A single ThinPrep® slide may not be representative in all head and neck fine needle aspirate specimens

Objectives:  Ideally, head and neck aspiration should be performed by trained aspirators within the setting of a one-stop clinic, where smeared material is available for immediate assessment. However, this may not always be possible for practical reasons and the use of liquid-based techniques in head and neck cytology is increasing. Although liquid-based cytology has been extensively validated for use in gynaecological cytology, no studies have investigated whether or not a single ThinPrep ^® slide is representative for head and neck aspirate specimens. We performed a prospective audit of head and neck fine needle aspiration specimens processed by the ThinPrep ^® method to investigate whether a single ThinPrep ^® slide was representative.
Methods:  A prospective audit of 115 consecutive head and neck aspirates was carried out. A single ThinPrep ^® slide was prepared and a diagnosis recorded. The remainder of the specimen was then spun down and prepared as a cell block. The ThinPrep ^® and cell block diagnoses were compared.
Results:  In 36 cases (31%), the cell block provided additional information that contributed to the diagnosis. In 14 (12%), the cell block was regarded as essential to the diagnosis.
Conclusions:  A single ThinPrep^® slide may not provide representative diagnostic material in all head and neck aspirates. This should be taken into consideration when contemplating the use of liquid-based methods for non-gynaecological cytology.     

The effect of lubricant contamination on ThinPrep® (Cytyc) cervical cytology liquid-based preparations

Objectives:  To assess the extent of lubricant use by smear-takers and the effect of lubricant contamination of ThinPrep^® processed cervical cytology samples.
Methods:  All primary care smear-takers were sent a questionnaire on lubricant type and frequency of use. Fifty cervical cytology samples were then contaminated with incremental amounts of K-Y^® jelly, 50 samples contaminated with incremental amounts of Aquagel^® and ten non-contaminated vials were processed using the ThinPrep^® T2000 processor followed by Papanicolaou staining. The morphological appearances of lubricant contamination were described microscopically and formal cell counts performed on all slides.
Results:  Seventy of 94 (74.5%) primary care smear-takers indicated lubricant use of whom 9/70 (12.8%) used Aquagel^® and 61/70 (87.2%) used K-Y^® jelly. K-Y^® jelly appeared as mucoid blue deposits in the slide background whereas Aquagel^® appeared as pink stringy background material. Cell counting showed a significant difference between Aquagel^® and K-Y^® jelly contaminated slides compared to the original non-contaminated preparations for all fields and the average fields ( P  < 0.001) with a significantly higher count for the original non-contaminated slides than the lubricant contaminated groups.
Conclusion:  Lubricant contamination of ThinPrep^® cervical cytology samples may result in reduced cellularity of the subsequent slide. This study provides evidence-based data to support British Society for Clinical Cytology recommendations for no lubricant use when taking cervical samples.     

The role of liquid-based cytology in the investigation of thyroid lesions

Objective:  This study investigates the role of liquid-based cytology by ThinPrep^® technique in the detection of thyroid lesions.
Methods:  In all, 252 specimens from 157 patients for pre-operative evaluation of thyroid nodules, prepared by the ThinPrep^®, were examined. In all cases thyroidectomy followed the initial cytological evaluation. All cytological diagnoses were correlated to the histological ones.
Results:  According to our findings, a sensitivity of 87.80%, a specificity of 99.50%, a positive predictive value of 97.30%, a negative predictive value of 97.56% and an overall accuracy of 97.52% were observed in fine needle aspiration cytology in correlation to the histological diagnosis after thyroidectomy.
Conclusions:  ThinPrep^® technique is a valid method for the pre-operative cytological diagnosis of thyroid nodules, offering the possibility of ancillary techniques, such as immunocytochemical and molecular methods and can, therefore, be potentially complementary to histological evaluation for further investigation of follicular lesions.     

Biosurfactant production by Pseudomonas sp. and its role in aqueous phase partitioning and biodegradation of chlorpyrifos

Aim:  To study the effect of biosurfactant on aqueous phase solubility and biodegradation of chlorpyrifos.
Methods and Results:  A Pseudomonas sp. (ChlD), isolated from agricultural soil by enrichment culture technique in the presence of chlorpyrifos, was capable of producing biosurfactant (rhamnolipids) and degrading chlorpyrifos (0·01 g l⁻¹). The partially purified rhamnolipid biosurfactant preparation, having a CMC of 0·2 g l⁻¹, was evaluated for its ability to enhance aqueous phase partitioning and degradation of chlorpyrifos (0·01 g l⁻¹) by ChlD strain. The best degradation efficiency was observed at 0·1 g l⁻¹ supplement of biosurfactant, as validated by GC and HPLC studies.
Conclusion:  The addition of biosurfactant at 0·1 g l⁻¹ resulted in more than 98% degradation of chlorpyrifos when compared to 84% in the absence of biosurfactant after 120-h incubation.
Significance and Impact of the Study:  This first report, to the best of our knowledge, on enhanced degradation of chlorpyrifos in the presence of biosurfactant(s), would help in developing bioremediation protocols to counter accumulation of organophosphates to toxic/carcinogenic levels in environment.     

Longevity of the freshwater anostracan Streptocephalus mackini (Crustacean: Anostraca) in relation to food (Chlorella vulgaris) concentration

SUMMARY 1. Temporary ponds are inhabited by a variety of invertebrates, of which anostracans are an important group. We studied the lifetables of male and female anostracan Streptocephalus mackini at 3 algal concentrations (0.5 × 10⁶, 1.0 × 10⁶ and 1.5 × 10⁶ cells mL⁻¹).
2. Regardless of sex, S. mackini showed better survivorship at lower food levels. The longest average lifespan observed was 85 ± 2 days for males fed Chlorella at 0.5 × 10⁶ cells mL⁻¹.
3. Both net reproductive rate and generation time decreased with increasing food level. The highest net reproductive rate was about 120 cysts per female. The longest generation time of about 40 days, observed at 0.5 × 10⁶ cells mL⁻¹, was more than three times that at 1.5 × 10⁶ cells mL⁻¹.
4. The rate of population increase ( r ) was nearly the same (0.31 ± 0.06) at high (1.5 × 10⁶ cells mL⁻¹) and intermediate (1.0 × 10⁶ cells mL⁻¹) food levels. The r -value at low food level (0.5 × 10⁶ cells mL⁻¹ of Chlorella ) was 0.20 ± 0.01 per day.     

Seasonal patterns of viruses, bacteria and dissolved organic carbon in a riverine wetland

SUMMARY 1. Viral and bacterial abundances were studied in relation to environmental attributes over an annual period, for both planktonic and attached (sediment, aquatic macrophyte and submerged wood) habitats, in a riverine wetland.
2. Annual mean abundance of planktonic viruses ranged from 2.3 × 10⁵−3.8 × 10⁵ particles mL⁻¹ and varied according to sampling site. Significant seasonal patterns in viral abundance were evident and appeared to be linked to variations in bacterial abundance, dissolved organic carbon and inorganic nutrients.
3. Annual mean abundance of viruses associated with surfaces ranged from 1.3 × 10⁶ particles cm⁻² on aquatic macrophytes to 1.1 × 10⁷ particles cm⁻² on wood and also showed seasonal patterns. The difference in viral dynamics among the different sites emphasizes the importance of considering habitat diversity within wetlands when examining microbial communities.     

High-risk HPV detection in specimens collected in SurePath preservative fluid: comparison of ambient and refrigerated storage

Objective:  With moves to introduce human papillomavirus (HPV) triage at sentinel sites in England, it is essential that optimal storage and transport conditions are determined for efficient HPV detection using residual liquid-based cytology specimens.
Methods:  Two cytology laboratories with comparable workloads sent residual cervical cytology specimens collected in BD Surepath™ Preservative Fluid to the Specialist Virology Centre for HPV testing. Storage and transport of specimens was at ambient (site A) or refrigerated (site R) temperatures. The effect of temperature on the ability to detect high-risk human papillomavirus (HR-HPV) using Digene Hybrid Capture^® 2 High-Risk HPV DNA Test (hc2) and Roche AMPLICOR^® HPV Test (AMPLICOR) was assessed. All specimens with discordant results were tested using Roche Linear Array HPV Genotyping test.
Results:  A total of 796 residual cytology specimens, with cytology ranging from normal to severe dyskaryosis, were provided (399 from site A and 397 from site R). Ambient storage and transit of cervical specimens in SurePath medium did not appear to affect significantly the suitability of the specimen for HPV testing, as measured by the concordance of the HR-HPV screening assays for ambient versus refrigerated specimens and by the proportion of specimens which tested invalid.
Conclusion:  Residual cytology specimens in SurePath medium, stored and transported at ambient temperature, appear suitable for HR-HPV detection by AMPLICOR beyond the manufacturer's recommended time and potentially up to four weeks.     

Potassium dependence and phytoplankton ecology: an experimental study

SUMMARY 1. Unialgal cultures of three species common in the freshwater phytoplankton were used to test limitation of specific growth rate and final yield in defined media of low K⁺ concentration (range <0.3–6 μmol L⁻¹ or mmol m⁻³).
2. Growth rate of the diatom Asterionella formosa was independent of K⁺ concentration above 0.7 μmol L⁻¹. Final yield was dependent on initial concentration when accompanied by K⁺ depletion below this concentration, but not by lesser depletion with more residual K⁺. Analyses of particulate K in the biomass indicated a mean final cell content of 2.8 μmol K 10⁻⁸ cells, approximately 1.0% of the organic dry weight.
3. Less detailed work with the diatom Diatoma elongatum showed no dependence of growth rate or final yield upon the initial K⁺ concentration in the range 0.8–3.2 μmol L⁻¹. The phytoflagellate Plagioselmis nannoplanctica suffered net mortality in the lowest concentration tested, 0.8 μmol L⁻¹.
4. Comparison with the range of K⁺ concentration in natural fresh waters, including a depletion induced by an aquatic macrophyte, suggests that K⁺ is unlikely to limit growth of phytoplankton. Nevertheless, there can be correlation of K⁺ with lake trophy.     

Mg2+-free buffer elevates transformation efficiency of Vibrio parahaemolyticus by electroporation

Aims:  The ability to transform Vibrio spp. is limited by the extracellular nuclease that their cells secrete. The reported transformation efficiency of this organism is 10²–10⁵ transformants per microgram DNA. We tried different buffers and conditions, aiming to elevate its transformation efficiency.
Methods and Results:  MgCl₂ and sucrose are often included in the washing and/or electroporation buffers to stabilize the cell membrane. However, Mg²⁺ is required for production and activity of the extracellular nuclease. A simple electroporation buffer lacking Mg²⁺ was found to increase transformation efficiency dramatically, to levels 50-fold more than the buffers containing Mg²⁺. To maintain the stability of the cell membranes, Mg²⁺ was replaced with high concentrations of sucrose, from 272 to 408 mmol l⁻¹. With the new buffers, the transformation efficiency of Vibrio parahaemolyticus was increased to 2·2 × 10⁶ transformants per microgram DNA.
Conclusions:  Mg²⁺ in the buffer adversely affected transformation of V. parahaemolyticus by electroporation. The cell membranes of vibrio can be stabilized by high concentration of sucrose when Mg²⁺ is absent.
Significance and Impact of the Study:  A greater transformation efficiency can facilitate the genetic analysis of an organism and its pathogenicity. Buffers lacking Mg²⁺ can be used for other nuclease-producing organisms.     

Prospective study of urine cytology screening for BK polyoma virus replication in renal transplant recipients

Objective:  BK virus (BKV) may be associated with interstitial nephritis in renal transplant recipients and this can lead to irreversible chronic allograft dysfunction. Early diagnosis of BKV nephropathy determines its progress because no specific antiviral therapy exists. Urine cytology, detection of viral DNA in urine or blood and renal biopsy are the main diagnostic tools. The purpose of this study was to evaluate the use of urine cytology for diagnosis of BKV replication in renal graft recipients.
Patients and methods:  We studied 32 de novo renal transplant recipients prospectively with sequential urine samples for a period of 1 year. Thin-Prep methodology was used to prepare the slides. Cytology results were correlated with polymerase chain reaction (PCR) in urine and blood.
Results:  Decoy cells indicative of BKV infection were detected in 14 (7.3%) of the 190 urine samples derived from 11 recipients. In three cases with positive decoy cells, BK viraemia and viruria were simultaneously identified. In a further three cases, BKV active replication was confirmed in urine by both cytology and PCR.
Conclusions:  Urine cytology is an easy and rapid method of detecting decoy cells in cases where renal biopsy is not possible. However, the low incidence of detection of decoy cells in the present study, together with poor correlation with PCR results, questions its sensitivity and specificity in diagnosing BKV reactivation.     

Spinal cord degeneration in C57BL/6N mice following induction of experimental parkinsonism with MPTP

We examined neurodegeneration in spinal cord (SC) and role of such extra-nigral degeneration in MPTP-induced experimental parkinsonism in C57BL/6N mice. HPLC-photodiode array analysis confirmed presence of the active neurotoxin MPP⁺ in SC after single injection of MPTP (25 mg/kg, i.p.). Mitochondrial enzyme monoamine oxidase-B (MAO-B) responsible for in vivo conversion of MPTP to MPP⁺ was inhibited in SC by pre-treatment with l -deprenyl, a specific inhibitor of MAO-B. Besides in vitro conversion of MPTP to MPP⁺ occurred by SC mitochondrial preparation, which was inhibited by l -deprenyl implicating SC as a specific target of MPTP-neurotoxicity. Double immunofluorescent labeling and spectrofluorimetric assay via kynuramine oxidation showed MAO-B expression and activity in SC neurons. Localization of dopamine transporter immunoreactivity in SC along with specific uptake of ³H-MPP⁺ by SC synaptosomal preparation further confirmed SC as target of MPTP-neurotoxicity. Compared with control, increased neuronal death on the seventh day in SC of mice injected with MPTP (2 × 25 mg/kg, at 6 h interval) strongly suggested SC degeneration in pre-symptomatic phase of MPTP-induced experimental parkinsonism. Such extra-nigral neurodegeneration in Parkinson's disease indicated novel molecular mechanism preceding nigrostriatal degeneration and suggested designing broad therapeutic intervention for this complex movement disorder.     

Sucrose phosphorylase of the rumen bacterium Pseudobutyrivibrio ruminis strain A

Aims:  To verify the taxonomic affiliation of bacterium Butyrivibrio fibrisolvens strain A from our collection and to characterize its enzyme(s) responsible for digestion of sucrose.
Methods and Results:  Comparison of the 16S rRNA gene of the bacterium with GenBank showed over 99% sequence identity to the species Pseudobutyrivibrio ruminis . Molecular filtration, native electrophoresis on polyacrylamide gel, zymography and thin layer chromatography were used to identify and characterize the relevant enzyme. An intracellular sucrose phosphorylase with an approximate molecular mass of 52 kDa exhibiting maximum activity at pH 6·0 and temperature 45°C was identified. The enzyme was of inducible character and catalysed the reversible conversion of sucrose to fructose and glucose-1-P. The reaction required inorganic phosphate. The K _m for glucose-1-P formation and fructose release were 3·88 × 10⁻³ and 5·56 × 10⁻³ mol l⁻¹ sucrose, respectively – while the V _max of the reactions were −0·579 and 0·9  μ mol mg protein⁻¹ min⁻¹. The enzyme also released free glucose from glucose phosphate.
Conclusion:  Pseudobutyrivibrio ruminis strain A utilized sucrose by phosphorolytic cleavage.
Significance and Impact of the Study:  Bacterium P. ruminis strain A probably participates in the transfer of energy from dietetary sucrose to the host animal.     

Effect of heat treatment on Cronobacter spp. in reconstituted, dried infant formula: preparation guidelines for manufacturers

Aim:  To explore safe guidelines for manufacturers and consumers to prepare, handle and store dry infant formula (DIF) to protect infants against Cronobacter spp.
Methods and Results:  Selected strains (2.45, FSM 293, ATCC-12868, FSM-271) screened from 68 strains of Cronobacter spp . were used to study growth and survival in commercial DIF. Prototype growth patterns in Enterobacteriaceae enrichment broth (EEB) containing a cocktail comprised of ATCC 12868, ATCC 29004, ATCC 29544 and ATCC 51329 showed a rapid increase in cell count (2·0 log₁₀ to 6·2 log₁₀ CFU ml⁻¹). Infant formula provided a better protective environment for the cells of Cronobacter strains than did buffered peptone water . Experiments on survival in inoculated (10⁴–10⁶ CFU ml⁻¹) reconstituted infant formula (RIF), preparation temperature, the effect of preparation volume (one-serving or two-serving) and effect of storage at room temperature for up to 10 h provided information to develop consumer guidelines for DIF preparation and handling.
Conclusions:  Reconstituted DIF in water at >70°C in larger volumes, minimizing storage time before feeding and storing unused reconstituted formulate at <4°C, may reduce the risk of Cronobacter infection in infants.
Significance and Impact of the Study:  Meningitis, necrotizing enterocolitis and bacteremia in premature babies has been linked to contaminated milk powder and DIF; better handling practices may improve the safety of these foods for neonates.     

Response surface methodology to optimize the nutritional parameters for enhanced production of jasmonic acid by Lasiodiplodia theobromae

Aims:  To find out the cumulative effect of the nutritional parameters and to enhance the production of jasmonic acid (JA) in static fermentation by Lasiodiplodia theobromae using response surface methodology (RSM).
Method and Results:  Malt extract, sucrose, NaNO₃ and MgSO₄.7H₂O were analysed by a 30-trial central composite design using RSM for optimizing their concentrations in the medium and the effect of their mutual interaction on JA production. Sucrose and NaNO₃ were found highly significant in influencing the JA production. Malt extract and MgSO₄.7H₂O showed an effect on the JA production in interaction with other variables. When the optimum values of the parameters obtained through RSM (19·95 g l⁻¹ malt extract, 50 g l⁻¹ sucrose, 7·5 g l⁻¹ NaNO₃ and 3·51 g l⁻¹ MgSO₄.7H₂O) were applied, 32% increase in JA production (299 mg l⁻¹) was observed in comparison with 225 mg l⁻¹ of JA produced with same media components not analysed by RSM and subsequently validated the statistical model.
Conclusions:  Increase in JA production was achieved by optimizing the nutritional parameters.
Significance and Impact of the Study:  This is the first report of using RSM for optimizing a medium for JA production. It resulted in an increase in JA production without augmentation of costly additives.     

Could nuclear matrix protein 22 (NMP22) play a role with urine cytology in screening for bladder cancer? – experience at Kuwait University

Objectives:  This prospective study was undertaken to evaluate nuclear matrix protein (NMP22) compared to urine cytology in the detection of bladder cancer and also to determine whether indexing suspicious cytology to NMP22 could enhance the clinical utility of cytology.
Methods:  Cytological findings of voided urine collected prior to a cystoscopic biopsy were correlated with urine NMP22 assay in 46 patients attending the urology clinic in Mubarak Al-Kabeer Hospital. The patients were clinically categorized into newly diagnosed cases of transitional cell carcinoma (TCC), recurrent TCC, TCC in remission and controls.
Results:  Using histological diagnosis as the gold standard the sensitivity and specificity of NMP22 were 78% and 43% respectively and of cases with malignant urine cytology were 30% and 87% respectively. If suspicious and malignant cytology were combined as positive results the sensitivity increased significantly to 87% while the specificity decreased but not significantly to 74%. Suspicious or malignant cytology enhanced by positive NMP22 gave a sensitivity of 70% and specificity of 87% neither of which was significantly different from cytology alone. There were three false positive cases on cytology and 13 false positive cases on NMP22 assay. There were three false negative cytology and five false negative NMP22 cases but only one was false negative for both, resulting in a high sensitivity (96%) but low specificity (30%) if either positive NMP22 or malignant or suspicious cytology was taken as a positive result.
Conclusion:  Combining NMP22 with malignant or suspicious cytological result improved sensitivity for the detection of bladder cancer but with a major decrease in specificity, suggesting a potential role in screening rather than diagnosis.     

Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine

Aims:  Brettanomyces / Dekkera bruxellensis is a particularly troublesome wine spoilage yeast. This work was aimed at characterizing its behaviour in terms of growth and volatile compound production in red wine.
Methods and Results:  Sterile red wines were inoculated with 5 × 10³ viable cells ml⁻¹ of three B. bruxellensis strains and growth and volatile phenol production were followed for 1 month by means of plate counts and gas chromatography-mass spectrometry (GC-MS) respectively. Maximum population levels generally attained 10⁶–10⁷ colony forming units (CFU) ml⁻¹ and volatile phenol concentrations ranged from 500 to 4000 μg l⁻¹. Brettanomyces bruxellensis multiplication was also accompanied by the production of organic acids (from C₂ to C₁₀), short chain acid ethyl-esters and the 'mousy off-flavour' component 2-acetyl-tetrahydropyridine.
Conclusions:  Different kinds of 'Brett character' characterized by distinct metabolic and sensory profiles can arise in wine depending on the contaminating strain, wine pH and sugar content and the winemaking stage at which contamination occurs.
Significance and Impact of the Study:  We identified new chemical markers that indicate wine defects caused by B. bruxellensis. Further insight was provided into the role of some environmental conditions in promoting wine spoilage.     

An alternative real-time PCR method for the detection of thermotolerant Bacillus sensu lato contaminants in naturally-contaminated gelatine

Aims:  Comparison of an internally-controlled real-time PCR assay with the current plate-based assay for the detection of Bacillus sensu lato contaminants in gelatine.
Methods and Results:  A comprehensive TaqMan^® probe was designed allowing the real-time PCR assay to be fully inclusive for the gelatine-contaminating Bacillus s.l. species. An internal amplification control was implemented at 500 copies per reaction without impact on target detection. Specific and selective detection of target cells was achieved with a quick and simple DNA preparation procedure. No significant difference (Kappa value = 0·94) was observed between the performance of the real-time PCR and the current plate-based method on naturally contaminated gelatines ( n  = 162). Relative accuracy, relative sensitivity and relative specificity were 97·5%.
Conclusions:  The real-time PCR assay is an adequate alternative of the current plate-based assay.
Significance and Impact of the Study:  The real-time PCR assay decreased the time between sample collection and result from 2 days to 2 h. The gelatine-producing industry can ensure gelatine quality in a much faster way.     

Female control of the embryonic environment in a terrestrial amphipod, Mysticotalitrus cryptus (Crustacea)

1. In amphipod crustaceans the ventral chamber plays an integral role in a number of physiological processes and in the female forms the marsupium in which eggs are brooded. The ventral chamber can be viewed as a pre-adaptation to the colonization of land by the family Talitridae. The hypothesis that the female of the terrestrial species, Mysticotalitrus cryptus , can control the osmotic concentration of the marsupial fluid ([MF]) bathing the eggs, thereby buffering the brood from potential physiological stresses presented by the terrestrial environment, is examined.
2. [MF] was maintained significantly higher than the concentration of the external medium ([Medium]) on both dechlorinated tap-water and 400 mOsm kg^–1 media. In each case, [MF] was intermediate to [Medium] and the concentration of the female haemolymph ([Haem]): when [Medium] = 40 mOsm kg^–1, [MF] = 277 mOsm kg^–1 and [Haem] = 590 mOsm kg^–1, respectively, and when [Medium] = 413 mOsm kg^–1, [MF] = 516 mOsm kg^–1 and [Haem] = 722 mOsm kg^–1, respectively.
3. Evidence is provided that females produce urine that is isosmotic with the haemolymph and that the urine is directed, by capillarity, into the marsupium via cuticular channels. It is suggested that this urine plays a role in controlling [MF] in combination with other behavioural mechanisms.
4. Some preliminary observations are presented on the ontogeny of embryonic osmoregulation in M. cryptus which suggest that osmoregulatory ability improves with developmental stage. There is also limited evidence for the ability of the late embryonic stages to hypo-osmoregulate on concentrated media, even though adults lack this capacity.
5. The results are discussed in relation to the colonization of the terrestrial environment by the Talitridae.     

Long-term effects of successive Ca(OH)2 and CaCO3 treatments on the water quality of two eutrophic hardwater lakes

1. Whole-lake experiments were conducted in two hardwater lakes (Halfmoon and Figure Eight) in Alberta, Canada, to investigate the effectiveness of repeated lime (slaked lime: Ca(OH)₂ and/or calcite: CaCO₃) treatments (5–78 mg L^–1) for up to 7 years.
2. Randomized intervention analysis of intersystem differences between the experimental and three reference lakes demonstrated a decline in euphotic total phosphorus and chlorophyll a concentrations in the experimental lakes after repeated lime treatments.
3. After the second lime application to Halfmoon Lake, mean winter total phosphorus release rates (TPRR) decreased to < 1 mg m^–2 day^–1 compared with 3.6 mg m^–2 day^–1 during the winter after initial treatment. In the final year of lime application, mean summer TPRR decreased to 4.5 mg m^–2 day^–1 compared with 7.6 mg m^–2 day^–1 in the pre-treatment year.
4. Mean macrophyte biomass declined and species composition was altered at 1 and 2 m depths in Figure Eight Lake during lime application. Over the first 6 years of treatment, macrophyte biomass at 2 m declined by 95% compared with concentrations recorded during the initial treatment year. In the last year of the study, macrophyte biomass at 2 m reached initial treatment concentrations, which coincided with the greatest water transparency. Over the treatment period, macrophyte species shifted from floating to rooted plants.
5. Multiple lime applications can improve water quality in eutrophic hardwater lakes for periods of up to 7 years.