The Gothenburg H70 Birth cohort study 2014-16: design, methods and study population.

To improve health care for older persons, we need to learn more about ageing, e.g. identify protective factors and early markers for diseases. The Gothenburg H70 Birth Cohort Studies (the H70 studies) are multidisciplinary epidemiological studies examining representative birth cohorts of older populations in Gothenburg, Sweden. So far, six birth cohorts of 70-year-olds have been examined over time, and examinations have been virtually identical between studies. This paper describes the study procedures for the baseline examination of the Birth cohort 1944, conducted in 2014-16. In this study, all men and women born 1944 on specific dates, and registered as residents in Gothenburg, were eligible for participation (n = 1839). A total of 1203 (response rate 72.2%; 559 men and 644 women; mean age 70.5 years) agreed to participate in the study. The study comprised sampling of blood and cerebrospinal fluid, psychiatric, cognitive, and physical health examinations, examinations of genetics and family history, use of medications, social factors, functional ability and disability, physical fitness and activity, body composition, lung function, audiological and ophthalmological examinations, diet, brain imaging, as well as a close informant interview, and qualitative studies. As in previous examinations, data collection serves as a basis for future longitudinal follow-up examinations. The research gained from the H70 studies has clinical relevance in relation to prevention, early diagnosis, clinical course, experience of illness, understanding pathogenesis and prognosis. Results will increase our understanding of ageing and inform service development, which may lead to enhanced quality of care for older persons.

Higher CSF interleukin-6 and CSF interleukin-8 in current depression in older women. Results from a population-based sample.

OBJECTIVE: The literature regarding cerebrospinal fluid (CSF) cytokines in geriatric depression is sparse. The aim of this study was to examine associations between CSF interleukin-6 (IL-6), interleukin-8 (IL-8) and depression in a population-based sample of older women who were followed for 17 years. METHODS: 86 dementia-free women aged 70-84 years who participated in the Prospective Population Study of Women in Gothenburg, Sweden took part in a lumbar puncture in 1992-3. CSF IL-6 and CSF IL-8 were measured. Psychiatric symptoms were rated with the Comprehensive Psychopathological Rating Scale at baseline and at three subsequent face-to-face examinations. Depression (major or minor) was diagnosed in accordance with DSM-IV/DSM-IV research criteria. RESULTS: At baseline, women with ongoing major (n=10) or minor depression (n=9) had higher levels of CSF IL-6 (p=0.008) and CSF IL-8 (p=0.007) compared with those without depression (n=67). Higher CSF IL-8 was related to higher MADRS score (p=0.003). New cases of depression were observed in 9 women during follow-ups. No associations between CSF cytokine levels and future depression could be shown in women without depression at baseline. CONCLUSION: Higher levels of CSF IL-6 and IL-8 were associated with current depression in this population-based sample. CSF IL-6 and CSF IL-8 may play a role in depression in late life.

Effect of hydrothermally carbonized char application on trace gas emissions from two sandy soil horizons.

The application of biochar to soil is a potential tool for the long-term sequestration of C and a possible mitigation of greenhouse gas (GHG) emissions. Among the various processes available to produce biochar, hydrothermal carbonization is one technique that is suitable for moist feedstock like digestates from biogas production. The aim of this study was to investigate the stability of C and emissions of NO after the addition of (i) digested wheat ( L.) straw (digestate) and (ii) hydrothermally carbonized (HTC) char of wheat straw as well as (iii) HTC char of digested wheat straw to two soil horizons that differed in C content. The HTC chars were obtained from wheat straw and digested wheat straw that were hydrothermally carbonized at 230°C for 6 h. The digestate and HTC chars were mixed with soil and incubated in 125-mL vessels. The GHG emissions of CO and NO were measured at regular intervals. Additionally, after 108 d, N was applied in the form of NHNO equivalent to 100 kg N ha. After 500 d of incubation, the digestate had lost 34% of C, while the soil mixture with the corresponding HTC char lost 12% of C in the form of CO from the topsoil. The estimated bi-exponential half-life of the recalcitrant C was more than 50% longer for the carbonized material than for the untreated digestate. The NO emissions from both HTC chars were significantly reduced compared with untreated digestate. The reductions were up to 64% for the topsoil and 60% for the subsoil samples. These laboratory results show that HTC holds the potential to increase the C stability of fermented and carbonized biomasses and to reduce NO emissions.

Lower CSF interleukin-6 predicts future depression in a population-based sample of older women followed for 17 years.

OBJECTIVE: The literature regarding cerebrospinal fluid (CSF) cytokines in geriatric depression is sparse. The aim of this study was to examine associations between CSF interleukin-6 (IL-6) and related proinflammatory cytokines and current and future depression in a population-based sample of older women who were followed for 17 years. METHODS: 83 non-demented women aged 70-84 years who participated in the Prospective Population Study of Women in Gothenburg, Sweden took part in a lumbar puncture in 1992-3. CSF- IL-6, interleukin-1ß (IL-1ß), interleukin- 8 (IL-8) and tumor necrosis factor-α (TNF-α) were measured. Psychiatric symptoms were rated with the Comprehensive Psychopathological Rating Scale at baseline and at three subsequent face-to-face examinations. Depression (major or minor) was diagnosed in accordance with DSM-IV/DSM-IV research criteria. RESULTS: At baseline, women with ongoing depression had lower levels of IL-6 (p<0.04), IL-8 (p<0.05) and TNF-α (p<0.05) compared with those without depression. In women without depression at baseline, lower CSF IL-6 levels predicted depression at one or more follow-up examination (p<0.03). Results from the generalized linear mixed logistic model using all baseline and follow-up data on depression status and Mini Mental State Examination score showed a significant relationship between IL-6 and depression (p=0.005 OR 0.370 CI [0.184-0.744]). CONCLUSION: Lower levels of CSF IL-6 were associated with current depression and with future depression during a follow-up of almost two decades. Our findings suggest that lower levels of CSF IL-6 may be related to depression vulnerability in later life.

Cerebrospinal fluid biomarkers of axonal and synaptic degeneration in a population-based sample.

BACKGROUND: Neurofilament light (NfL) and neurogranin (Ng) are promising candidate AD biomarkers, reflecting axonal and synaptic damage, respectively. Since there is a need to understand the synaptic and axonal damage in preclinical Alzheimer's disease (AD), we aimed to determine the cerebrospinal fluid (CSF) levels of NfL and Ng in cognitively unimpaired elderly from the Gothenburg H70 Birth Cohort Studies classified according to the amyloid/tau/neurodegeneration (A/T/N) system. METHODS: The sample consisted of 258 cognitively unimpaired older adults (age 70, 129 women and 129 men) from the Gothenburg Birth Cohort Studies. We compared CSF NfL and Ng concentrations in A/T/N groups using Student's T-test and ANCOVA. RESULTS: CSF NfL concentration was higher in the A-T-N+ group (p=0.001) and the A-T+N+ group (p=0.006) compared with A-T-N-. CSF Ng concentration was higher in the A-T-N+, A-T+N+, A+T-N+, and A+T+N+ groups (p<0.0001) compared with A-T-N-. We found no difference in NfL or Ng concentration in A+ compared with A- (disregarding T- and N- status), whereas those with N+ had higher concentrations of NfL and Ng compared with N- (p<0.0001) (disregarding A- and T- status). CONCLUSIONS: CSF NfL and Ng concentrations are increased in cognitively normal older adults with biomarker evidence of tau pathology and neurodegeneration.

Cognitive Performance and Cerebrospinal Fluid Markers in Preclinical Alzheimer's Disease: Results from the Gothenburg H70 Birth Cohort Studies.

BACKGROUND: We have previously shown that older adults with preclinical Alzheimer's disease (AD) pathology in cerebrospinal fluid (CSF) had slightly worse performance in Mini-Mental State Examination (MMSE) than participants without preclinical AD pathology. OBJECTIVE: We therefore aimed to compare performance on neurocognitive tests in a population-based sample of 70-year-olds with and without CSF AD pathology. METHODS: The sample was derived from the population-based Gothenburg H70 Birth Cohort Studies in Sweden. Participants (n = 316, 70 years old) underwent comprehensive cognitive examinations, and CSF Aß-42, Aß-40, T-tau, and P-tau concentrations were measured. Participants were classified according to the ATN system, and according to their Clinical Dementia Rating (CDR) score. Cognitive performance was examined in the CSF amyloid, tau, and neurodegeneration (ATN) categories. RESULTS: Among participants with CDR 0 (n = 259), those with amyloid (A+) and/or tau pathology (T+, N+) showed similar performance on most cognitive tests compared to participants with A-T-N-. Participants with A-T-N+ performed worse in memory (Supra span (p = 0.003), object Delayed (p = 0.042) and Immediate recall (p = 0.033)). Among participants with CDR 0.5 (n = 57), those with amyloid pathology (A+) scored worse in category fluency (p = 0.003). CONCLUSION: Cognitively normal participants with amyloid and/or tau pathology performed similarly to those without any biomarker evidence of preclinical AD in most cognitive domains, with the exception of slightly poorer memory performance in A-T-N+. Our study suggests that preclinical AD biomarkers are altered before cognitive decline.

Degradability of raw and post-processed chars in a two-year field experiment.

The object of the present work was to analyse the degradation dynamics of four chars and a digestate applied to a sandy soil as well as possible initial priming effects on the mineralisation of soil organic carbon in a two-year field experiment. For that purpose, soil carbon content, soil respiration and the corresponding carbon isotopic abundances were repeatedly measured throughout two consecutive vegetation periods. In order to quantify and separate the amount and the degradation of the substrate-derived carbon and to assess soil priming effects, isotopic mixing models were applied to soil-derived and substrate-derived carbon, and to the respired CO2. Pyrolysis char was degraded with decreasing intensity over time with an estimated half-life of about 80years. HTC (HydroThermal Carbonisation) char showed a high degradation during the first year but, during the second year, the remaining recalcitrant pool was degraded much slower with a half-life between 49 and 61years. Digestate was degraded at a constant intensity with a half-life of about 14years. When the chars were fermented before being applied to the soil, the initial degradation of HTC char was reduced, but on the two-year scale, the degradation of both chars was higher than for untreated chars, yielding a half-life between 11 and 15years, comparable to digestate. The results showed considerable stability of the untreated pyrolysis and HTC chars under field conditions, and moreover, no net influence of chars as well as of digestate on the degradation of soil organic carbon after two years.

Toxicity screening of biochar-mineral composites using germination tests.

This study assessed the properties and toxicity (water cress germination trials) of 38 waste-derived, novel biochar-mineral composites (BMCs) produced via slow pyrolysis and hydrothermal carbonization (hydrochars). The biochars were produced from sewage sludge and compost-like output (CLO) by varying the type of mineral additive (zeolite, wood ash and lignite fly ash), the mineral-to-feedstock ratio and the carbonization process. While pure hydrochars completely inhibited germination of water cress, this effect was ameliorated by mineral additives. Seedlings grew best in pyrolysis chars and while wood ash addition decreased plant growth in many cases, 1:10 addition to CLO doubled germination rate. The factors responsible for the phytotoxicity can be attributed to pH, salinity and organic contaminants. Importantly, while pure minerals inhibited germination, conversion of minerals into BMCs reduced their inhibitory effects due to buffered release of minerals. Overall, mineral wastes (e.g., combustion ashes) and waste biomass can be used safely as sources of nutrients and stable organic carbon (for soil carbon sequestration) when converted into specific biochar-mineral composites, exploiting synergies between the constituents to deliver superior performance.

Prevalence of preclinical Alzheimer disease: Comparison of current classification systems.

OBJECTIVE: To determine the prevalence of preclinical Alzheimer disease (AD) according to current classification systems by examining CSF from a representative general population sample of 70-year-olds from Gothenburg, Sweden. METHOD: The sample was derived from the population-based H70 Gothenburg Birth Cohort Studies in Gothenburg, Sweden. The participants (n = 322, age 70 years) underwent comprehensive neuropsychiatric, cognitive, and somatic examinations. CSF levels of ß-amyloid (Aß)42, Aß40, total tau, and phosphorylated tau were measured. Preclinical AD was classified according to criteria of the A/T/N system, Dubois 2016, National Institute on Aging-Alzheimer's Association (NIA-AA) criteria, and International Working Group-2 (IWG-2) criteria. Individuals with Clinical Dementia Rating score >0 were excluded, leaving 259 cognitively unimpaired individuals. RESULTS: The prevalence of amyloid pathology was 22.8%, of total tau pathology was 33.2%, and of phosphorylated tau pathology was 6.9%. With the A/T/N system, the prevalence of A+/T-/N- was 13.1%, A+/T-/N+ was 7.3%, A+/T+/N+ was 2.3%, A-/T-/N+ was 18.9%, and A-/T+/N+ was 4.6%. When the Dubois criteria were applied, the prevalence of asymptomatic at risk for AD was 36.7% and of preclinical AD was 9.7%. With the NIA-AA criteria, the prevalence of stage 1 was 13.1% and stage 2 was 9.7%. With the IWG-2 criteria, the prevalence of asymptomatic at risk for AD was 9.7%. The APOE ε4 allele was associated with several of the categories. Men more often had total tau pathology, A+/T-/N+, preclinical AD according to Dubois 2016, asymptomatic at risk for AD according to the IWG-2 criteria, and NIA-AA stage 2. CONCLUSION: The prevalence of pathologic AD markers was very common (46%) in a representative population sample of 70-year-olds. The clinical implications of these findings need to be scrutinized further in longitudinal studies.

Cerebrospinal Fluid Fatty Acid-Binding Protein 3 is Related to Dementia Development in a Population-Based Sample of Older Adult Women Followed for 8 Years.

BACKGROUND: Increased fatty acid-binding protein 3 (FABP-3) levels have been reported in neurodegenerative diseases, including Alzheimer's disease (AD). Cerebrospinal fluid (CSF) FABP-3 has therefore been proposed as a putative marker for dementia. Population-based studies examining whether CSF FABP-3 predicts later development of dementia are lacking. OBJECTIVE: The aim of this study was to examine CSF levels of FABP-3 in relation to later development of dementia in elderly women and in relation to Aß42, T-tau, P-tau181, and CSF: serum albumin ratio. METHODS: 86 non-demented women aged 70-84 years who participated in the Prospective Population Study of Women in Gothenburg, Sweden took part in a lumbar puncture in 1992-93. CSF-FABP-3, Aß42, T-tau, P-tau181, and the CSF: serum albumin ratio were measured at baseline. Participants were examined with a neuropsychiatric exam at baseline and at follow-up in 2000. Dementia was diagnosed in accordance with DSM-III-R criteria. RESULTS: Between 1992 and 2000, 8 women developed dementia (4 AD, 3 vascular dementia, 1 mixed vascular dementia and AD). Higher levels of CSF-FABP-3 at baseline were related to development of dementia (OR 1.36 CI [1.05-1.76] p = 0.022) and the subtype AD (OR 1.38 CI [1.06-1.82), p = 0.019) during follow-up. FABP-3 correlated with CSF T-tau (r = 0.88, p <  0.001), P-tau181 (r = 0.619, p <  0.001), and CSF:serum albumin ratio (r = 0.233, p = 0.031), but not with Aß42 (r = -0.08, p = 0.444)CONCLUSION:CSF FABP-3 may be an early marker for later development of dementia, probably related to neuronal degeneration, but independent of Aß metabolism.

Calcium supplementation and risk of dementia in women with cerebrovascular disease.

OBJECTIVE: To determine whether calcium supplementation is associated with the development of dementia in women after a 5-year follow-up. METHODS: This was a longitudinal population-based study. The sample was derived from the Prospective Population Study of Women and H70 Birth Cohort Study in Gothenburg, Sweden, and included 700 dementia-free women aged 70-92 years. At baseline in 2000-2001, and at follow-up in 2005-2006, the women underwent comprehensive neuropsychiatric and somatic examinations. A CT scan was performed in 447 participants at baseline. Information on the use and dosage of calcium supplements was collected. Dementia was diagnosed according to DSM-III-R criteria. RESULTS: Women treated with calcium supplements (n = 98) were at a higher risk of developing dementia (odds ratio [OR] 2.10, 95% confidence interval [CI] 1.01-4.37, p = 0.046) and the subtype stroke-related dementia (vascular dementia and mixed dementia) (OR 4.40, 95% CI 1.54-12.61, p = 0.006) than women not given supplementation (n = 602). In stratified analyses, calcium supplementation was associated with the development of dementia in groups with a history of stroke (OR 6.77, 95% CI 1.36-33.75, p = 0.020) or presence of white matter lesions (OR 2.99, 95% CI 1.28-6.96, p = 0.011), but not in groups without these conditions. CONCLUSIONS: Calcium supplementation may increase the risk of developing dementia in elderly women with cerebrovascular disease. Because our sample was relatively small and the study was observational, these findings need to be confirmed.

Rice husks and their hydrochars cause unexpected stress response in the nematode Caenorhabditis elegans: reduced transcription of stress-related genes.

Currently, char substrates gain a lot of interest since soils amended with such substrates are being discussed to increase in fertility and productivity, water retention, and mitigation of greenhouse gases. Char substrates can be produced by carbonization of organic matter. Among different process conditions, temperature is the main factor controlling the occurrence of organic and inorganic contaminants such as phenols and furfurals, which may affect target and non-target organisms. The hydrochar produced at 200 °C contained both furfural and phenol with concentrations of 282 and 324 mg kg(-1) in contrast to the 300 °C hydrochar, which contained only phenol with a concentration of 666 mg kg(-1). By washing with acetone and water, these concentrations were significantly reduced. In this study, the potential toxic effects of hydrochars on the free-living nematode Caenorhabditis elegans were investigated via gene transcription studies using the following four matrices: (i) raw rice husk, (ii) unwashed rice char, (iii) acetone/water washed rice char, and (iv) the wash water of the two rice chars produced at 200 and 300 °C via hydrothermal carbonization (HTC). Furthermore, genetically modified strains, where the green fluorescent protein (GFP) gene sequence is linked to a reporter gene central in specific anti-stress regulations, were also exposed to these matrices. Transgenic worms exposed to hydrochars showed very weak, if any, fluorescence, and expression of the associated RNAs related to stress response and biotransformation genes was surprisingly downregulated. Similar patterns were also found for the raw rice husk. It is hypothesized that an unidentified chemical trigger exists in the rice husk, which is not destroyed during the HTC process. Therefore, the use of GFP transgenic nematode strains cannot be recommended as a general rapid monitoring tool for farmers treating their fields with artificial char. However, it is hypothesized that the observed reduced transcriptional response with the subsequent lack of energy-consuming stress response is an energy-saving mechanism in the exposed nematodes. If this holds true in future studies, this finding opens the window to an innovative new field of stress ecology.

Effects of different biochars and digestate on N2O fluxes under field conditions.

Field studies that have investigated the effects of char materials on the emission of nitrous oxide (N2O) are still scarce. Therefore, we conducted a field trial with bio- and hydrochars and measured N2O emissions for one whole year. It was hypothesised that the incorporation of chars reduces the emissions of N2O. Chars were produced by pyrolysis and hydrothermal carbonisation (HTC) using either maize silage or wood residues as feedstock. In addition, after production chars were post-treated with digestate in order to accelerate the ageing process of the chars. Chars and digestate were applied to the soil to raise the C content. Emissions of N2O were measured weekly and soil samples for inorganic nitrogen (N) and soil water-content were taken once a month. Additionally, the abundance of functional marker genes from denitrification (nosZ) was determined in October 2012 and in June 2013. The treatment with pure digestate emitted the most N2O compared to the control and char treatments. However, this was significant only in one case. There were no great differences between the char treatments due to high spatial variability and gene abundance of nosZ did not differ between treatments. Overall, emissions of N2O were relatively low. This was attributed to the heterogeneous distribution of the chars and the sandy soils that did not favour the production of N2O. To conclude, the emissions of N2O were mainly influenced by temperature and precipitation and to a minor extent by the type of char and post-treatment.

A 9-year prospective population-based study on the association between the APOE*E4 allele and late-life depression in Sweden.

BACKGROUND: It is well established that there is an association between the apolipoprotein E (APOE) ε4 allele (APOE*E4) and Alzheimer's disease. It is less clear whether there is also an association with geriatric depression. We examined the relationship between APOE*E4 and 5-year incidence of depression in a Swedish population-based sample of older adults without dementia and excluding older adults who developed dementia within 4 years after the diagnosis of depression. METHODS: In 2000-2001, 839 women and men (age range, 70-92 years; mean age, 73.8 years) free from dementia and depression underwent neuropsychiatric and neuropsychological examinations and genotyping of the APOE*E4 allele. Follow-up evaluations were conducted in 2005 and 2009.The association between APOE*E4 allele and 5-year incidence of depression was examined, while avoiding possible confounding effects of clinical or preclinical dementia by excluding participants who had dementia at study entry, subsequently developed dementia during the 9-year follow-up period, or had a decline in Mini-Mental State Examination score of ≥5 points. RESULTS: Among subjects without depression at study entry and without dementia or significant cognitive decline during the subsequent 9 years, APOE*E4 was prospectively associated with more severe depressive symptoms (b = 1.56, p = .007), incident minor depression (odds ratio = 1.99 [confidence interval = 1.11-3.55], p = .020), and any depression (odds ratio = 1.75 [confidence interval = 1.01-3.03], p = .048). CONCLUSIONS: The presence of the APOE*E4 allele predicted future depression in this Swedish population study, even after excluding depressed individuals who later developed dementia, suggesting that the APOE*E4 allele could potentially identify people at high risk for clinically significant depression.

Does low-dose acetylsalicylic acid prevent cognitive decline in women with high cardiovascular risk? A 5-year follow-up of a non-demented population-based cohort of Swedish elderly women.

OBJECTIVE: The aim of this study was to examine whether low-dose acetylsalicylic acid (ASA) influences the rate of cognitive change in elderly women. DESIGN: Prospective, population-based cohort study. SETTING: The city of Gothenburg, Sweden, including those living in private households as well as in residential care. PARTICIPANTS: The sample was derived from the Prospective Population Study of Women and from the H70 Birth Cohort Study in Gothenburg, Sweden. Both samples were obtained from the Swedish Population Register, based on birth date, and included 789 (response rate 71%) women aged 70-92 years. After the exclusion of individuals with dementia and users of warfarin, clopidogrel or heparin at baseline, 681 women were examined. Among all participants, 95.4% (N=601) had a high cardiovascular risk (CVD), defined as 10% or higher 10-year risk of any CVD event according to the Framingham heart study and 129 used low-dose ASA (75-160 mg daily) at baseline. After 5 years a follow-up was completed by 489 women. PRIMARY OUTCOME AND SECONDARY OUTCOME MEASURES: Cognitive decline and dementia incidence in relation to the use of low-dose ASA and cardiovascular risk factors. Cognition was measured using the Mini Mental State Examination (MMSE), word fluency, naming ability and memory word tests. Dementia was diagnosed according to the DSM-III-R criterion. As secondary outcome incidence of stroke and peptic ulcer in relation to low-dose ASA use was studied. RESULTS: Women on regular low-dose ASA declined less on MMSE at follow-up than those not on ASA. This difference was even more pronounced in those who had ASA at both examinations (p=0.004 compared with never users; n=66 vs n=338). All other cognitive tests showed the same trends. There were no differences between the groups regarding short-term risk for dementia (N=41). CONCLUSION: Low-dose ASA treatment may have a neuroprotective effect in elderly women at high cardiovascular risk.

Hydrothermal carbonization of anaerobically digested maize silage.

Hydrochars were prepared by hydrothermal carbonization (HTC) of maize silage previously treated at 55 °C in a two-stage solid-state reactor system. The HTC was carried out in a 1-L stirred pressure reactor with pH regulation by citric acid. The treated silage carbonized at relatively mild conditions (190 °C, 2 h), and the hydrochars showed mainly amorphous macro-size features with a carbon content of 59-79% (ash-free, dry) and a higher heating value of 25-36 MJ kg⁻¹. Temperature was the main influencing factor. The surface area according to Brunauer-Emmett-Teller (BET) analysis was highest at 190 °C (12.3 m²) g⁻¹). Based on these results, the hydrochars are potentially interesting for applications such as an alternative fuel or a soil conditioner.

Stress responses and metal tolerance of Chlamydomonas acidophila in metal-enriched lake water and artificial medium.

Chlamydomonas acidophila faces high heavy-metal concentrations in acidic mining lakes, where it is a dominant phytoplankton species. To investigate the importance of metals to C. acidophila in these lakes, we examined the response of growth, photosynthesis, cell structure, heat-shock protein (Hsp) accumulation, and metal adsorption after incubation in metal-rich lake water and artificial growth medium enriched with metals (Fe, Zn). Incubation in both metal-rich lake water and medium caused large decreases in photosystem II function (though no differences among lakes), but no decrease in growth rate (except for medium + Fe). Concentrations of small Hsps were higher in algae incubated in metal-rich lake-water than in metal-enriched medium, whereas Hsp60 and Hsp70A were either less or equally expressed. Cellular Zn and Fe contents were lower, and metals adsorbed to the cell surface were higher, in lake-water-incubated algae than in medium-grown cells. The results indicate that high Zn or Fe levels are likely not the main or only contributor to the low primary production in mining lakes, and multiple adaptations of C. acidophila (e.g., high Hsp levels, decreased metal accumulation) increase its tolerance to metals and permit survival under such adverse environmental conditions. Supposedly, the main stress factor present in the lake water is an interaction between low P and high Fe concentrations.