg/L) and MP (0-10%) were used for aggregate size and diversity analysis by structural equation modeling addressing the effects of dissolved organic matter (DOM) and test animals (D. magna) on the particle aggregation. The main aggregation drivers were DOM and filtration by the daphnids, although the total concentration of suspended solids and MP contribution also promoted it. Moreover, the daphnids introduced microorganisms to the exposure system, resulting in their propagation and biofilm formation on the test particles, which affected the daphnid response to the exposure (Paper IV). The bacterial diversity was mostly affected by the aggregate size and diversity, although the total suspended solids and DOM also contributed, promoting mainly the non-adhering cells, whereas particle-associated biofilms were affected by MP. The adverse effects on Daphnia were induced by small aggregate size, high DOM and biofilm diversity, with no direct MP effects.

These findings demonstrate that test organisms, DOM, mineral particles, and MP can affect aggregation and bacterial diversity in the exposure experiments over a few days resulting in non-stable exposure conditions. Moreover, the aggregate diversity and biofilm composition can be the proximate drivers of the test organism responses, with the indirect and often minor role of MP. Assessing PSD and biofilms in ecotoxicity testing of MP and, most likely, any solid waste particles, such as black carbon, will facilitate interpreting the results and developing assays towards ecologically relevant hazard assessment. 

Ökande oro väcks över miljöriskerna med mikroplaster (MP) och deras interaktioner med biota, vilket har utlöst en rad effektstudier. Nuvarande metodologi inom partikelektoxikologi är emellertid inte rustad att utvärdera MP-effekter. En intressant aspekt av mikroplaster är deras tendens att aggregera och interagera med olika ämnen i sin omgivande miljö, inklusive bakterier. De ännu olösta metodologiska utmaningarna inom MP-ektoxikologi är aggregering och biofilmbildning. För att utveckla adekvata protokoll för farobedömning behöver vi därför förstå hur partikelbeteende och mikrobiell kolonisering påverkar testarternas respons på MP.

Denna avhandling syftade till att förbättra vår förståelse för MP-beteende i ekotoxikologiska tester, med fokus på samspelet mellan aggregering, biofilmer och effekter på testorganismer. Först genomfördes en metaanalys och syntes av resultat från 20 publicerade studier om MP-effekter på alg-tillväxt för att identifiera material- och partikelegenskaper som är förknippade med tillväxthämning (Paper I). Resultaten visade bristande experimentell kontroll över partikelaggregering, sedimentation och bildning av alg-biofilm, vilket resulterade i motsägelsefulla resultat mellan studierna, även för samma polymerer och partikelstorlekar, samt otillräckligt stöd för tillväxthämning på grund av MP-exponeringen.

Dessa utmaningar hanterades genom att tillämpa experimentell design med lerpartiklar som är typiska för naturliga miljöer, bakterier och mätning av aggregering som partikelstorleksfördelning (PSD) i tester med Daphnia magna som modellorganism i inkubationssystem som förhindrar sedimentation (Paper II-IV). I Artikel II odlades naturliga biofilmer på MP- och lerpartiklar som användes i experimenten vid höga koncentrationer av suspenderat material (10-1000 mg/L) och dödlighet hos Daphnier som slutpunkt. Vi fann att MP direkt stimulerade aggregering i partikelblandningen och indirekt påverkade överlevnaden hos Daphnier eftersom större aggregat var förknippade med lägre dödlighet. Dessutom hade biofilmen positiva effekter på aggregatstorlek och Daphniernas överlevnad. I Paper III användes miljömässigt relevanta nivåer av suspenderat material (0,1-10 mg/L) och MP (0-10%) för en detaljerad analys av aggregatstorlek och mångfald genom PLS-SEM-modellering; effekterna av löst organiskt material (DOM) och testdjur (D. magna) på partikelaggregeringen behandlades också. Huvuddrivkrafterna för aggregering var DOM och filtrering av Daphnier, även om MP och den totala koncentrationen av suspenderat material också främjade aggregatstorlek och mångfald. Dessutom införde Daphnierna mikroorganismer i exponeringssystemet, vilket resulterade i biofilmbildning på testpartiklarna, vilket påverkade Daphniernas respons på exponeringen (Paper IV). Bakteriediversiteten påverkades mest av aggregatstorlek och topologi, även om det totala suspenderade materialet och DOM också bidrog, främst genom att öka antalet icke-häftande celler, medan partikelassocierade biofilmer påverkades av MP. De skadliga effekterna på Daphnier inducerades av små aggregatstorlekar, högt DOM och diversitet i biofilm, utan direkta MP-effekter. Dessa resultat visar att testorganismer, DOM, mineralpartiklar och MP kan påverka aggregering och bakteriediversitet i exponeringsförsöken under några dagar och resultera i instabil exponering. Dessutom kan diversitet i aggregat och biofilmsammansättning vara de direkta drivkrafterna bakom testorganismernas respons, med MP:s indirekta och oftast mindre betydande roll. Att bedöma PSD och biofilmer i ekotoxicitetstester av MP och förmodligen även av andra fasta avfallspartiklar, som sot, kommer att underlätta tolkningen av resultaten och utvecklingen av tester mot ekologiskt relevanta faro-bedömningar.