Long-term laboratory culture causes contrasting shifts in tolerance to two marine pollutants in copepods of the genus Tigriopus.

Organismal chemical tolerance is often used to assess ecological risk and monitor water quality, yet tolerance can differ between field- and lab-raised organisms. In this study, we examined how tolerance to copper (Cu) and tributyltin oxide (TBTO) in two species of marine copepods, Tigriopus japonicus and T. californicus, changed across generations under benign laboratory culture (in the absence of pre-exposure to chemicals). Both copepod species exhibited similar chemical-specific changes in tolerance, with laboratory maintenance resulting in increased Cu tolerance and decreased TBTO tolerance. To assess potential factors underlying these patterns, chemical tolerance was measured in conjunction with candidate environmental variables (temperature, UV radiation, diet type, and starvation). The largest chemical-specific effect was found for starvation, which decreased TBTO tolerance but had no effect on Cu tolerance. Understanding how chemical-specific tolerance can change in the laboratory will be critical in strengthening bioassays and their applications for environmental protection and chemical management.

Water-effect ratio of copper and its application on setting site-specific water quality criteria for protecting marine ecosystems of Hong Kong.

Generic water quality criteria (WQC) of a chemical are usually set based on results generated from toxicity tests which were conducted using standard laboratory water with well-controlled physiochemical properties. However, in natural aquatic environments, physiochemical characteristics, such as salinity, total suspended solid, total organic carbon and the co-existence of chemical contaminants, often vary spatially and temporally. These parameters can, in turn, alter the bioavailability of target chemicals and, thus, influence their toxicity to marine organisms. To account for site specificity, the US Environmental Protection Agency's water-effect ratio (WER = site water-LC50 / laboratory water-LC50) procedure can be applied to derive site-specific WQC. Most past studies, however, were conducted for freshwater systems. Here, for the first time, the WER of copper (Cu) was determined for three marine water control zones (WCZs) in Hong Kong: Victoria Harbour, Deep Bay and Southern WCZs. Samples of water were collected from three locations within each WCZ, while acute toxicities to the marine diatom Skeletonema costatum, intertidal copepod Tigriopus japonicus and larvae of marine medaka Oryzias melastigma were determined in site or laboratory (artificial seawater) waters. Results of this study showed that conservative final WER relative coefficients for Cu ranged from 0.57 to 0.73 for the three WCZs, and water from some locations caused >30% mortality in the fish larvae in the controls (without Cu addition). These results suggested that current generic WQC for Cu are likely under-protective for marine organisms in the three areas, and it should be tightened by multiplying it with site-specific WER to offer better protection to marine biodiversity and integrity of the ecosystem.

Binary mixture toxicities of triphenyltin with tributyltin or copper to five marine organisms: Implications on environmental risk assessment.

Triphenyltin (TPT) often coexists with tributyltin (TBT) and Cu in coastal waters worldwide. The combined toxic effect of TPT and TBT has always been assumed to be additive without any scientific proof, and the combined effect of Cu and TPT on marine organisms has not been vigorously studied. This study, therefore, investigated the acute toxicity of binary mixture of TPT/Cu and TPT/TBT to five selected marine species including Thalassiosira pseudonana, Skeletonema costatum, Tigriopus japonicus, Brachionus koreanus and Oryzias melastigma. The interaction between TPT and TBT or Cu was modeled antagonistic based on concentration addition (CA) model, while it was synergistic according to response addition (RA) model. Both model well predicted the toxicity of binary mixtures to the five organisms. As for the environmental risk assessment, CA overestimated the toxicity in most cases and thus is a more conservative model than RA model for assessing the toxicity of these chemical mixtures.

Temperature-dependent physiological and biochemical responses of the marine medaka Oryzias melastigma with consideration of both low and high thermal extremes.

This study aimed to investigate temperature effect on physiological and biochemical responses of the marine medaka Oryzias melastigma larvae. The fish were subjected to a stepwise temperature change at a rate of 1 °C/h increasing or decreasing from 25 °C (the control) to six target temperatures (12, 13, 15, 20, 28 and 32 °C) respectively, followed by a 7-day thermal acclimation at each target temperature. The fish were fed ad libitum during the experiment. The results showed that cumulative mortalities were significantly increased at low temperatures (12 and 13 °C) and at the highest temperature (32 °C). For the survivors, their growth profile closely followed the left-skewed 'thermal performance curve'. Routine oxygen consumption rates of fish larvae were significantly elevated at 32 °C but suppressed at 13 and 15 °C (due to a high mortality, larvae from 12 °C were not examined). Levels of heat shock proteins and activities of malate dehydrogenase and lactate dehydrogenase were also measured in fish larvae exposed at 15, 25 and 32 °C. The activities of both enzymes were significantly increased at both 15 and 32 °C, where the fish larvae probably suffered from thermal discomfort and increased anaerobic components so as to compensate the mismatch of energy demand and supply at these thermal extremes. Coincidently, heat shock proteins were also up-regulated at both 15 and 32 °C, enabling cellular protection. Moreover, the critical thermal maxima and minima of fish larvae increased significantly with increasing acclimation temperature, implying that the fish could develop some degrees of thermal tolerance through temperature acclimation.

Variation in tolerance to common marine pollutants among different populations in two species of the marine copepod Tigriopus.

Geographical variation in chemical tolerance within a species can significantly influence results of whole animal bioassays, yet a literature survey showed that the majority of articles using bioassays did not provide detail on the original field collection site of their test specimens confounding the ability for accurate replication and comparison of results. Biological variation as a result of population-specific tolerance, if not addressed, can be misinterpreted as experimental error. Our studies of two marine copepod species, Tigriopus japonicus and Tigriopus californicus, found significant intra- and inter-specific variation in tolerance to copper and tributyltin. Because both species tolerate copper concentrations orders of magnitude higher than those found in coastal waters, difference in copper tolerance may be a by-product of adaptation to other stressors such as high temperature. Controlling for inter-population tolerance variation will greatly strengthen the application of bioassays in chemical toxicity tests.

Acute and chronic toxicities of zinc pyrithione alone and in combination with copper to the marine copepod Tigriopus japonicus.

Zinc pyrithione (ZnPT) is a widely used booster biocide in combination with copper (Cu) in antifouling paints as a substitute for tributyltin. The co-occurrence of ZnPT and Cu in coastal marine environments is therefore very common, and may pose a higher risk to marine organisms if they can result in synergistic toxicity. This study comprehensively investigated the combined toxicity of ZnPT and Cu, on the marine copepod Tigriopus japonicus, for the first time, based on both 96-h acute toxicity tests using adult copepods and chronic full-life cycle tests (21 d) using nauplii <24-h old. As ZnPT has been reported to be easily trans-chelated to copper pyrithione (CuPT) in the presence of Cu, the acute toxicities of CuPT alone and in combination with Cu on adult copepods were also assessed. Our results showed that ZnPT and Cu exhibited a strong synergistic toxic effect on the copepod in both acute and chronic tests. During the acute test, the mortalities of adult copepods increased dramatically even with an addition of Cu at concentrations as low as 1-2 µg/L compared with those exposed to ZnPT alone. Severe chronic toxicities were further observed in the copepods exposed to ZnPT-Cu mixtures, including a significant increase of naupliar mortality, postponing of development from naupliar to copepodid and from copepodid to adult stage, and a significant decrease of intrinsic population growth when compared with those of copepods exposed to ZnPT or Cu alone. Such synergistic effects might be partly attributable to the formation of CuPT by the trans-chelation of ZnPT and Cu, because CuPT was found to be more toxic than ZnPT based on the acute toxicity results. Mixtures of CuPT and Cu also led to synergistic toxic effects to the copepod, in particular at high Cu concentrations. A novel non-parametric response surface model was applied and it proved to be a powerful method for analysing and predicting the acute binary mixture toxicities of the booster biocides (i.e., ZnPT and CuPT) and Cu on the copepod. To better protect precious marine resources, it is necessary to revise and tighten existing water quality criteria for biocides, such as ZnPT and CuPT, to account for their synergistic effects with Cu at environmentally realistic levels.

Temperature-dependent toxicities of four common chemical pollutants to the marine medaka fish, copepod and rotifer.

We hypothesize that chemical toxicity to marine ectotherms is the lowest at an optimum temperature (OT) and it exacerbates with increasing or decreasing temperature from the OT. This study aimed to verify this hypothetical temperature-dependent chemical toxicity (TDCT) model through laboratory experiments. Acute toxicity over a range of temperatures was tested on four commonly used chemicals to three marine ectotherms. Our results confirmed that toxicities, in terms of 96-h LC50 (median lethal concentration; for the marine medaka fish Oryzias melastigma and the copepod Tigriopus japonicus) and 24-h LC50 (for the rotifer Brachionus koreanus), were highly temperature-dependent, and varied between test species and between study chemicals. The LC50 value of the fish peaked at 20 °C for copper (II) sulphate pentahydrate and triphenyltin chloride, and at 25 °C for dichlorophenyltrichloroethane and copper pyrithione, and decreased with temperature increase or decrease from the peak (i.e., OT). However, LC50 values of the copepod and the rotifer generally showed a negative relationship with temperature across all test chemicals. Both copepod and rotifer entered dormancy at the lowest temperature of 4 °C. Such metabolic depression responses in these zooplanktons could reduce their uptake of the chemical and hence minimize the chemical toxicity at low temperatures. Our TDCT model is supported by the fish data only, whereas a simple linear model fits better to the zooplankton data. Such species-specific TDCT patterns may be jointly ascribed to temperature-mediated changes in (1) the physiological response and susceptibility of the marine ectotherms to the chemical, (2) speciation and bioavailability of the chemical, and (3) toxicokinetics of the chemical in the organisms.

Acute and chronic toxicities of Irgarol alone and in combination with copper to the marine copepod Tigriopus japonicus.

Irgarol 1051 has been widely used as a booster biocide in combination with copper (Cu) in antifouling paints. The combined toxicity of Irgarol with Cu on marine organisms, however, has not been fully investigated. This study investigated the acute and chronic toxicities of binary mixtures of Irgarol and CuSO(4) to the marine copepod Tigriopus japonicus. The acute combined toxicity of Irgarol and Cu was simple additive as revealed by two response surface models and their contours. However, based on chronic full life-cycle tests, when Irgarol was combined with Cu at an environmentally realistic concentration (10 µg L(-1)), a slightly synergistic effect was observed at a high Irgarol concentration (940 µg L(-1)), as shown by a significant increase in larval mortality. As Cu contamination is widespread in coastal environments, our results entail the importance of considering the combined toxic effect of the booster biocide and Cu for setting ecologically realistic water quality criteria.

Acute toxicities of five commonly used antifouling booster biocides to selected subtropical and cosmopolitan marine species.

Since 1990s, various booster biocides have been increasingly used as substitutes of organotins. However, knowledge about their toxicities on tropical/sub-tropical marine species is significantly lacking. This study comprehensively investigated the acute toxicities of copper, tributyltin (TBT), and five commonly used booster biocides including Irgarol, diuron, zinc pyrithione (ZnPT), copper pyrithione (CuPT) and chlorothalonil on the growth or survival of 12 marine species in which eight of them are native species of subtropical Hong Kong. We found that Irgarol was more toxic than TBT on the growth of autotrophic species. The toxicity of CuPT was comparable to that of TBT on almost all test species, while it showed higher toxicity than TBT on medaka fish larvae. As the usage of these biocides is expected to further increase worldwide, accurate assessments of their ecological risks are required for better informed decision on their management. This study provided useful datasets for such purposes.

Comparison of the Qwiklite algal bioluminescence test with marine algal growth rate inhibition bioassays.

Although marine algal bioassays based on growth rate inhibition over 72-96 h have been widely used to assess the toxicity of contaminants in waters and sediments, changes in pH over the test duration can lead to changes in contaminant speciation and consequently an under- or over-estimation of toxicity. In addition, high cell densities are used in order to obtain a detectable response, further reducing the tests' environmental relevance in marine waters. There is a need for rapid acute tests with ecologically relevant test endpoints that may be used as surrogates for longer-term chronic tests. This study compares the sensitivity and reproducibility of a rapid marine dinoflagellate (Pyrocystis lunula) bioluminescence test (QwikLite) with standard algal growth rate bioassays (Nitzschia closterium and Entomoneis c.f. punctulata) using ammonia and several antifouling agents (tributyltin [TBT], copper, and diuron) as reference toxicants. QwikLite was of similar sensitivity to ammonia as standard algal growth rate tests, but was less sensitive to copper, diuron and TBT, with 24-h EC50 values of 10 +/- 1.1 mg N/L, 0.128 +/- 0.021 mg Cu/L, 19 +/- 13 mg diuron/L, and 0.226 +/- 0.028 mg TBT/L. Inter-test precision using different batches of P. lunula was generally acceptable. On the basis of NOEC values, QwikLite was more sensitive to copper and ammonia at 25 degrees C than at 21 degrees C. QwikLite shows promise as a rapid, inexpensive screening test for acute toxicity of contaminants in marine environments.

Synergistic toxic effects of zinc pyrithione and copper to three marine species: Implications on setting appropriate water quality criteria.

Zinc pyrithione (ZnPT) is widely applied in conjunction with copper (Cu) in antifouling paints as a substitute for tributyltin. The combined effects of ZnPT and Cu on marine organisms, however, have not been fully investigated. This study examined the toxicities of ZnPT alone and in combination with Cu to the diatom Thalassiosira pseudonana, polychaete larvae Hydroides elegans and amphipod Elasmopus rapax. Importantly, ZnPT and Cu resulted in a strong synergistic effect with isobologram interaction parameter lambda>1 for all test species. The combined toxicity of ZnPT and Cu was successfully modelled using the non-parametric response surface and its contour. Such synergistic effects may be partly due to the formation of copper pyrithione. It is, therefore, inadequate to assess the ecological risk of ZnPT to marine organisms solely based on the toxicity data generated from the biocide alone. To better protect precious marine resources, it is advocated to develop appropriate water quality criteria for ZnPT with the consideration of its compelling synergistic effects with Cu at environmentally realistic concentrations.

Copper toxicity in the marine copepod Tigropus japonicus: low variability and high reproducibility of repeated acute and life-cycle tests.

The intertidal copeopod Tigriopus japonicus, which is abundant and widely distributed along the coasts of Western Pacific, has been suggested to be a good marine ecotoxicity testing organism. In this study, a series of experiments were conducted to investigate the reproducibility and variability of copper (Cu) sensitivity of T. japonicus so as to evaluate its potential to serve as an appropriate test species. To understand the seasonal variation of Cu sensitivity, individuals of T. japonicus were collected from the field in summer and winter, and subjected to standard 96 h acute (static renewal) toxicity tests. 96 h-LC50 values of T. japonicus collected from the two seasons were marginally different (p = 0.05), with an overall coefficient of variation (CV) of 33%. Most importantly, our results indicated that chronic Cu sensitivity of T. japonicus was highly reproducible. The CVs of intrinsic rates of increase in the population of the control and Cu treatment (10 microg Cu l(-1)) groups were only 10-11% between 10 runs of a standardised complete life-cycle test. Moreover, different Cu(II) salts generally resulted in a similar 96 h-LC50 value while Cu(I) chloride was consistently slightly less toxic than Cu(II) salts. Given such a high reproducibility of toxic responses, it is advocated to use T. japonicus as a routine testing organism.

Toxicities of antifouling biocide Irgarol 1051 and its major degraded product to marine primary producers.

Irgarol 1051 (2-methythiol-4-tert-butylamino-6-cyclopropylamino-s-triazine) is an algaecide commonly used in antifouling paints. It undergoes photodegradation which yields M1 (2-methylthio-4-tert-butylamino-6-amino-s-triazine) as its major and most stable degradant. Elevated levels of both Irgarol and M1 have been detected in coastal waters worldwide; however, ecotoxicity effects of M1 to various marine autotrophs such as cyanobacteria are still largely unknown. This study firstly examined and compared the 96 h toxicities of Irgarol and M1 to the cyanobacterium Chroococcus minor and two marine diatom species, Skeletonema costatum and Thalassiosira pseudonana. Our results suggested that Irgarol was consistently more toxic to all of the three species than M1 (96 h EC50 values: C. minor, 7.71 microug L(-1) Irgarol vs. > 200 microg L(-1) M1; S. costatum, 0.29 microg L(-1) Irgarol vs. 11.32 microg L(-1)M1; and T. pseudonana, 0.41 microg L(-1) Irgarol vs. 16.50 microg L(-1)M1). Secondly, we conducted a meta-analysis of currently available data on toxicities of Irgarol and M1 to both freshwater and marine primary producers based on species sensitivity distributions (SSDs). Interestingly, freshwater autotrophs are more sensitive to Irgarol than their marine counterparts. For marine autotrophs, microalgae are generally more sensitive to Irgarol than macroalgae and cyanobacteria. With very limited available data on M1 (i.e. five species), M1 might be less toxic than Irgarol; nonetheless this finding warrants further confirmation with additional data on other autotrophic species.