Ecological changes as a plausible explanation for differences in uptake of contaminants between European perch and eelpout in a coastal area of the Baltic Sea.

Unexpected increasing trends in the concentration of contaminants in European perch (Perca fluviatilis) and in activity of ethoxyresorufin-O-deethylase (EROD) in European perch and eelpout (Zoarces viviparus) have been observed at a Swedish coastal reference site. This study uses data from different sources to investigate plausible explanations. The results showed that a change in diet and an improved overall condition coincide with an increase in mercury in European perch. Furthermore, an increase in several organic contaminants in European perch coincided with the introduction of an invasive deep-burrowing polychaete, which likely contributed to the release of contaminants through bioturbation. The increase in EROD-activity in both species seems to be related to contaminants that reach the fish through the water rather than the diet. The results show that for contaminants that are taken up via the diet, trends in contamination can be opposite for different species of fish in the same area.

A gene to organism approach--assessing the impact of environmental pollution in eelpout (Zoarces viviparus) females and larvae.

A broad biomarker approach was applied to study the effects of marine pollution along the Swedish west coast using the teleost eelpout (Zoarces viviparus) as the sentinel species. Measurements were performed on different biological levels, from the molecular to the organismal, including measurements of messenger RNA (mRNA), proteins, cellular and tissue changes, and reproductive success. Results revealed that eelpout captured in Stenungsund had significantly higher hepatic ethoxyresorufin O-deethylase activity, high levels of both cytochrome P4501A and diablo homolog mRNA, and high prevalence of dead larvae and nuclear damage in erythrocytes. Eelpout collected in Göteborg harbor displayed extensive macrovesicular steatosis, whereby the majority of hepatocytes were affected throughout the liver, which could indicate an effect on lipid metabolism. Results also indicate that eelpouts collected at polluted sites might have an affected immune system, with lower mRNA expression of genes involved in the innate immune system and a higher number of lymphocytes. Biomarker assessment also was performed on livers dissected from unborn eelpout larvae collected from the ovary of the females. No significant differences were noted, which might indicate that the larvae to some extent are protected from effects of environmental pollutants. In conclusion, usage of the selected set of biological markers, covering responses from gene to organism, has demonstrated site-specific biomarker patterns that provided a broad and comprehensive picture of the impact of environmental stressors.

Comparison of population level and individual level endpoints to evaluate ecological risk of chemicals.

Ecological risk assessments (ERA) of chemicals are often based on mortality and reproduction of individuals. To protect populations, fixed safety factors are applied to the data. However, the relationship between individuals and populations cannot easily be described by predefined numbers. The use of population models may reduce uncertainty and, hence, the risk for erroneous assessments. However, introducing models also introduces additional complexity. Therefore, it is desirable to keep the models as simple as possible. The objective of the present study was to determine whether simple risk equations or matrix models can improve ERA compared to traditional endpoints. To examine this, complex models that included environmental stochasticity and density dependence were used to simulate population level risk based on dose-response data for five chemicals. The risk, measured as probability for pseudo extinction and recovery time, was then compared to risk estimates based on individual level data (acute and chronic), risk equations, and simple matrix models. The results showed that the simple matrix models reduced uncertainty by more than 88% and 76% compared to acute and chronic data, respectively. Also the simple risk equation reduced uncertainty considerably (80% and 61% compared to acute and chronic data, respectively).

Utility of population models to reduce uncertainty and increase value relevance in ecological risk assessments of pesticides: an example based on acute mortality data for daphnids.

Traditionally, ecological risk assessments (ERA) of pesticides have been based on risk ratios, where the predicted concentration of the chemical is compared to the concentration that causes biological effects. The concentration that causes biological effect is mostly determined from laboratory experiments using endpoints on the level of the individual (e.g., mortality and reproduction). However, the protection goals are mostly defined at the population level. To deal with the uncertainty in the necessary extrapolations, safety factors are used. Major disadvantages with this simplified approach is that it is difficult to relate a risk ratio to the environmental protection goals, and that the use of fixed safety factors can result in over- as well as underprotective assessments. To reduce uncertainty and increase value relevance in ERA, it has been argued that population models should be used more frequently. In the present study, we have used matrix population models for 3 daphnid species (Ceriodaphnia dubia, Daphnia magna, and D. pulex) to reduce uncertainty and increase value relevance in the ERA of a pesticide (spinosad). The survival rates in the models were reduced in accordance with data from traditional acute mortality tests. As no data on reproductive effects were available, the conservative assumption that no reproduction occurred during the exposure period was made. The models were used to calculate the minimum population size and the time to recovery. These endpoints can be related to the European Union (EU) protection goals for aquatic ecosystems in the vicinity of agricultural fields, which state that reversible population level effects are acceptable if there is recovery within an acceptable (undefined) time frame. The results of the population models were compared to the acceptable (according to EU documents) toxicity exposure ratio (TER) that was based on the same data. At the acceptable TER, which was based on the most sensitive species (C. dubia), the maximum reduction in population size was 13% and the maximum time to recovery was 4 d (both for D. magna). This information is clearly more informative for risk management than a risk ratio. For one of the species, D. pulex, a more complex model, which included sublethal effects on reproduction, was set up. The results of this model were in good agreement with a previous microcosm study and indicated that a traditional TER was overprotective.

An approach for developing simple generic models for ecological risk assessments of fish populations.

It has been argued that current approaches for ecological risk assessment (ERA) do not provide value relevance for risk managers and that the uncertainty in the predictions is large. One important reason for this is that current approaches are based on individual-level endpoints, but the environmental protection goals are defined on the population level. Population models may be useful tools to link the individual to the population and thus increase value relevance and reduce uncertainty. However, this requires that simple models and guidance on how to use them are made available for risk assessors. In the present study, an approach for developing simple generic models for the ERA of fish populations is presented. Fish have high ecological and economic value and are frequently used in ERA. It is, therefore, highly desirable to develop better methods for ERA of fish populations. The models were based on five types of life histories, and they were set up to be useful in a number of different management scenarios based on different environmental protection goals. A decision framework was set up for three levels of models (continuous, time limited, and varying exposure) and three levels of environmental protection. Data from previously published studies were tested for two of the environmental protection levels. The models provided maximum acceptable concentrations (MAC) that fell between the MACs of traditional ERA based on acute or chronic data, respectively.

A comparison of simple and complex population models to reduce uncertainty in ecological risk assessments of chemicals: example with three species of Daphnia.

Ecological risk assessments (ERA) are mostly based on effects on survival (S) and fertility (F) of individuals. However, the protection goals are most often defined on the population or community levels. It has been argued that population models can be a useful link between the individual and the population in ERA. However, for population models to be efficiently and routinely used in ERA, the level of model complexity that is needed has to be clearly determined. In the present study, complex age classified matrix population models and simple 2-stage models were developed for three species of Daphnia. The population growth rate (λ) from the simple 2-stage model correlated strongly to the results of the complex matrix model, which included density dependence and temporary reductions in S and F. This shows that the information that can be provided by more complex models also can be relatively well predicted with the simpler model. The output of the complex matrix population models were also compared to the reductions in S that were used in the models. This was done because acute mortality is the most commonly used estimate of toxic effects. The results showed that λ from the 2-stage model correlated stronger to the endpoints of the matrix model than S did in all cases except for pulsed exposures, where S and λ correlated equally well.

Using biological data from field studies with multiple reference sites as a basis for environmental management: the risks for false positives and false negatives.

Field surveys of biological responses can provide valuable information about environmental status and anthropogenic stress. However, it is quite usual for biological variables to differ between sites or change between two periods of time also in the absence of an impact. This means that there is an obvious risk that natural variation will be interpreted as environmental impact, or that relevant effects will be missed due to insufficient statistical power. Furthermore, statistical methods tend to focus on the risks for Type-I error, i.e. false positives. For environmental management, the risk for false negatives is (at least) equally important. The aim of the present study was to investigate how the probabilities for false positives and negatives are affected by experimental set up (number of reference sites and samples per site), decision criteria (statistical method and α-level) and effect size. A model was constructed to simulate data from multiple reference sites, a negative control and a positive control. The negative control was taken from the same distribution as the reference sites and the positive control was just outside the normal range. Using the model, the probabilities to get false positives and false negatives were calculated when a conventional statistical test, based on a null hypothesis of no difference, was used along with alternative tests that were based on the normal range of natural variation. Here, it is tested if an investigated site is significantly inside (equivalence test) and significantly outside (interval test) the normal range. Furthermore, it was tested how the risks for false positives and false negatives are affected by changes in α-level and effect size. The results of the present study show that the strategy that best balances the risks between false positives and false negatives is to use the equivalence test. Besides tests with tabulated p-values, estimates generated using a bootstrap routine were included in the present study. The simulations showed that the probability for management errors was smaller for the bootstrap compared to the traditional test and the interval test.

Biomarker analyses in caged and wild fish suggest exposure to pollutants in an urban area with a landfill.

An unexpectedly high frequency of skeletal deformations in brown trout has previously been observed in the brook Vallkärrabäcken in southern Sweden. Environmental pollutants from storm water and leachate from an old landfill have been suggested as responsible for the observed deformations. Biomarkers in farmed rainbow trout, placed in tanks with water supplied from the brook, were used to investigate if exposure to pollutants may induce toxic responses in fish. Furthermore, biomarkers were also measured in wild brown trout that were caught in the brook. The most important finding was that the hepatic ethoxyresorufin-O-deethylase (EROD) activity was five to seven times higher for rainbow trout and brown trout in exposed areas compared to reference sites (P<0.001). Analyses of bile in rainbow trout showed that the concentration of PAH-metabolites was two to three times higher (P<0.001) in the exposed areas. However, due to their smaller size and the feeding status, only insufficient amounts of bile could be retrieved from the wild brown trout. The study provides evidence for pollution in parts of Vallkärrabäcken. It is therefore possible that the previously observed high frequency of skeletal damage have been caused by pollutants. The methodology with farmed rainbow trout in flow through tanks worked well and provided more information about the occurrence of pollutants in Vallkärrabäcken than the data from brown trout. The main reasons for this were that the size and the feeding status of the fish could be controlled. This allowed a total of 21 biomarkers to be analyzed in farmed rainbow trout compared to only five in wild brown trout. Furthermore, the use of farmed fish eliminates the risk of migration, which may otherwise bias the data when wild fish are used.

Spatial and annual variation to define the normal range of biological endpoints: an example with biomarkers in perch.

A signal from a biological endpoint can be considered evidence of environmental impact only if it is known that similar signals are unlikely to occur naturally. Using multiple reference sites, the normal range can be estimated. This can be defined as the span that includes 95% of the reference sites. If an investigated site is outside this range, it is interpreted as evidence of impact. The choice of reference sites is thus important for the outcome of the study. Here, biomarker levels in perch (Perca fluviatilis) at a potentially impacted site were compared to different types of reference data. The potentially impacted site was located close to a densely populated area. Four reference sites were located in relatively undisturbed areas on the Swedish Baltic coast. Furthermore, historical data from one of the reference sites were included. The present study showed that multiple reference sites are needed to avoid the risk of interpreting natural variation as impact. The number of reference sites needed depends on the desired level of statistical power. An alternative to multiple reference sites may be to use historical data to estimate the natural variation. However, historical data can include temporal variation due to factors that may not be relevant for the hypothesis that is tested, e.g., climatic variation or changed background levels of pollution.

Experiences from a biomarker study on farmed rainbow trout (Oncorhynchus mykiss) used for environmental monitoring in a Swedish river.

Biomarkers in fish may serve as a useful tool for evaluating the pollution load in the environment and for early warning signals about new environmental threats. By employing a strategy with fish that are caged or reared in tanks, problems linked to migration and feeding status can be reduced or eliminated. Such a strategy, however, also may introduce other confounding factors linked to, for example, hierarchical behavior or disease outbreaks. In the present study, rainbow trout (Oncorhynchus mykiss) were reared in plastic tanks at four sites in the Göta älv river system (plus one external reference) in western Sweden during 2006 and 2007. Because of low population density and high water turnover in the area, pollution levels are expected to be low. Therefore, this should be a good test for the sensitivity of the methodology. Several significant differences were found between sites as well as between years, such as ethoxyresorufin-O-deethylase (EROD) activity, polycyclic aromatic hydrocarbon (PAH) metabolites in bile, and concentration of inorganic ions in blood plasma, but it also was seen that factors other than pollution could be contributing to these differences. The condition factor (CF) varied between sites, possibly because of differences in feeding resulting from variations in water turbidity. Furthermore, even the small differences in CF that were found within sites correlated significantly to several of the biomarkers. It was shown that PAHs likely are the most important EROD inducers in Göta älv and that variation over time is greater than variation between sites. Because CF differed between sites despite a standardized feeding ration, starving of the fish during the exposure period should be considered for future studies.

Analyses of perch (Perca fluviatilis) bile suggest increasing exposure to PAHs and other pollutants in a reference area on the Swedish Baltic coast.

Kvädöfjärden on the Swedish Baltic coast has been used as a reference station in the Swedish National Marine Monitoring Programme since 1988. During this time an increasing trend in ethoxyresorufin-O-deetylase (EROD) activity and a decreasing trend in gonad size for female perch have been observed. It is known that EROD activity respond to exposure to certain groups of pollutants, including polycyclic aromatic hydrocarbons (PAHs). In the present study, perch bile from two years with high (2006) and low (2003) EROD levels, respectively, were analyzed. The concentration of PAH metabolites in bile was semi-quantified using fixed wavelength fluorescence. Furthermore, toxicity of the bile was tested on Daphnia magna. The total concentration of PAH metabolites and toxicity to D. magna was higher in 2006 than in 2003. Furthermore, there was a higher proportion of combustion-type PAHs in bile from 2006. The concentration of PAH metabolites in bile correlated to the EROD activity of the fish from which the bile was taken. There was no correlation between the concentrations of PAH metabolites and bile toxicity. Therefore, other pollutants than PAHs may also have been elevated in 2006. The results suggest that increasing exposure to PAHs can be causing the time trend in EROD activity. It is known that PAHs can have a negative effect on gonad development. Therefore, the decreasing trend in gonad size may also be caused by increasing exposure to PAHs.

Evaluation of long-term biomarker data from perch (Perca fluviatilis) in the Baltic Sea suggests increasing exposure to environmental pollutants.

Since 1988, biomarkers in female perch (Perca fluviatilis) have been analyzed at a reference site on the Swedish Baltic coast. Strong time trends toward increasing hepatic ethoxyresorufin-O-deethylase (EROD) activity and reduced gonadosomatic index (GSI) have been observed. This could be caused by pollutants as well as other factors, such as increasing water temperature or reduced mean age of sampled fish. Correlation analyses were used to find the most probable explanation for the time trends. The time trends were still significant for EROD (p < 0.001) and GSI (p < 0.001) when the correlations were controlled for age. Furthermore, increasing water temperature could not explain the time trends. Exposure to pollutants through runoff from land was found to be probable, because mean flow rate in a nearby river during the last 20 d before sampling correlated to EROD activity (p < 0.01). In addition, the sum of EROD activities during the life time of the perch (ERODlife) correlated significantly with GSI (p < 0.001). This suggests that perch exposed to more EROD-inducing chemicals during their lifetime have reduced or delayed gonad development. The time trend in GSI and the correlation between ERODlife and GSI were supported by data from a site in the Bothnian Bay (northern Baltic Sea; p < 0.05). The results indicate that increased rain fall (climate change) can affect the distribution and bioavailability of chemicals in coastal areas. The link between EROD and gonad size supports the common assumption that biochemical biomarkers can act as early warning signals for effects on higher levels, which commonly is difficult to show. The significant results can probably be attributed to the unique 20-year data set.

Fixed wavelength fluorescence to detect PAH metabolites in fish bile: increased statistical power with an alternative dilution method.

Polyaromatic hydrocarbons (PAHs) are a group of environmental toxicants mainly emitted from diffuse sources. Fixed wavelength fluorescence of fish bile is a simple screening method that allows environmental monitoring of PAH exposure to fish. One drawback with this method is that the results can be biased by the presence of a so-called inner filter effect. This effect can be reduced by dilution of the bile samples. However, bile samples differ in density and extensive dilution may cause increased measurement error. An alternative method is to adjust the dilution rate to the density of each bile sample. Here, both methods have been applied to bile samples collected from caged rainbow trout. The bile density was estimated using biliverdin. The results show that the variance within experimental groups is reduced when the dilution rate is adjusted to the bile density, resulting in increased statistical power to detect differences when applied for environmental monitoring.

Influence of feeding procedure on biomarkers in caged rainbow trout (Oncorhynchus mykiss) used in environmental monitoring.

Biomarkers in feral fish can be a useful tool for environmental monitoring of aquatic ecosystems. Drawbacks, however, are that suitable fish species are not always available and that natural variations can bias the results. An alternative strategy is to use farmed fish placed in cages. There is, however, still a risk that factors other than pollution level could have an impact on the biomarkers and the observed responses in the fish. The present study evaluates the effects of feeding procedure on biomarkers in caged fish. Two feeding rations (2% and 8% weekly feeding) have been examined for a large number of biomarkers in caged rainbow trout (Oncorhynchus mykiss). Significant effects of feeding rations were found on hepatic ethoxyresorufin-O-deethylase (EROD) and catalase activity, PAH metabolites in bile, plasma ion concentrations and the presence of immature red blood cells. The influence on EROD activity and PAH metabolites seems to be caused by elevated uptake of pollutants when feeding ratios are higher. The effects on other biochemical and physiological variables are more likely caused by stress due to insufficient feeding. According to these results, valid comparison of fish groups in environmental monitoring requires standardized feeding levels.

The effect of different holding conditions for environmental monitoring with caged rainbow trout (Oncorhynchus mykiss).

Biomarkers in fish can be a useful tool for environmental monitoring of aquatic ecosystems when diffuse pollution is becoming more important and new chemicals are being created continuously. There are, however, a number of drawbacks with this method. Because of environmental variability, health status of wild fish populations may differ between years, leading to unrepresentative results in long term studies. Furthermore, genetic or adaptive differences between populations complicate the interpretation of studies on different sites. The use of farmed fish, placed in cages, can reduce these problems. However, experimental conditions are likely to differ between sites. For practical reasons it may, e.g., be neccesary to use different types of caging. Here, the use of net cages and flow through tanks has been compared for a number of biomarkers. Rainbow trout (Oncorhynchus mykiss) were placed in net cages and flow through tanks in the river Göta Alv, in western Sweden, during three different periods in 2004 and 2005. No differences between types of caging were found for any biomarker. Therefore, the results suggest that net cages and flow through tanks can be used and compared in environmental monitoring using biomarkers in caged rainbow trout. However, efforts should be taken to reduce differences in experimental conditions, e.g., light intensity and feeding levels.

Population-level effects of male-biased broods in eelpout (Zoarces viviparus).

Effects of environmental pollutants are most obvious when mortality is increased. However, there are other nonlethal factors that may affect population size significantly. Endocrine disruption as a mechanism of action for pollutants recently has received much attention. Observations of effects likely caused by endocrine disruptors in pulp mill effluents have been made on several fish species, e.g., male-biased broods in eelpout (Zoarces viviparus). Fewer females represent a lower fecundity and could have dramatic effects on the population. In this study, a population viability analysis of the effects of skewed primary sex ratios in the eelpout was conducted using a female-based matrix population model. The model is age-structured with one deterministic version and another that incorporates environmental stochasticity. The model showed that the deterministic and stochastic growth rates in an undisturbed population (50% female fry) were 1.172 and 1.075, respectively, compared to 1.097 and 1.007 using the lowest proportion of female fry observed (38.7%). When primary sex ratios were more male-biased, the probability of pseudoextinction increased. Model simulations showed that the probability of a decreased population size to 5% of the initial within a 100-year time horizon was 44.7% with 38.7% female fry compared to only 7.7% for an undisturbed population.