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Invertebrate life cycle responses to PAC exposure

Invertebrate life cycle responses to PAC exposure
Miriam León Paumen


Faculty of Science, Mathematics and Computer Science, University of Amsterdam, 1018 XE Amsterdam, THE NETHERLANDS.


Polycyclic Aromatic Compounds (PACs) have been classified as priority toxic substances by the European Commission. PACs frequently determine the need for soil and sediment remediation, since total PAC concentrations in the environment are often far above background levels and in some cases still increasing.

PACs accumulated in soils and sediments usually occur as a complex mixture, consisting of homocyclic hydrocarbons and, in smaller fractions, a myriad of heterocyclic structures. Although they are present in fairly high concentrations, heterocycles are not included in PAC risk assessment, and are rarely included in ecotoxicological test schemes. Hence, heterocycles deserve much more scientific attention than they have received so far. Azaarenes and their stable degradation products, the most ubiquitous group of heterocycles, have therefore been chosen as ‘model’ heterocyclic toxicants by our research group.

PAC environmental risk assessment is performed using a limited set of merely homocyclic structures. The occurrence of heterocyclic structures is thus neglected, while they are ubiquitous and as toxic as their homocyclic analogues. Aiming to diminish the uncertainties that complicate PAC effect prediction and hinder the reliability of PAC risk assessment, in this thesis:
  • life cycle effects of homo- and heterocyclic PACs on soil and sediment inhabiting invertebrates were determined
  • the application of Kow based prediction models to establish general patterns and exceptions in the outcome of life cycle PAC toxicity experiments was validated
  • multi-generation effects of a homocyclic PAC on a soil inhabiting invertebrate were established and compared to effects of single generation exposure and
  • the validity of the present PAC risk assessment was reviewed by examining the implications of the generated long-term toxicity data.
For the experiments described in chapter 2, eleven PACs were chosen as test compounds: six homocyclic PACs, three azaarenes and two Phase I azaarene transformation products. For the experiments performed in chapters 3 and 4, a subset of three three-ring PAC isomer pairs (two homocycles, their two azaarene analogues and the two main azaarene transformation products) was used. In the multi- generation experiment (chapter 6), the homocylic PAC phenanthrene was used as test compound. Four test organisms were chosen to perform the life cycle experiments: two soil inhabiting invertebrates, the springtail Folsomia candida and the oligochaete Enchytraeus crypticus, and two sediment inhabiting invertebrates, the oligochaete Lumbriculus variegatus and the non-biting midge Chironomus riparius.

In chapter 2, the chronic effects of the eleven PACs on the two terrestrial invertebrates were determined, using reproduction and survival as endpoints. The results demonstrated that as far as narcosis induced mortality is concerned, effects of both homocyclic and heterocyclic PACs were well described by the relationship between estimated acute pore water LC50 values and logKow. In contrast, specific effects on reproduction varied between species and between compounds as closely related as isomers, showing up as deviations from the relationship between pore water effect concentrations and logKow.

In chapter 3, the chronic effects of the six selected PACs on the emergence of the midge Chironomus riparius were evaluated. Twenty eight-day LC50 values and 50% emergence times (Emt50) were determined. Concentration response relationships were observed for phenanthrene, acridine, phenanthridine and acridone. Anthracene and phenanthridone had no effect on total emergence, but did cause a delay in emergence. As observed for the soil organisms, calculated porewater LC50 values correlated well with logKow values, suggesting narcosis as mode of action. In contrast, effect concentrations for delay in male emergence (Emt50) deviated from narcosis, suggesting a sex-related specific mode of action of PACs during long term exposure.

Chronic toxicity experiments using sediment or soil deal with the uncertainty of uncontrolled exposure, because in the spiked substrate various processes modify the availability of the test substance to the test organisms and hamper an accurate determination of exposure concentrations. The study presented in chapter 4 aimed therefore to monitor the availability of the selected PACs to the oligochaete Lumbriculus variegatus during 28 days of exposure to spiked sediments, in order to obtain reliable chronic effect concentrations for reproduction. During the sediment toxicity tests, available PAC concentrations in pore water (estimated using Solid Phase Microextraction) decreased more than total PAC concentrations in the sediment. Relating effects to PAC concentrations in pore water and in organisms showed that the two homocyclic compounds caused narcotic effects during chronic exposure, but only one of the four tested heterocyclic PACs caused narcotic effects. The transformation product phenanthridone was not toxic at the tested concentrations, whereas EC50 values for the parent compound phenanthridine and the isomer acridone were below the estimated limit for narcosis, suggesting a specific mode of action. These results demonstrated the unpredictable (isomer) specific chronic toxicity of azaarenes and their transformation products.

To elucidate consistent patterns in PAC toxicity to soil and sediment inhabiting invertebrates, in chapter 5 we examined our complete experimental dataset (chapters 2-4), consisting of twenty-one chronic effect concentrations for the four invertebrates exposed to six selected PACs. In order to determine if effect concentrations were well predicted by existing toxicity- Kow relationships describing narcosis, chronic pore water effect concentrations were plotted jointly against logKow. Fifteen of the twenty- one effect concentrations (71%) fitted well on the acute LC50-logKow relationship, showing that narcosis was the main mode of action for the majority of the tested homo- and heterocyclic PACs during chronic exposure. Toxicity of all tested compounds to soil organisms was well described by the effect- Kow relationship. However, for the sediment invertebrates exposed to some of the tested heterocyclic PACs deviations from narcosis were identified, related to specific physicochemical properties of the test compounds and/or species specific sensitivities. It was concluded therefore that existing toxicity- Kow relationships describing narcosis in some cases underestimate chronic PAC toxicity to sediment inhabiting invertebrates.

Results of life-cycle toxicity experiments are supposed to be indicative for long- term effects of exposure to toxicants. Several studies, however, have shown that adaptation or extinction of populations exposed for several generations may occur. Therefore, in chapter 6, a multi-generation experiment was performed to determine if the effects of the PAH phenanthrene on survival and reproduction of the springtail Folsomia candida exposed for ten consecutive generations would progressively increase. LC50 values for the first four generations were similar, as expected for a narcotic compound. In the fourth generation, however, springtails exposed to a concentration similar to the EC50 for one generation showed internal phenanthrene concentrations in the range known to cause mortality; no reproduction took place, and the population went extinct. From the fifth generation onwards, survival and reproduction were not affected by the remaining exposure concentrations. Apparently, up to a certain threshold concentration the springtails were able to metabolize phenanthrene, as shown by the lack of adverse effects and the lack of adaptation. During multi-generation exposure, the graded concentration-response relationship changed into an all-or nothing response with a defined threshold concentration. Together with the worsening of effects, this raises concerns about the use of single-generation studies to tackle long-term population effects of environmental toxicants.

Due to the lack of chronic toxicity data, risk assessment for homocyclic PACs can not be performed according to the best available method. Also, PAC heterocycles are neglected in actual PAC risk assessment. Therefore, in chapter 7 a risk limit derivation was performed using the life cycle toxicity data from chapters 2, 3 and 4. The derivation showed that the use of chronic effect concentrations to derive risk limits was a clear step forward. It was also shown that risk limits for the tested heterocycles were in general in the same range as risk limits for their homocyclic analogues, meaning that heterocycles could be directly incorporated to the current PAC risk assessment. However, sediment risk limits for the tested heterocycles were one order of magnitude lower than soil risk limits, showing that benthic species will be at risk more often due to PAC contamination than soil inhabiting species. In the second part of chapter 7, implications of our results for the main uncertainties regarding PAC risk assessment were discussed. It was concluded that some of these uncertainties, like PAC availability and the occurrence of PACs in mixtures, can be tackled with the tools available at the moment. In contrast, the unexpected changes occurring during multi-generation exposure showed the necessity of mechanistic research. Understanding the mechanisms underlying PAC toxicity together with the results of long term exposure experiments may diminish the uncertainties that complicate PAC effect prediction, and may improve the reliability of PAC risk assessment.