Organotin contamination in the Atlantic Ocean off the Iberian Peninsula in relation to shipping

Imposex in female snails, a bioindicator of TBT contamination, and the presence of organotins in snails' tissue and sediments were studied at nine sites off the western Iberian Peninsula. The study was part of a European project (acronym HIC-TBT) co-financed by the EU-LIFE programme, intending to investigate and communicate the impact of organotins from ships in marine ecosystems. Snails and sediments were sampled during two cruises in May/June 1999 and in January 2000 in areas of high, intermediate and low-shipping density. Imposex was found in female snails from several sampling sites, some of which had an imposex incidence of 100%. Differences in sensitivity were found between species; hence comparison of imposex levels between locations where different species were collected was not straightforward. Total organotin concentrations in sediments (sum of butyl and phenyltin compounds) ranged from 21 to 185 ng Sn g(-1) with higher values for most sites sampled in the vicinity of shipping lanes. Organotin concentration in snails' tissue ranged from <5 to 196 ng Sn g(-1), which are similar to those found in snails from other offshore areas contaminated by TBT. Overall, these results give further support to the recent ban on the use of organotin based antifouling paints to all ship size.     

Transfer and effects of cadmium in an experimental food chain involving the snail Helix aspersa and the predatory carabid beetle Chrysocarabus splendens

The transfer and the toxic effects of Cd were studied in an experimental food chain involving the snail Helix aspersa as prey organism and one of its natural predators, the carabid beetle Chrysocarabus splendens. Juvenile snails were fed plant-based food enriched with 0, 10, 50 and 100 microg g(-1) of Cd, then were offered as prey to beetle larvae from egg hatching to pupation stage. Cd concentrations in snail tissues increased with increasing Cd concentration in food and with duration of exposure. Bioaccumulation factors ranged from 1.87 to 3.39, showing that H. aspersa snails, even in their early life stages, belong to macroconcentrator species for Cd. No significant reduction of snail consumption by beetles was found in exposed groups. Cd concentrations in beetle larvae remained very low (lower than 1 microg g(-1) for all groups), demonstrating a very effective regulation capacity in beetle larvae. However, Cd concentrations in highest exposed groups were higher than those found in control groups. Cd contents in adult beetles were lower than in larvae, showing a loss of Cd during metamorphosis. Despite the low Cd concentrations found in beetles, their exposure to Cd contaminated snails led to 31% of mortality, which occurred only during pupation and for the highest exposure level. No clear sublethal effects were found. These results showed that snails inhabiting heavily polluted areas may represent a risk of secondary poisoning for predatory invertebrates and provided quantitative data on the transfer of Cd between two compartments of a terrestrial food chain.     

Should apple snail Pomacea canaliculata (Caenogastropoda, Ampullariidae) be used as bioindicator for BDE-209?

Apple snail Pomacea canaliculata has been reported to accumulate polybrominated diphenyl ethers (PBDEs) and was recently proposed as PBDE bioindicator. This work investigates the ability of P. canaliculata to accumulate BDE-209 by dietary exposure under controlled experimental conditions. A 30-day long enrichment feeding assay was carried out using 30 adult apple snails, placed in individual aquaria. Food was enriched at three BDE-209 concentrations (400, 4,700, and 8,300 μg g^?1 lipid weight). Correlation between BDE-209 values in food and snail tissue were estimated according to Stockholm Convention suggested criteria for chemicals with K_OW >5. All animals survived with no evident physical alterations, and all of them accumulated BDE-209. BDE-209 levels in tissue samples increased exponentially with the exposure concentration. The bioaccumulation factor vs. food concentration plot showed a peculiar pattern, in which at intermediate concentrations the snails accumulated less BDE-209 than expected. Our results suggest that P. canaliculata would present a detoxification mechanism for BDE-209 different from the most commonly reported metabolic pathways.     

Butyltin compounds in sediment and molluscs from the shipping strait between Denmark and Sweden

The distribution of tributyltin (TBT) contamination in the subtidal zone of the waters between Denmark and Sweden was investigated in relation to major international shipping lanes. Sediment and different benthic molluscs were sampled in transects along and perpendicularly to the shipping lanes in the Sound (?resund) and the Kattegat/Skagerrak region. The samples were analysed for TBT and its degradation products, dibutyltin (DBT) and monobutyltin (MBT), using GC-PFPD. In sediments, the TBT concentration ranged from <1 to 19 ng g(-1) dry weight (dw) with a strong correlation between the TBT concentration and the organic fraction in sediment (r(2)=0.90) in the samples collected in the Sound, where the highest concentrations were found. This relationship was not observed in the samples from the Kattegat because the TBT concentration in most sediment samples was below the limit of detection. In the molluscs, TBT and its degradation products were detected in all samples from the entire area with concentrations ranging from 8.1 ng g(-1) dw in Buccinum undatum to 1316 ng g(-1) dw in Nuculana pernula. The deposit-feeding bivalve N. pernula was found to have a particularly high accumulation potential for TBT. In addition, a strong correlation between TBT concentrations in sediment and N. pernula was found. Therefore this species seems to be an ideal organism for monitoring sediment contamination. The TBT concentration in N. pernula was found to decrease gradually along the shipping lanes from the Sound, through the Kattegat and into the Skagerrak.     

Occurrence and seasonal variation of alkylphenols in marine organisms from the coast of Taiwan

The occurrence and distribution of alkylphenols (APs, i.e., 4-tert-octylphenol (4-t-OP) and 4-nonlyphenol isomers (4-NP)) in oysters (Crassostrea gigas), snails (Thais clavigera), coastal water and coastal sediments of the western coast of southern Taiwan are investigated. Alkylphenols were present in all matrices of interest: in water, ranging from 61 to 370 ng/l, in sediments, ranging from 27 to 190 ng/g, and in biota samples (i.e., oyster and snail), ranging from 20 to 5190 ng/g. Statistical analysis indicated that the probability distribution of most determined concentrations in oyster and snail samples was log-normal distribution. The bioaccumulation of APs resulted in a seasonal variation with respect to their compositions and concentrations in oyster and snail samples. For oysters, concentrations of APs in winter exceeded those in summer. In contrast, those in snails in summer exceeded those in winter. Estimated biomagnification factors of APs from snails to oysters ranged from 1.4 to 4.3 in summer, and 0.5 to 0.8 in winter on the dry weight basis. The results suggested that seasonally mediated physiological changes, such as dilution caused by growth, biotransformation and metabolism, may affect the bioaccumulation of APs in according to season and organism.     

The influence of environmental factors on the molluscicidal activity of Euphorbia milii latex.

The present study was undertaken to investigate the influence of biotic (snail size and presence of food during the test) and abiotic factors (temperature, water hardness and concentration of organic materials) on the molluscicidal activity of Euphorbia milii latex. Bioassays were conducted with B. glabrata (10 snails per concentration) and snail lethality was evaluated after 24 hr and 48 hr of exposure to lyophilized latex solutions. Neither the degree of water hardness, nor the presence of food during the test affected latex-induced snail mortality. The snail size had a minor influence on E. milii-induced snail lethality. Newly-hatched (shell diameter < or = 1 mm) as well as young (3-8 mm) snails were slightly less susceptible than older (10-25 mm) mollusks. On the other hand, the molluscicidal effect of E. milii latex was modified by environmental factors such as temperature (i.e., LC50 and LC90 values were halved for every 10 degrees C rise in temperature) and concentration of organic materials in the water (i.e. the higher the concentration of organic matter, the higher the LC50 value). The efficacy of E. milii latex as a molluscicide can be modified by factors such as water temperature and concentration of organic materials, and to a lesser extent by snail size.     

Copper desorption in flooded agricultural soils and toxicity to the Florida apple snail (Pomacea paludosa): implications in Everglades restoration

Copper (Cu) desorption and toxicity to the Florida apple snail were investigated from soils obtained from agricultural sites acquired under the Comprehensive Everglades Restoration Plan. Copper concentrations in 11 flooded soils ranged from 5 to 234 mg/kg on day 0 and from 6.2 to 204 mg/kg on day 28 (steady-state). The steady-state Cu concentration in overlying water ranged from 9.1 to 308.2 microg/L. In a 28-d growth study, high mortality in snails occurred within 9 to 16 d in two of three soil treatments tested. Growth of apple snails over 28 d was affected by Cu in these two treatments. Tissue Cu concentrations by day 14 were 12-23-fold higher in snails exposed to the three soil treatments compared to controls. The endangered Florida snail kite and its main food source, the Florida apple snail, may be at risk from Cu exposure in these managed agricultural soil-water ecosystems.     

Study of fractionation and potential mobility of metal in sludge from pyrite mining and affected river sediments: changes in mobility over time and use of artificial ageing as a tool in environmental impact assessment

Sludge from mining exploitation can be a source of land and water contamination in the adjacent zone. Accidents such as the break of waste mining pools in the Aznalcollar Mine (Seville, Spain) in 1998 produce important ecological disasters. In this work is presented a study of the evolution of aqua regia leachable concentration and mobility of metals in sediment samples of Guadiamar River basin from the accident date up to 2001. The application of BCR standard extraction procedures provides valuable information about the mobility and toxicity of the spill and the metal mobility in Guadiamar River polluted sediments. As a rule, themobility of several metals in the initial sludge (sulphides) is low, except for copper. Otherwise, the results of aqua regia leachable concentration and mobility of metal obtained for sediments samples indicate that the cleaning and inertisation works carried out in the zone have been adequate but insufficient, being the metal levels observed in the zone are higher than natural levels. To establish the initial impact of the spill and the mobility changes with time, mineralogical composition of the pyritic sludge and its evolution after the natural weathering and after the induction, of accelerated ageing processes by light and temperature was studied in the laboratory. Oxidation of initial sulphides to sulphates was observed. Both environmental and laboratory oxidation of the metallic sulphides increase the mobility of all metals, especially of copper, zinc and lead. The proposed laboratory procedure allows to predict the changes in mobility and therefore in toxicity that can occur at short or long term after exposure of sludge at environ-mental conditions. The mobility results in sludge and sediment samples are interpreted in terms of the mineral composition of the samples.     

Repartition du virus poliomyelitique a l'interface eau de mer-sediment d'un systeme experimental

The distribution between sea water and sediment of a polio virus has been studied under an experimental system. Throughout 7 days of incubation, a significant statistical difference exist between the viral concentration of sea water in times T₀ and T₇ (P～0,99) and the viral concentration of sea water samples and sediments in times T₇ (P～0,95). this for any type of sediment.     

The effects of endosulfan on the great ramshorn snail Planorbarius corneus (Gastropoda, Pulmonata): a histopathological study

In this study the great ramshorn snail (Planorbarius corneus), one of the most abundant gastropod of Turkish limnic systems, was investigated to determine the histopathological effects of endosulfan on the digestive gland, foot and mantle under laboratory conditions. Samples were collected from small artificial pools in Karaot at Gelendost-Isparta (southwest of Turkey), where agricultural activities are widespread. The snails were exposed to two sublethal concentrations of endosulfan (0.4 and 0.8 mg/l) for periods of 10, 20 and 30 days. Fifteen snails were kept in 2.5 l glass jars containing dechlorinated tap water and exposed under semi-static test (daily exchange of test water). All the testing was carried out on adult specimens, and snails were maintained on a photothermal period with 16 light hours at 22 +/- 2 degrees C. The histopathological examinations revealed the following changes: amoebocytes infiltration, dilatation in hemolymphatic spaces between the tubules, degeneration of cells, abnormal lumen, necrosis of cells and atrophy in the connective tissue of digestive gland; desquamation of the epithelium cells, changes in the number of mucocytes and protein gland cells, lipid vacuolus and atrophy of the columnar muscle fibers of the foot and mantle tissues. Pycnotic state of cells was also seen in the mantle tissues. Endosulfan caused significant histopathological alterations in the digestive gland, foot and mantle tissues of the snail, irrespective of concentrations of the pesticide and its exposure periods. The results are discussed, particularly in comparison to those of other aquatic organisms.     

Spatial distribution of polychlorinated biphenyls (PCBs) and polybrominated biphenyl ethers (PBDEs) in an e-waste dismantling region in Southeast China: Use of apple snail (Ampullariidae) as a bioindicator

Fengjiang is a large e-waste dismantling site located in southeast China. In this paper, apple snail and soil samples were collected from this e-waste dismantling site and 25 vicinal towns to investigate the contamination status, spatial distributions and congener patterns of polychlorinated biphenyls (PCBs) and polybrominated biphenyl ethers (PBDEs). Total PCB concentrations in apple snails (3.78-1812 ng g⁻¹ dry weight (dw)) were significant higher than that in soil samples (0.48-90.1 ng g⁻¹ dw). PBDE (excluding BDE 209) concentrations in apple snail and soil samples ranged from 0.09 to 27.7 ng g⁻¹ dw and 0.06 to 31.2 ng g⁻¹ dw, respectively. Concentrations of PCBs and PBDEs in snails and soils correlated negatively with the distance from Fengjiang. Both the concentrations and profiles of the pollutants were significantly correlated (p < 0.05) between the snail and soil samples, indicating the suitability of apple snail as a reliable bioindicator for PCBs and PBDEs contamination in this region. Relatively high concentrations of PCBs and PBDEs at locations far from e-waste dismantling sites implied that these pollutants have been transported to surrounding regions.     

The influence of environmental factors on the molluscicidal activity of Euphorbia milii latex

Abstract

The present study was undertaken to investigate the influence of biotic (snail size and presence of food during the test) and abiotic factors (temperature, water hardness and concentration of organic materials) on the molluscicidal activity of Euphorbia milii latex. Bioassays were conducted with B. glabrata (10 snails per concentration) and snail lethality was evaluated after 24 hr and 48 hr of exposure to lyophilized latex solutions. Neither the degree of water hardness, nor the presence of food during the test affected latex‐induced snail mortality. The snail size had a minor influence on E.milii‐induced snail lethality. Newly‐hatched (shell diameter ≤ 1mm) as well as young (3–8 mm) snails were slightly less susceptible than older (10–25 mm) mollusks. On the other hand, the molluscicidal effect of E.milii latex was modified by environmental factors such as temperature (i.e. LC₅₀ and LC₉₀ values were halved for every 10°C rise in temperature) and concentration of organic materials in the water (i.e. the higher the concentration of organic matter, the higher the LC₅₀ value). The efficacy of E.milii latex as a molluscicide can be modified by factors such as water temperature and concentration of organic materials, and to a lesser extent by snail size.     

The effects of engineered nanoparticles on survival, reproduction, and behaviour of freshwater snail, Physa acuta (Draparnaud, 1805)

Increasing uses of engineered nanoparticles (ENPs) in commercial products and industrial applications has eventually resulted to their releases into atmospheric, terrestrial, and aquatic environments. However, knowledge gaps in ENPs toxicity, fate, and behaviour currently limit our ability to quantify risk assessment of materials with nanoscale dimensions, and therefore, the extent of the resultant environmental impacts remains unknown. In the present study, we evaluated the effects of γ-alumina, α-alumina, modified TiO(2) (M-TiO(2)), and commercial TiO(2) (C-TiO(2)) ENPs on the survival, behaviour, and early life stages of the freshwater snail Physa acuta (Draparnaud). The toxicity evaluation was carried out after spiking commercial sand with ENPs concentrations of 0.005, 0.05, or 0.5 gk g(-1). Our findings suggest that increases of γ-alumina and α-alumina concentrations at sub-lethal level concentrations caused significant reduction in the embryo growth rate and embryo hatchability. In addition, these ENPs induced observable developmental deformities of the embryos. In addition, toxicity evaluations using acute 96-h and chronic 28-d tests showed exposure duration may be a significant factor in ENPs-induced toxicity. Therefore, long-term exposure of aquatic organisms to ENPs - potentially can alter certain ecological populations at different trophic levels - and may compromise the entire aquatic ecological functionality. The percentage hatchlings in test chambers containing 0.5 gk g(-1) γ-alumina and α-alumina concentration was 50% less to those observed in the controls. Our results suggest the embryonic growth and hatchability tests are useful endpoints in chronic sediment toxicity tests for determining the toxic thresholds of ENPs in sediment environment. Although no snail mortalities were observed during the static 96-h test containing sediment spiked with different concentrations of M-TiO(2), C-TiO(2), γ-alumina and α-alumina - the antioxidant enzymatic assay results indicated a significant change in antioxidant levels which altered peroxidation at 0.05 or 0.5 gk g(-1)concentrations for both γ-alumina and α-alumina.     

Monitoring of the booster biocide dichlofluanid in water and marine sediment of Greek marinas

Dichlofluanid (N-dichlorofluoromethylthio-N'-dimethyl-N-phenylsulphamide) is used as booster biocide in antifouling paints. The occurrence of dichlofluanid and its metabolite DMSA (N'-dimethyl-N-phenyl-sulphamide) was monitored in seawater and marine sediment from three Greek marinas. Seawater and sediment samples were collected at three representative positions and one suspected hotspot in each marina and shipped to the laboratory for chemical analysis. As part of the project, an analytical method had been developed and validated. Furthermore, some additional experiments were carried out to investigate the potential contribution of paint particle bound dichlofluanid on the total concentration in the sediment. As expected, given its known high hydrolytic degradation rate, no detectable concentrations of dichlofluanid were measured in any of the seawater samples. DMSA was detected in seawater samples at very low concentrations varying from <3 ng l(-1) (LOD) to 36 ng l(-1). During method validation, it had already been demonstrated that dichlofluanid is unstable in sediment and can therefore only be determined as its metabolite DMSA. In a separate experiment, in which marine sediment was spiked with artificial paint particles containing dichlofluanid and then analysed according to the validated method, it was demonstrated that if there is any dichlofluanid originating from paint particles, this would be determined as DMSA. No DMSA was detected in any of the sediment samples. It could therefore be concluded that there were no significant concentrations of dichlofluanid in the sediment samples.     

Distribution characteristics of nonylphenolic chemicals in Masan Bay environments, Korea

To understand the distribution characteristics of nonylphenolics and sterols, samples such as in creek water, sea surface water, waste water treatment plant (WWTP) effluent water, sediment and mussel were collected and analyzed. The principal analytes are nonylphenol (NP), nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), coprostanol (5beta) and cholestanol (5alpha). All these target pollutants showed 100% detection frequency in all of the samples analyzed. Total concentration of nonylphenolic compounds ranged from 334 to 3628ngl(-1) (average: 1331ngl(-1)) in creek water, from 15 to 36400ngl(-1) (average: 1013ngl(-1)) in sea surface water, from 131 to 2811ngg(-1) dry weight (average: 581ngg(-1) dry weight) in sediment and from 50.5 to 289ngg(-1) dry weight (average: 139ngg(-1) dry weight) in mussel. For water samples, levels of nonylphenolics determined in summer season were higher than those in spring season. Among them, nonylphenol and NP1EO was dominant in creek water and seawater, respectively. The highest concentration was recorded in sediment near a WWTP effluent outlet. And high levels of nonylphenolics and sterols were found in about 3km area surrounding WWTP effluent outlet. Coefficient of linear regression (R(2)) for NP in mussel and in sediment was 0.90. Similarly good correlation (R(2)=0.98) was obtained between concentration in water and in mussel indicating that a steady state has been reached in this bay. The calculated bio concentration factor (BCF=2990) for NP in Masan Bay agrees well with reported values in the literature.     

Organotins and imposex in the rock shell, Thais clavigera, from oyster mariculture areas in Taiwan

The amounts of organotin compounds such as butyltins [sigma BTs: tributyl- (TBT), dibutyl- (DBT), monobutyl- (MBT)] and phenyltins [sigma PhTs: triphenyl- (TPhT), diphenyl- (DPhT) and monophenyl- (MPhT)] in rock shells, Thais clavigera, from the northern (Shiangsan), central (Lukang) and southern (Chiku) Taiwan oyster mariculture areas varied with season and location. High values of sigma BTs (mainly TBT) and low values of sigma PhTs (mainly TPhT) were found during winter (January 1999) at Shiangsan, while high amounts of sigma PhTs (mainly TPhT) and low sigma BTs (mainly TBT) were found during summer (August 1998) at Lukang. The snails were also affected by imposex. Imposex at Shiangsan, Lukang and Chiku increased from 67.1, 59.3 and 36.7% in summer to 100, 100 and 80% in winter, respectively. Relative penis size indices similar trend at Chiku site. No male displayed rock shells were found at Shiangsan and Lukang in winter. Linear correlations between pseudopenis length versus TBT (r = 0.7655, P < 0.001), DBT (r = 0.4253, P < 0.05), MBT (r = 0.5865, P < 0.01) and TPhT (r = -0.6160, P < 0.01) were obtained significant. Among 200 samples, significant positive correlations between length (pseudopenis/shell) ratio of female versus TBT (r = 0.6944, P < 0.005) and sigma BTs (r = 0.6413, r < 0.01) were also observed. The weak correlations between length (pseudopenis/shell) ratio of female versus DBT (r = 0.3085) and MBT (r = 0.4240, P < 0.1) and TPhT (-0.4917, P < 0.05; negative correlation) could indicate that DBT, MBT and TPhT have little or no effect on the development of imposex in rock shells.     

Gyrineum natator: a potential indicator of imposex along the Indian coast

Imposex related studies have used neogastropods as sentinel organism. In this effort, we elucidate the potential of a mesogastropod, Gyrineum natator, for imposex monitoring on the central west coast of India. Observations were made with specimens collected from Mumbai (three stations), Ratnagiri (two stations), Goa (one station) and Mangalore (one station). The analyzed populations at all four sites were affected by imposex. The six different stages of imposex in this organism are illustrated through schematic and scanning electron micrographs. In view of the anticipated ban on TBT based antifouling paints this baseline information can serve as an indicator for environmental monitoring in this region.     

Chemical and bacterial changes during laboratory conditioning of formulated and natural sediments

Within the framework of toxicity testing using formulated sediment, a conditioning treatment prior to toxic contamination has been examined. This preliminary step enables the bacterial colonisation of the sediment, the initiation of organic matter degradation, and the establishment of stable biological and physico-chemical conditions. The treatment involved in keeping the formulated sediment under water in conditions similar to that chosen for toxicity tests. The behaviour of a formulated sediment was compared with a natural sediment. The monitoring of physico-chemical and biological parameters of sediment and water column was carried out over a 30-day incubation in two laboratories. The parameters of pH and redox, dissolved organic carbon (DOC), NH4 and NO2, total organic carbon (TOC) were measured. The bacterial community was characterised by the determination of bacterial density, in total bacteria number or colony forming units (CFU), several exoenzymatic activities (P-glucosidase, xylosidase, leucine-amino-peptidase phosphatase and sulfatase activities), and three gas productions (CO2, N2O and CH4). The same experiment was carried out with a natural sediment. A 10- to 15-day conditioning allowed a physico-chemical stabilisation and corresponded to kinetic changes in hydrolysis activities. As compared to data of the natural sediment, the biological activity of the formulated sediment showed a different dynamic with lower activity levels. For both sediments, an important decrease of activities levels was observed after 15 days because of a substrate limitation. The work showed that a preliminary conditioning treatment of a formulated sediment provides the stabilisation of parameters that can affect toxicant bioavailability. Additional research is needed to determine the real influence of conditioning on the bioavailability of contaminants. The possible advisability of organic matter input, to maintain the sediment bacterial activity, has to be studied.     

Toxicity of anti-fouling biocides to encysted metacercariae of Echinoparyphium recurvatum (Digenea: Echinostomatidae) and their snail hosts

The toxicity of the anti-fouling biocides tributyltin (TBTO), copper, and Irgarol 1051 (irgarol) at a nominal concentration of 10 microg/l over a 30 day period were investigated against the viability of metacercarial cysts of the digenean parasite Echinoparyphium recurvatum resident within the body of two common freshwater snails, Lymnaea peregra and Physa fontinalis. Reduced parasite viability was found under most exposures in both snail species. However a greater effect of toxicant exposure was found in cysts within P. fontinalis compared to those in L. peregra. This was associated with an increased mortality of the host snail. Among all tested biocides, TBTO exposures induced the highest mortality to both the parasite and their hosts. These results suggest that parasite viability is interlinked with survival of the host snail. The mechanisms of differing toxicity between host species and its relevance to successful parasite transmission to the next host are discussed.     

Behaviour of EDTA in marine microcosms

Laboratory-scale microcosm tests were carried out in sea water with and without sediment to investigate the importance of ultraviolet (UV) light and microbes in the temperature-dependent degradation of metal complexes of ethylenediaminetetraacetic acid (EDTA). After 17 weeks, 44% and 48% of the original EDTA (initial concentration 385 microg/L) were converted at 10 degrees C and 22 degrees C, respectively. The degradation was more influenced by UV light than by sediment microbes, although the latter were very important. At both temperatures, absorption of EDTA to the sediment phase reached a maximum in the initial stage of the experiments (<4 weeks): at 10 degrees C about 4% and at 22 degrees C about 7% of the original EDTA.