Magnus Breitholtz

Oestrogens have no hormonal effect on the development and reproduction of the harpacticoid copepod Nitocra spinipes.

In recent years, reports have described endocrine-disruptive effects of environmental oestrogens in fish, but little is known about similar effects in crustaceans. The objective of the present study was therefore to examine whether the oestrogens 17-beta-oestradiol, 17-alpha-ethynylestradiol and diethylstilbestrol (DES), could affect mortality, larval development rate, fecundity and sex ratio in the sexually reproducing harpacticoid copepod Nitocra spinipes. Newly released nauplii (<24-h old) were exposed to 1/1,000, 1/100 and 1/10 (nominal concentrations) of each oestrogens 96 h-LC50 value for < or = 18 days at 22 +/- 1 degrees C. The percentage of gravid females and the number of developed copepodites were both reduced at 0.03 mg l(-1) DES, although the latter response was not significant. None of the other two oestrogens induced any measurable effects. Since the only observed significant response appeared at a DES concentration no more than 10 times below the 96 h-LC50 value, there is no evidence of endocrine-disruptive activity in N. spinipes exposed to oestrogens.

Effects of four synthetic musks on the life cycle of the harpacticoid copepod Nitocra spinipes

A full life-cycle (</=26 days exposure) toxicity test with the harpacticoid copepod Nitocra spinipes was used to study the effects of one nitro musk (musk ketone) as well as three polycyclic musks (Tonalide, Celestolide and Galaxolide). A subchronic individual life-table endpoint, the larval development rate, was recorded after 7-8 days exposure of juveniles and was significantly decreased in copepods exposed to sublethal concentrations of musk ketone, Celestolide and Galaxolide. However, none of the Tonalide concentrations had any effect on larval development. The lowest Galaxolide concentration (0.02 mg/l), which affected juvenile development, was about 100 times below the adult 96-h-LC(50)-value of 1.9 mg/l (95% confidence interval: 1.4-2.7 mg/l). However, none of the four musks had any agonistic or antagonistic activity in the ecdysteroid-sensitive Drosophila melanogaster B(II)-cell line. This indicates that the decrease in larval development rate was due to pharmacological effects rather than steroid receptor-mediated endocrine disruption. A modified Euler-Lotka equation was used to calculate a population-level endpoint, the intrinsic rate of natural increase (r(m)), from individual life-table endpoints, i.e. mortality rate, time of release of first brood, sex ratio, the fraction of ovigerous females among all females as well as the number of nauplii per ovigerous female. The second highest musk ketone concentration (0.1 mg/l) was the only treatment, which significantly affected r(m) (***P<0.001). At the highest musk ketone (0.3 mg/l) and Celestolide (0.3 mg/l) concentrations, all copepods were dead at the end of the exposures. This shows that a sensitive individual life-table endpoint is protective over the population-level endpoint r(m). Though we think that it is necessary to obtain population-level endpoints from standardised toxicity test, for ecologically successful risk characterisation of synthetic musks as well as other chemicals. The results from the present study show that it is possible to obtain population-level data from the full life-cycle test with N. spinipes. However, there seems to be little risk that synthetic musks are harmful to copepods at present environmental concentrations.

Effects of three PBDEs on development, reproduction and population growth rate of the harpacticoid copepod Nitocra spinipes

The current knowledge concerning effects of polybrominated diphenyl ethers (PBDEs) on aquatic organisms is very limited. A full life-cycle (< or =26 days exposure) ecotoxicity test with the particle-feeding copepod Nitocra spinipes was therefore used to study effects of BDE-47, -99 and -100 on larval development rate (LDR) and population growth rate (r(m)). LDR significantly decreased in copepods exposed for 6 days to nominal concentrations > or =0.013 mg/l BDE-47 and > or =0.03 mg/l BDE-99. Large concentration ratios (< or =338) between adult acute and juvenile subchronic endpoints were observed. Exposure over the full life cycle (< or =26 days) showed that r(m) in general was a less sensitive endpoint than LDR. Still, the r(m) in copepods exposed to 0.04 mg/l BDE-47 was significantly reduced compared to the controls (***P<0.001). Partitioning experiments with 14C-BDE-47 and 14C-BDE-99 in the test system showed that the major fractions (approximately 50-80%) were associated to particulate material. Our findings indicate that development and reproduction in N. spinipes are sensitive to the tested PBDEs and that ingestion of particle-adsorbed PBDEs most likely is the predominant route of exposure in N. spinipes. However, to further improve the usefulness of laboratory effect levels of PBDEs and other lipophilic substances for environmental risk assessment, it is important to develop ecotoxicological tools, which can evaluate and rate the toxic contribution from different matrices, such as suspended particles, sediment, food, water etc.

Brominated flame retardants: Activities in a crustacean development test and in an ecdysteroid screening assay

Brominated flame retardants (BFRs) were investigated for toxic effects both in vivo and in vitro in two invertebrate bioassays. Subchronic effects of tetrabromobisphenol A (TBBPA), tribromophenol (TBP), and four polybrominated diphenyl ethers ([PBDEs]; BDE-28, BDE-47, BDE-99, and BDE-100) on larval development of the marine copepod Acartia tonsa were studied. For TBBPA and TBP 5-d effective median concentration (EC50) values for inhibition of the larval development rate were 125 and 810 microg/L, respectively, whereas the PBDEs were much more potent with 5-d EC50 in the low microg/L range (1.2 microg/L for BDE-100; 4.2 microg/L for BDE-99; 13 microg/L for BDE-28; and 13 microg/L for BDE-47). These concentrations were up to two orders of magnitude below the 48-h LC50 for acute adult toxicity (108 microg/L for BDE-28; 400 microg/L for TBBPA; 520 microg/L for BDE-100; 705 microg/L for BDE-99; 1,500 microg/L for TBP; and 2,370 microg/L for BDE-47). To distinguish between general toxicological and endocrine-mediated toxic effects, the BFRs were assessed in vitro for ecdysteroid agonistic/antagonistic activity with the ecdysteroid-responsive Drosophila melanogaster B(II)-cell line. The pentabrominated diphenyl ethers BDE-99 and BDE-100 showed weak ecdysteroid antagonistic activity. Thus, these PBDEs may be regarded as potential endocrine disrupters in invertebrates. The combination of in vitro assays and subchronic biotests with ecologically important crustacean species is a rapid and cost-effective tool when screening for sublethal effects of BFRs and other chemicals.

Sucralose – An ecotoxicological challenger?

The non-calorie sweetener sucralose - sucrose containing three chlorine atoms - is intensively sweet and has become a popular substitute for sugar. Its widespread use, exceptional stability in combination with high water solubility have thus resulted in contamination of recipient waters. Earlier studies on sucralose in aquatic organisms indicate low bioaccumulation potential and negligible acute/chronic toxicity, but the close structural resemblance with sucrose in combination with the importance of sugar in nature, warrant a more detailed ecotoxicological assessment. The aim of this investigation was therefore to study behavioural and physiological effects of sucralose in crustaceans. Our results show that both physiology and locomotion behaviour were affected by exposure to sucralose. In Daphnia magna, the behavioural response was manifested as altered swimming height and increased swimming speed, whereas in gammarids the time to reach food and shelter was prolonged. Regardless if these behavioural responses were initiated via traditional toxic mechanisms or stimulatory effects, they should be considered as a warning, since exposed organisms may diverge from normal behaviour, which ultimately can have ecological consequences.

Inhibition of larval development of the marine copepod Acartia tonsa by four synthetic musk substances.

A nitro musk (musk ketone) and three polycyclic musks (Tonalide, Galaxolide and Celestolide) were tested for acute and subchronic effects on a marine crustacean, the calanoid copepod Acartia tonsa. Sublethal effects on A. tonsa larvae were investigated with a rapid and cost effective bioassay, which is based on the easily detectable morphological change from the last nauplius to the first copepodite stage during copepod larval development. The inhibition of larval development after 5 days exposure was a very sensitive endpoint, with 5-d-EC(50)-values as low as 0.026 mg/l (Tonalide), 0.059 mg/l (Galaxolide), 0.066 mg/l (musk ketone) and 0.160 mg/l (Celestolide), respectively. These values were generally more than one order of magnitude below the 48-h-LC(50)-values found for adults, which were 0.47 mg/l (Galaxolide), 0.71 mg/l (Celestolide), 1.32 mg/l (musk ketone) and 2.5 mg/l (Tonalide). Since the synthetic musks strongly inhibited larval development in A. tonsa at low nominal concentrations, they should be considered as very toxic. The larval development test with A. tonsa is able to provide important aquatic toxicity data for the evaluation of synthetic musks, for which there is little published ecotoxicological information available regarding Crustacea. It is suggested that subchronic and chronic copepod toxicity tests should be used more frequently for risk assessment of environmental pollutants.

An evaluation of free water surface wetlands as tertiary sewage water treatment of micro-pollutants.

Increased attention is currently directed towards potential negative effects of pharmaceuticals and other micro-pollutants discharged into the aquatic environment via municipal sewage water. A number of additional treatment technologies, such as ozonation, have therefore been suggested as promising tools for improving the removal efficiency of pharmaceuticals in existing Sewage Treatment Plants (STPs). Constructed wetlands are also capable of removing a variety of micro-pollutants, including some pharmaceuticals, and could hence be a resource efficient complement to more advanced treatment technologies. The purpose of the present study was therefore to increase the knowledge base concerning the potential use of constructed wetlands as a treatment step to reduce emissions of organic micro-pollutants from municipal sewage effluents. Under cold winter conditions, incoming and outgoing waters from four Swedish free water surface wetlands, operated as final treatment steps of sewage effluent from municipal STPs, were sampled and analyzed for levels of a set of 92 pharmaceuticals and 22 inorganic components as well as assessed using subchronic ecotoxicity tests with a macro-alga and a crustacean. Sixty-five pharmaceuticals were detected in the range from 1 ng L(-1) to 7.6 μg L(-1) in incoming and outgoing waters from the four investigated wetlands. Although the sampling design used in the present study lacks the robustness of volume proportional to 24h composite samples, the average estimated removal rates ranged from 42% to 52%, which correlates to previous published values. The effects observed in the ecotoxicity tests with the macro-alga (EC(50)s in the range of 7.5-46%) and the crustacean (LOECs in the range of 11.25-90%) could not be assigned to either pharmaceutical residues or metals, but in general showed that these treatment facilities release water with a relatively low toxic potential, comparable to water that has been treated with advanced tertiary treatments. From the present study it can be concluded that constructed wetlands may provide a complementary sewage treatment option, especially where other treatment is lacking today. To fully remove micro-pollutants from sewage effluent, however, other more advanced treatment technologies are likely needed.

Improving environmental risk assessment of human pharmaceuticals.

This paper presents 10 recommendations for improving the European Medicines Agencys guidance for environmental risk assessment of human pharmaceutical products. The recommendations are based on up-to-date, available science in combination with experiences from other chemical frameworks such as the REACH-legislation for industrial chemicals. The recommendations concern: expanding the scope of the current guideline; requirements to assess the risk for development of antibiotic resistance; jointly performed assessments; refinement of the test proposal; mixture toxicity assessments on active pharmaceutical ingredients with similar modes of action; use of all available ecotoxicity studies; mandatory reviews; increased transparency; inclusion of emission data from production; and a risk management option. We believe that implementation of our recommendations would strengthen the protection of the environment and be beneficial to society. Legislation and guidance documents need to be updated at regular intervals in order to incorporate new knowledge from the scientific community. This is particularly important for regulatory documents concerning pharmaceuticals in the environment since this is a research field that has been growing substantially in the last decades.

Expression of ecdysteroids and cytochrome P450 enzymes during lipid turnover and reproduction in Calanus finmarchicus (Crustacea: Copepoda)

The marine copepod Calanus finmarchicus is the most abundant zooplankton species in the northern regions of the Atlantic Ocean and the Barents Sea. Very little is known about molecular regulation of hormone metabolism, moulting and reproduction in copepods. To investigate these processes, we sampled adult male and female copepods (females at three distinct reproductive stages) and copepodites stage five (CV) from the culture at SINTEF/NTNU Sealab. Copepods were individually photographed, analyzed biometrically (body size, length and lipid storage size) and for ecdysteroid concentrations. In addition, we analyzed copepods for gene expression of three putative cytochrome P450 enzymes possibly involved in ecdysteroid regulation: CYP301A1, CYP305A1 and CYP330A1. The CV group exhibited the highest ecdysteroid concentrations and the largest lipid storage size, and a significant positive correlation was found between these parameters. Also, two of the P450 enzymes (CYP305A1 and CYP330A1) were more highly expressed at CV than at the adult stage, suggesting that these P450 enzymes are involved in ecdysteroid synthesis and lipid storage regulation. The expression of CYP330A1 was higher in newly moulted females than in females that had produced eggs. In addition, we observed that ecdysteroid concentrations were higher in females with large egg sacs, suggesting that ecdysteroids may be involved in egg maturation and reproduction. The CYP301A1 was more highly expressed in males and post-spawning females, and may be involved in ecdysteroid degradation since these groups also exhibited the lowest ecdysteroid concentrations.

Evidence of population genetic effects of long-term exposure to contaminated sediments-a multi-endpoint study with copepods.

In the environment, pollution generally acts over long time scales and exerts exposure of multiple toxicants on the organisms living there. Recent findings show that pollution can alter the genetics of populations. However, few of these studies have focused on long-term exposure of mixtures of substances. The relatively short generation time (ca. 4-5 weeks in sediments) of the harpacticoid copepod Attheyella crassa makes it suitable for multigenerational exposure studies. Here, A. crassa copepods were exposed for 60 and 120 days to naturally contaminated sediments (i.e., Svindersviken and Trosa; each in a concentration series including 50% contaminated sediment mixed with 50% control sediment and 100% contaminated sediment), and for 120 days to control sediment spiked with copper. We assayed changes in F(ST) (fixation index), which indicates if there is any population subdivision (i.e., structure) between the samples, expected heterozygosity, percent polymorphic loci, as well as abundance. There was a significant decrease in total abundance after 60 days in both of the 100% naturally contaminated sediments. This abundance bottleneck recovered in the Trosa treatment after 120 days but not in the Svindersviken treatment. After 120 days, there were fewer males in the 100% naturally contaminated sediments compared to the control, possibly caused by smaller size of males resulting in higher surface: body volume ratio in contact with toxic chemicals. In the copper treatment there was a significant decrease in genetic diversity after 120 days, although abundance remained unchanged. Neither of the naturally contaminated sediments (50 and 100%) affected genetic diversity after 120 days but they all had high within treatment F(ST) values, with highest F(ST) in both 100% treatments. This indicates differentiation between the replicates and seems to be a consequence of multi-toxicant exposure, which likely caused selective mortality against highly sensitive genotypes. We further assayed two growth-related measures, i.e., RNA content and cephalothorax length, but none of these endpoints differed between any of the treatments and the control. In conclusion, the results of the present study support the hypothesis that toxicant exposure can reduce genetic diversity and cause population differentiation. Loss of genetic diversity is of great concern since it implies reduced adaptive potential of populations in the face of future environmental change.