Christian Blaise

Biomarkers in zebra mussels (Dreissena polymorpha) for the assessment and monitoring of water quality of the St Lawrence River (Canada)

Five biomarkers (MT: metallothionein-like proteins, EROD: ethoxyresorufin ortho-dééthylase, DNA strand breaks, LPO: peroxidation of lipids, VG: vitellogenin-like protiens) were measured in the soft tissues of zebra mussels (Dreissena polymorpha) in order to assess the spatial variation of exposure to contaminants along the St Lawrence River (Canada). Fifteen mussels >25 mm shell length were analyzed from each of the 13 sampling sites. Significant differences between sites were noted for all biomarkers, but the general level of variability was low. Three biomarkers (DNA, LPO and VG) exhibited a similar pattern of spatial variation while MT and EROD had distinct and specific patterns. MT had the strongest discriminating power and EROD showed the largest range of variation among sites. Highest biomarker responses were measured in specimens from local contaminated sites such as harbors and industrial sectors. A positive relationship was found between MT and copper (Cu), but no significant correlation was observed between other biomarker responses and the levels of ten trace metals bioaccumulating in the zebra mussels tissues. Results indicate that the measurement of biomarker responses is technically feasible. The performance of each biomarker is assessed in the context of the role and advantages of selecting a battery of biomarkers for detecting contamination problems. The use of zebra mussels as a sentinel species for biomonitoring potential toxic effects in situ is discussed.

Ecotoxicity of selected nano-materials to aquatic organisms.

Present knowledge concerning the ecotoxic effects of nano‐materials is very limited and merits to be documented more fully. For this purpose, we appraised the toxicity of nine metallic nano‐powders (copper zinc iron oxide, nickel zinc iron oxide, yttrium iron oxide, titanium dioxide, strontium ferrite, indium tin oxide, samarium oxide, erbium oxide, and holmium oxide) and of two organic nano‐ powders (fullerene‐C60 and single‐walled carbon nanotube or SWCNT). After a simple process where nano‐powders (NPs) were prepared in aqueous solution and filtered, they were then bioassayed across several taxonomic groups including decomposers (bacteria), primary producers (micro‐algae), as well as primary and secondary consumers (micro‐invertebrates and fish). Toxicity data generated on the 11 NPs reflected a wide spectrum of sensitivity that was biological level‐, test‐, and endpoint‐specific. With all acute and chronic tests confounded for these 11 NPs, toxicity responses spanned over three orders of magnitude: >463 mg/L (24 h LC50 of the invertebrate Thamnoplatyurus platyurus for fullerene‐C60) ÷ 0.3 mg/L (96 h EC50 of the invertebrate Hydra attenuata for indium tin oxide), that is a ratio of 1543. On the basis of the MARA (Microbial Array for Risk Assessment) assay toxic fingerprint concept, it is intimated that NPs may have different modes of toxic action. When mixed in a 1:1 ratio with a certified reference material (CRM) sediment, two solid phase assays and an elutriate assay, respectively, showed that five NPs (copper zinc iron oxide, samarium oxide, erbium oxide, holmium oxide, and SWCNT) were able to increase both CRM sediment toxicity and its elutriate toxicity. This initial investigation suggests that chemicals emerging from nanotechnology may pose a risk to aquatic life in water column and sediment compartments and that further studies on their adverse effects are to be encouraged.

Determination of vitellogenin-like properties in Mya arenaria hemolymph (Saguenay Fjord, Canada): A potential biomarker for endocrine disruption

The level of vitellogenin‐like (Vg‐like) proteins in the hemolymph of the soft‐shell clam (Mya arenaria) was indirectly determined using an alkali‐labile phosphate (ALP) measurement assay. Biochemical characterization of hemolymph from actively reproducing clams showed the presence of Zn, Ca and of substantial amounts of ALP, all of which are indicative of fish serum vitellogenin, reported to be a Zn and Ca containing glycolipophosphoprotein. ALP assays undertaken on different phosphate‐containing compounds (phosphate‐buffer saline, diphospholipids and 12,000×g supernatants of Vg‐rich trout ovary homogenates) showed little interference by the first two substrates and confirmed the specificity of the ALP assay as an indirect method of determining Vg‐like proteins in clam hemolymph. Moreover, clams injected with 17β‐estradiol and two known xenoestrogens (nonylphenol and pentachlorophenol) significantly induced hemolymph ALP levels. Field work revealed significant differences in hemolymph ALP levels from clams collected at specific intertidal stations of the Saguenay Fjord during temporal (May to October) and spatial (June) surveys in 1997, which appear to indicate that (anti‐)estrogenic chemicals may be present in the Saguenay Fjord. Our preliminary results thus show that this simple and rapid ALP assay holds promise as a biomarker of estrogen disruption with molluscan shellfish as bioindicators. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 455–465, 1999

Evaluation of estrogenic effects of municipal effluents to the freshwater mussel Elliptio complanata.

Municipal effluents are an important source of estrogens to the aquatic environment. The purpose of this study was to examine the estrogenicity of municipal effluents to the indigenous freshwater mussel, Elliptio complanata. First, estradiol-binding sites in gonad homogenates were characterized to determine the binding affinity and specificity of estrogens. Mussels were exposed to increasing concentrations of a municipal effluent for 96 h at 15 degrees C. In another experiment, mussels were placed in cages and submerged for 62 days at 1.5 km upstream and 5 km downstream of a municipal effluent plume in the St. Lawrence River. Mussels were harvested for assessment of vitellogenin-like proteins in the hemolymph and determination of total lipid, carbohydrate and protein in the gonad. The presence of specific estrogen-binding sites was found in both male and female gonads. Binding of estradiol to cytosol proteins reached saturation, yielding a dissociation constant of 0.4 nM. Vitellogenin (Vg) levels increased significantly in both the hemolymph and the gonad after exposure to the effluent. Moreover, females appeared to be more sensitive than males to producing Vg. Mussels exposed in situ to contaminated surface waters had higher levels of Vg at the downstream site, again, females had higher levels of Vg than did males. On the other hand, lipid and sugar levels in male gonads were significantly increased at the downstream site. Moreover, mussels at the downstream site had decreased shell growth length and increased total and soft tissue weights. We conclude that municipal effluents contain bio-available xenoestrogens at levels sufficient to elicit effects in freshwater mussels.

An ecotoxicological characterization of nanocrystalline cellulose (NCC)

Abstract The pulp and paper industry in Canada is developing technology for the production and use of nanocrystalline cellulose (NCC). A key component of the developmental work is an assessment of potential environmental risks. Towards this goal, NCC samples as well as carboxyl methyl cellulose (CMC), a surrogate of the parent cellulosic material, were subjected to an ecotoxicological evaluation. This involved toxicity tests with rainbow trout hepatocytes and nine aquatic species. The hepatocytes were most sensitive (EC20s between 10 and 200 mg/l) to NCC, although neither NCC nor CMC caused genotoxicity. In tests with the nine species, NCC affected the reproduction of the fathead minnow at (IC25) 0.29 g/l, but no other effects on endpoints such as survival and growth occurred in the other species at concentrations below 1 g/l, which was comparable to CMC. Based on this ecotoxicological characterization, NCC was found to have low toxicity potential and environmental risk.

Occurrence of alkylphenol polyethoxylates in the St. Lawrence River and their bioconcentration by mussels (Elliptio complanata).

A study was conducted in 1999 to determine the occurrence of alkylphenol polyethoxylates in the St. Lawrence River and their bioconcentration by mussels (Elliptio complanata). Concentrations of selected contaminants were measured in surface water, municipal effluent, sediments and mussels. Analyses were performed on 4-tert-octylphenol (4-t-OP), 4-n-nonylphenol (4-n-NP), nonylphenol polyethoxylates (NP(1-16)EO), nonylphenol-mono and di-ethoxycarboxylic acids (NP(1)EC and NP(2)EC), and octylphenol-mono and di-ethoxycarboxylic acids (OP(1)EC and OP(2)EC). Mussels (Elliptio complanata) taken from a reference lake were placed in cages and submerged for 62 days at two sites in the St. Lawrence River, 1.5 km upstream and 5 km downstream of the outfall of a municipal wastewater treatment plant. The results showed that many of the target chemicals were present in all matrices studied: in water, at ppt and ppb levels, and reaching ppm levels in sediments and mussels. Concentrations of these contaminants were higher in matrices sampled at the downstream site than in those drawn at the site upstream of the Montreal effluent outfall, especially in sediments. Likewise, the slight, but not significant, bioconcentration of certain alkylphenol polyethoxylates (AP(n)EO) in the mussels was more noticeable at the downstream site than at the upstream site.

Toxic effects and bioaccumulation of carbamazepine evaluated by biomarkers measured in organisms of different trophic levels

The antiepileptic drug carbamazapine (CBZ) readily persists in sewage-water treatment plant wastewaters and finds its way into receiving water bodies. Our study sought to examine the bioaccumulation and toxicity of CBZ using an experimental aquatic trophic chain composed of the green alga, Pseudokirchneriella subcapitata, the crustacean, Thamnocephalus platyurus, and the cnidarian, Hydra attenuata. Bioaccumulation of CBZ was estimated by liquid chromatography-tandem mass spectrometry and revealed bioaccumulation factors of 2.2 and 12.6, respectively, in algae and crustaceans. No significant bioaccumulation was observed in H. attenuata. In T. platyurus, a strong stimulation of global heme oxidase (HO) (76%), and glutathione-S-transferase activity (130%) but a drastic inhibition of cytochrome P450 3A-like activity was found which suggests alteration of enzyme activity by CBZ. However, in the hydranth H. attenuata, an increase in both global cytochrome and cytochrome P450 3A-like activity was found, while GST activity was inhibited. Lipid peroxidation was reduced in T. platyurus and H. attenuata suggesting that redox activity of the lipophilic CBZ was at play. This study highlighted the processes of carbamazepine toxicity transfer between trophic levels in aquatic organisms.

Hepatic metallothionein level and mixed function oxidase activity in fingerling rainbow trout (Oncorhynchus mykiss) after acute exposure to pulp and paper mill effluents

Hepatic metallothionein (MT) levels and mixed function oxidase (MFO) activity (7-ethoxyresorufin-o-deethylase or EROD) were measured in fingerling rainbow trout (Oncorhynchus mykiss) exposed to sublethal concentrations of 12 pulp and paper effluents, after completion of 96 h static acute lethality assays. Barring one primary-treated effluent where MFO levels were significantly depressed and two secondary-treated effluents where no significant MFO induction were observed, all other effluents triggered significant induction of MT and EROD, regardless of mill process/treatment or of effluent lethality and chemical characteristics. MT and EROD inductions were significant, however, at higher concentrations for secondary-treated effluents than for primary-treated ones. Lethal (96 h LC50s) to sublethal (MT and EROD lowest observable effect concentrations) ratios were variable and indicated that significant biochemical effects were present at effluent concentrations that were roughly 4–33 (MT) and 3–59 (EROD) times lower than the LC30. Enzyme induction ranged from 1.3 to 2.5-fold for MT and from 1.3 to 9.4-fold for EROD compared to controls. Limited chemical data available suggest that there were indeed classes of compounds present capable of inducing MT or EROD. Observed patterns of MT/MFO responses also suggest that contaminant interactions may have interfered with induction for some of the effluents studied. Refinements of this combined (sub)lethal bioassay procedure are envisaged to determine whether it can provide an efficient means of detecting hazardous chemicals in industrial wastewaters.

Delayed gametogenesis of Mya arenaria in the Saguenay fjord (Canada): a consequence of endocrine disruptors?

The Saguenay fjord located in Canada on the north coast of the Saint-Lawrence Estuary, is well known for its multiple contaminations following a linear distribution upstream to downstream. Mya arenaria is well established in the fjord and potentially exposed to persistent sediment contamination as an endobenthic bivalve. From May to October 1997, clams energy storage and utilization in the gonad was shown to be closely linked with reproduction. Wherever the sampling site location in the fjord, a seasonal pattern is evident for lipid and glycogen levels in the gonad, this, reflecting the vitellogenic process. Nevertheless, in comparison with energy status and gametogenesis of clams collected on the north coast of the Saint-Lawrence Estuary, clams located in the fjord have to face limited nutritive conditions resulting in a single and shorter reproductive period. Although environmental factors could explain differences in physiological condition and reproductive status observed between clams from the fjord and the estuary, our results can discriminate clams from the upper part of the fjord, according to a delayed gametogenesis, concomitant with significant higher gonad glycogen concentrations observed in June 1996 and 1997. A persistent dysfunction of a vitellogenic process is suspected to be due to the exposure to anti-estrogenic contaminants.

Alteration of the biochemical properties of female gonads and vitellins in the clam Mya arenaria at contaminated sites in the Saguenay Fjord

Vitellins (Vn) are the major egg yolk proteins that constitute an important energy reserve for mollusc embryos. The purpose of the present study was to examine whether the relative levels of sugars, lipids, phosphates, and labile IIb metals and calcium normally associated with Vn would differ in clam populations living at contaminated sites. Softshell clams (Mya arenaria) were collected at three sites in the area of the Saguenay Fjord: a marina, a municipal sewer outfall zone, and a reference site. The condition factor (weight:length ratio), metallothionein-like proteins, cytochrome P450 1A1 activity and DNA damage were all determined in the clams digestive gland. Levels of total sugars, lipids, alkali-labile phosphates, proteins, and labile zinc and calcium were determined in female gonad homogenates and in purified Vn. The results show that clam gonads at the contaminated site by a marina displayed a lower index of gonad activity than the reference site. In addition, the condition factor was significantly lower at the marina site as compared to the reference site, with a concomitant increase in DNA damage and metallothionein (MT) induction. In fact, the condition factor was significantly correlated with DNA damage (R = -0.413, P = 0.045) and MT levels (R = -0.622, P = 0.03). Homogenates of female gonads were found to contain higher levels of labile IIb metals and calcium, with lower lipid content at the marina site compared to the reference site, and much higher levels of alkali-labile phosphates (ALP) and calcium at the municipal outfall site. Vn from the marina site were significantly higher in labile IIb metals but lipid content appeared to be somewhat lower than at the reference site. Vn from the municipal site were found to be highly phosphorylated, with higher levels of lipids and calcium. These results suggest that the chemical composition of the gonads and Vn are altered in contaminated sites.