Niklas Hanson

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.

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.

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.

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.

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.

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.

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.

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

Abstract 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. Environ Toxicol Chem 2015;34:1511–1523.