Toxicological and Environmental Safety Data - Case Study
Introduction
AGRAGEL is a cross-linked copolymer of acrylic acid and acrylamide partially neutralized as potassium/ ammonium salt. The polymer is in its dry form a granulate and forms a gel-like material upon addition of water or aqueous solutions. Due to the cross-linking it is insoluble in water. Uptake of water is facilitated mainly by the negative carboxylic groups of the polymer and their hydration with water molecules. Complete solubilization is hindered because of the cross-linking of different polymer chains. Due to incomplete cross-linking small amounts (generally about 6 %) can be leached out of the polymer matrix by water, the so-called water extracts. To these the environment can be potentially exposed to besides the gel.
Specific analytical means for determination of AGRAGEL in a complex environmental matrix are not available besides of 14C-radio labeling in laboratory experiments. Depending on the method, determination of the polymer and its water extracts in particular may be quantified by the total organic carbon (TOC) parameter, later on referred to as 'mg carbon/l'. The water extracts comprise low (oligomers) to high molar mass molecules of up to approximately 2 x 106 dalton in molar mass. Besides this the polymer matrix still contains a small amount of residual acrylamide and acrylic acid all of which is in the salt form with potassium. The following data have been generated for the superabsorbent polymer AGRAGEL. The testing was performed after relevant exposure scenarios were identified and the necessary tests derived from intended and reasonably anticipated exposure situations. However, it should be taken into consideration that the data to follow were generated under laboratory conditions and have to be extrapolated to environmental conditions. Therefore, caution should be exercised in applying the generated figures for accurate estimation of actual environmental risks.
The presented toxicological and eco-toxicological studies were performed according to international recognized test methods and in compliance with the 'Principles of Good Laboratory Practice (GLP)'. Note: Not all of the different variations of the superabsorbent polymers of the AGRAGEL-series were tested with all of the following test systems due to ethical and economical reasons.
Toxicity Data
Acute Oral Toxicity (Limit-Test)
Approximate (A) LD50 > 5000 mg/kg body weight of up to 3 % (w/v) AGRAGEL as a gel in 0.9 % (w/v) saline applied with stomach tube to 5 male and 5 female rats each. No toxic symptoms could be observed; body weight development was normal during observation for 14 days after application; necropsy revealed no visible organ alterations, i.e. the test substance has to be regarded as essentially non-toxic after oral intake.
Acute Dermal Toxicity (Limit-Test)
Approximate (A) LD50 > 2000 mg/kg body weight of up to 3 % (w/v) of AGRAGEL as a gel in a 0.9 % (w/v) saline applied to the shorn skin of 5 male and 5 female rats. No toxic symptoms; body weight development was normal for 14 days after application; necropsy revealed no visible organ alterations; no deaths occurred, i.e. AGRAGEL is essentially non-toxic when applied to the skin.
Sub-acute Skin Compatibility
Repeated application of a 3 % (w/v) gel in 0.9 % (w/v) saline of AGRAGEL onto the shorn skin, without occlusion, of 10 female mice, revealed occasionally, very slight transient edemas accompanied with a slight increase in skin-fold thickness. The test substance was applied 3 times per week over 8 weeks. Body weight was within normal range; no systemic effects, due to the application of the test substance, were observed, i.e. the test substance, in its gelled state shows good skin compatibility.
Acute Eye Irritation
Application of 0.1 g of AGRAGEL into the conjunctival sac of the eyes of rabbits caused slight erythematic of eyes and adjacent mucous membranes. Further lesions were not observed. There were no systemic symptoms due to the application of the test substance. The observed slight irritative effect onto mucous membranes of the eyes are caused by the somewhat abrasive properties of the dry, cristalline granulate of AGRAGEL and its capacity to dry out the membranes due to the uptake of fluid. The watery suspension of AGRAGEL exerts no such effects on the eyes of rabbits.
Hens' Egg Test
The Hen's Egg Test is an alternative test method to reduce animal testing. With this method irritative substance effects on membranes such as mucus and eye can be determined. Chorion allantois membrane (CAM) testing in hen's eggs revealed only moderate irritative effects when 200 mg of the dry granulate of AGRAGEL were used. Application of a 3 % (w/v) gel led to no effects on the CAM. The observed effects are due to the drying- out properties of the granulate test substance and are therefore characterized as physical, not chemical effects.
Cell Toxicity
AGRAGEL was examined regarding its influence on mammalian cells in a cell culture system using a fibroblastic cell line derived from mice. The cells were incubated for 24 hours with an extract of AGRAGEL (15 g/l of medium) in concentrations up to 100 %. No adverse effects on the morphology or viability of the cells was observed.
Skin Sensitization AGRAGEL was tested with respect to its potential to sensitize animals after skin contact according to the maximization test of Magnusson and Kligman. Ten (10) male and ten (10) female guinea pigs were treated intradermally with a water extract [1.5 % (w/v)] and dermally with a 3.0 % (w/v) gel in 0.9 % (w/v) saline during the induction period. The challenge was executed with a 0.3 % (w/v) gel in 0.9 % (w/v) saline. No erythemas or edemas were observed, therefore, it is unlikely for AGRAGEL to exhibit a potential for skin sensitization.
Elimination following oral administration
A study was performed to provide information on the absorption, excretion and plasma kinetics of total radioactivity following single oral administration of 14C-AGRAGEL to rats.
Excretion of radioactivity was rapid with nearly all of the dose recovered within 48 h post dose. Feces was the major route of excretion accounting for? more than 90 % of the applied dose. A further 3 % of the dose was excreted via urine. Negligible amounts of radioactivity were retained in the carcass after 5 days. The presence of only very low concentrations of radioactivity in the plasma indicates as well, that the oral dose is poorly absorbed and the small amount of radioactivity which was absorbed was eliminated quickly.
Mutagenicity Test with Bacteria
For testing mutagenicity an Ames Test was executed with an extract of AGRAGEL [20 g/l in 0.9 % (w/v) saline with 10 % (v/v) ethanol] with and with-out metabolic activation by rat liver microsomes. Extracts of AGRAGEL were tested in 4 different strains of histidin requiring Salmonella typhimurium (base pair substitution and frame shift mutation) and 2 tryptophan requiring strains of Escheria coli for their ability to induce point mutations with and without the presence of a metabolic activation system. Up to 5000 micrograms/plate no mutagenic events could be observed. Furthermore, cytotoxicity was not detected up to 5000 micrograms/plate. Therefore, there was no indication of a mutagenic potential to bacteria of the extract of AGRAGEL up to the equivalent of 5000 micrograms/plate.
Mutagenicity Test with Mammalian Cells
Mouse Lymphoma L 5178 Y Cells were exposed With (S 9 mix from Aroclor induced rat hepatocytes) and without metabolic activation to an extract of AGRAGEL [20 g/l in 0.9 % (w/v) saline with 10 % (v/v) ethanol]. The test substance failed to induce point mutations at the HPRT locus up to the equivalent of 400 micrograms/ml.
Conclusions regarding Toxicology
AGRAGEL is devoid of any potential adverse effects provided direct contact to mucous membranes is avoided (by protective cloth and goggles). Usage of AGRAGEL has no negative effects on the health of users due to the low toxicity profile.
Ecotoxicity Data
Toxicity to Bacteria
Growth behavior and propagation of the microorganism Pseudomonas putida was determined with a saline (0.9 %) extract of AGRAGEL. Negative effects onto the growth behavior of the Pseudomonas putida were not observed, i.e. no cyto-toxic, cytostatic or biocidal effects are to be expected. The EC50-value for half maximum propagation is higher than the highest concentration tested, i.e. 1768 mg carbon/l which equals 8 g polymer/l. Therefore adverse effects on bacteria are not expected when relevant environmental exposure is considered.
Toxicity to Algae
Growth behavior of single cellular algae Scenedesmus subspicatus was determined with a soluble extract of AGRAGEL up to 910 mg carbon/ l (5 g AGRAGEL/l). From 20 mg carbon/l inhibition of growth was observed. The EC50-value which defines half maximum growth was 150 mg carbon/l which is the equivalent of 1 g polymer/l. Higher concentration led to further growth reduc- tion. The observed slight to moderate toxicity is thought to be no practical importance, when realistic environmental exposure conditions are taken into consideration.
Toxicity to Ciliates
Cells of the ciliate Tetrahymena pyriformus were incubated for 48 hours with the watery extract of AGRAGEL in concentration up to 1000 mg carbon/ l (corresponding 6 g/l polymer). No negative effects on growth behavior were observed. The EC50-value for half maximum cell propagation was greater than the highest concentration tested. Therefore biocidal effects are not expected under relevant environmental exposure conditions.
Toxicity to Waterpolyps
Cytotoxic effects and inhibition of reproduction of the waterpolyp Hydra litoralis were determined with a watery extract of AGRAGEL with concen- trations up to 400 mg carbon/l (5 g polymer/l). At low concentrations no cytotoxic effects were observed and the reproduction rate was increased in comparison to the control. Higher concentrations led to cytotoxic symptoms and a reduction on repro-duction. The EC50-value defined as the concentration of the test substance which restricts the reproduction rate by 50 % is approximately 140 mg carbon/l which equals 1.6 g polymer/l. Therefore adverse effects on waterpolyps are not expected under realistic environmental exposure conditions.
Toxicity to Daphnids
Acute effects of AGRAGEL on the swimming ability of the daphnids Daphnia magna for a 48 hours period was determined with a soluble extract of AGRAGEL at concentrations of up to 1600 mg carbon/l which is the equivalent of 10 g AGRAGEL /l. Up to 400 mg carbon/l no toxicity effects on the daphnids could be observed. The EC50-value for half maximum inability to swim is approximately 980 mg carbon/l which equals 6 g AGRAGEL/l. Therefore, under appropriate use conditions, no critical deleterious effects are ex-pected on daphnids.
Acute Toxicity to Fish
To determine acute lethal effects to fish the cold water species Leuciscus idus (golden orf) and the warm water species Brachydanio rerio (zebra fish) were exposed to watery extracts of AGRAGEL. The LC50-value which defines the mean lethal concentration is approximately 250 mg carbon/l which equals 3.6 g polymer/l for golden orf and higher than 300 mg carbon/l which equals 5 g polymer/l for zebra fish.
Chronic Toxicity to Fish
To determine chronic toxic effects the fish Brachydanio rerio (zebra fish) was exposed to watery extracts of AGRAGEL over a time period of 10 days. No deaths were observed at the highest concentration tested and the no-observable-effect level for the prolonged toxicity test with zebra fish is higher than 300 mg carbon/l which equals 5 g polymer/l. Therefore AGRAGEL has to be regarded as practically non-toxic to fish.
Toxicity to Birds
Acute toxicity of AGRAGEL to the bird Colinus virginianus (bob white quail) was determined by oral application. As no deaths or adverse clinical effects were observed at a dose level of 2000 mg/kg the LD50-value for half maximum lethality is greater than 2000 mg/kg. Therefore no critical effects to birds are expected under relevant exposure conditions.
Toxicity to Earthworms
To determine acute toxic effects to earthworms Eisenia foetida was exposed to AGRAGEL over a 14 day exposure period. The test soil was prepared at a limit concentration of 20 g AGRAGEL/kg soil. As no unusual behavior was noted throughout the study period and the cumulative percentage mortality was similar as in the control vessels the LC50-value for half maximum lethality is greater than 20 g/kg. Therefore adverse effects on earthworms are not expected under appropriate exposure conditions.
Cress Germination Test
The influence of AGRAGEL on garden cress (Lapidium sativum) was determined with water-soluble extracts of AGRAGEL up to a concentration of 993 mg carbon/l (7 g AGRAGEL/l). After 5 days development status and root length of plants treated with AGRAGEL were compared with plants without AGRAGEL. In concentrations of up to 3 g/l AGRAGEL had a positive influence on root growth and yielded a higher dry weight of the plants. Higher concentrations had a slight negative effect. Seedlings growth with a 1 % AGRAGEL gel were in good condition in general, with some decrease in root length vs. control plants, but with increases in additional roots. The EC50-value of 900 mg carbon/l (6 g/l polymer) and the positive results in germination in the gel yield no expectation of critical effects under relevant conditions of use.
Terrestrial Plants Growth Test
Phytotoxic effects of AGRAGEL onto emergence and growth of seedlings over a 20 day period were determined with application rates of 0, 5, 10 and 20 g/kg soil of the following plants:
Phaseolus aureus (mung bean),
Triticum aestivum (wheat),
Lactuca sativa (lettuce),
Lapidium sativum (cress),
Lycopersicon esculentum (tomato) and
Cucumis sativus (cucumber).
Uptake by Plants
The uptake and translocation of AGRAGEL in plants has been investigated in wheat, carrots and lettuce. AGRAGEL was incorporated into sandy loam topsoil at a target rate of 10 g/kg and the plants were grown from germination to maturity. Plants were harvested at intervals between sowing and maturity and analyzed. The percentage of AGRAGEL taken up into the plants was very low at all time points with the highest level in lettuce at maturity accounting for only 0.12 % of the applied radioactivity. Re-fixation of 14CO2 liberated by degradation of the polymer by soil microorganisms was not detected. In order to clarify, if the radioactivity taken up comprises monomer Acrylamide the lettuce plants were analyzed for their content of acrylamide additionally.
No acrylamide was detected with a limit of quantification of 0.5 go/kg.
To investigate the uptake of acrylamide in food crops several field trials with tomatoes, mushrooms, melons and maize were performed. The plants and fungi were grown to maturity on soil or substrate amended with AGRAGEL under standard growth conditions. Following harvest the mature fruits and crops were analyzed for their content of acrylamide. In none of the samples acrylamide was detected with a limit of quantification of 0.5 μg/kg.
Conclusions regarding Ecotoxicity
AGRAGEL exhibits no relevant eco-toxicity when realistic environmental exposure conditions are taken into account. The use of AGRAGEL is safe for the environment.
Ready Biodegradability
To determine the ready biodegradability of AGRAGEL in a Modified-Sturm-Test (CO2 evolution test) the water-soluble extract was exposed to activated sludge microorganisms at concentrations of 10 and 20 mg/l of dissolved organic carbon. The cumulative carbon dioxide release over a 28 day exposure period was used to calculate the percentages of biodegradation. As no significant levels of biodegradation were recorded AGRAGEL is not readily biodegradable under aqueous, aerobic conditions.
Biodegradation in compost
Biodegradability of AGRAGEL was tested under solid, aerobic conditions in a controlled composting test. The inoculums consisting of stabilized and mature compost was mixed with 10 % (w/w) AGRAGEL and incubated for 45 days at temperatures following a real composting temperature profile. Biodegradation was calculated by the percentage of solid carbon of the test compound which has been converted to carbon dioxide. As after two weeks of incubation the final conversion level of 3 % was reached AGRAGEL has to be regarded as poorly biodegradable under aerobic conditions in compost.
Degradation and conversion processes during composting
The fate regarding degradation and conversion of a linear copolymer of acrylic acid and acrylamide was determined in a composting study. The polymer - not cross-linked and thus soluble - represents the water soluble part of AGRAGEL.
After the composting of the radio labeled polymer for 45 days in a laboratory composting equipment the biodegradation rate (mineralization) was calculated from the amount of carbon dioxide produced. Further the compost was extracted with water and sodium hydroxide solution to determine the distribution characteristic of the polymer in the water soluble part and in the soluble and insoluble humus fractions of the compost. Composting resulted in a biodegradation rate of 2.4 %. The remaining water soluble part was 26 % and further 5.3 % were found in the soluble fraction of the humic acids. However 63 % of the applied radioactivity were detected in the insoluble sediment indicating a high binding rate of the polymer to the humus fraction. Unspecific binding of the polymer to the humus is excluded. The results indicate that the soluble part of AGRAGEL will be mineralized only partly during composting but the main fraction will become part of the humus matrix.
Biodegradation by fungi
The degradation of AGRAGEL by white-rot fungi, ubiquitous organisms which are capable to degrade the most recalcitrant biopolymer, lignin, was investigated. Liquid cultures of Phanerochaete chrysosporium were incubated with radio-labeled AGRAGEL as a gel under nutrient limited conditions and the rate of mineralization was monitored by quantifying the amount of 14CO2 produced. The insoluble polymer was de-polymerized within 3 weeks to water-soluble products by an extra-cellular enzymatic degradation system. The polymer degradation products were neither toxic to the fungus nor to other organisms as shown in toxicological tests with bacteria, algae, ciliates and daphnia. Over longer time periods up to 80 % of the water-soluble metabolites were incorporated in the fungal mycelial mat. Mineralization by the fungus occurred throughout the time course and although the overall amount of polymer mineralized was low, the results suggest that almost all of the polymer was degraded to fungal metabolites. The results have demonstrated that white-rot fungi are in principle capable of degrading AGRAGEL.
Biodegradation in soil by bacteria
To investigate the biodegradation by soil bacteria Radio-labeled AGRAGEL was incubated with intact soil for 2 years. The rate of mineralization was monitored by quantifying the amount of 14CO2 produced and the amount of radioactivity extractable as well as non-extractable from the soil was determined. Within the first few days a small increase in the CO2-evolution and a decrease in the extractable fraction was determined. The rate of mineralization was low throughout the time course with a slow but steady increase, and the cumulative mineralization rate after 2 years was 3 %. The amount of non-extractable radioactivity was high during the whole time period indicating a high adsorption capacity of AGRAGEL to soil. The results confirm that soil bacteria are in principle capable of degrading AGRAGEL as long as certain molar masses are not exceeded; this is valid for the smaller oligomers and monomers which account for approximately 3 % of the water-soluble fraction.
Biodegradation in soil by bacteria and fungi
The synergistic effect of soil bacteria and white-rot fungi on the biodegradation of AGRAGEL in soil was investigated. The soil microcosms were prepared by mixing soil with radio-labeled AGRAGEL and with sawdust inoculated with the fungus Phanerochaete chrysosporium. Control microcosms with either sterile soil but with fungus or with intact soil but un-inoculated sawdust was prepared as well. Microcosms were maintained at 37 °C for 76 days and mineralization rates were determined by the amount of 14CO2 produced. Solubilization of the polymer was estimated by the degree to which radioactivity distributed throughout the soil. While mineralization was minimal in microcosms with soil bacteria but without the fungus (0.35 %) significant mineralization (4.3 %) was determined in microcosms containing fungus but no bacteria. The highest mineralization was observed in microcosms containing soil bacteria and fungi accounting for 7.3 % in 76 days. AGRAGEL was solubilized in fungal-containing soil microcosms within 19 days demonstrated by monitoring the spread of radioactivity throughout the soil. Moreover, the amount of radioactivity observed was evenly spread throughout the microcosm within 76 days, suggesting that the polymer had been completely solubilized and spread within the fungal hyphae network, as it is incorporated by the fungus. Biodegradation of 14C-AGRAGEL was also studied in agricultural soil by two white rot fungi (Pleurotus ostreatus, Dichomitus sqalens), a brown rot fungus (Flammulina velutipes) and a saprophytic soil fungus (Agaricus bitorquis) using microcosms. The highest mineralization of the copolymer to 14CO2 was measured with Pleurotus ostreatus (8.8 % within 22 weeks in soil and 31 % within 28 weeks in a pure culture on wheat straw). This species also increased the portion of soluble components of AGRAGEL in soil significantly.
The results indicate that fungi initiate significant conversion and degradation processes, leading to an increase of the polymer fraction which can be mineralized by soil microorganisms. The synergistically cooperation of white-rot fungi and soil bacteria will lead to the degradation of AGRAGEL with time.
Distribution in Terrestrial Systems
To investigate the absorption and de-absorption characteristics of AGRAGEL the aqueous extractable components of radio-labeled 14C-AGRAGEL were incubated with four different soil types: sand, sandy loam, silt clay loam and clay loam. Significant adsorption of radioactivity resulted in all soil types following 16 h equilibration and correlated well with the soil organic matter content. In sand the lowest and in clay loam the highest adsorption was obtained.
Subsequent de-absorption was incomplete in all soil types. The magnitude of the Freundlich adsorption coefficient Kf indicates that AGRAGEL has a high capacity to adsorb to soil.
To determine the soil leaching potential AGRAGEL was applied as moistened gel to the surface of soil columns of each of the four soil types and leached for 5 days.
Migration throughout the columns was very low and leaching was in the order sand > sandy loam > silt clay loam > clay loam. The distribution coefficient for each of the soil types indicates that the potential for leaching is very low.
Heavy Metal Mobilization
A watery suspension of sludge and compost was incubated with AGRAGEL (0.1 % and 1 %) for 24 hours to investigate the heavy metal mobilization potential of AGRAGEL. After processing the concentrations of Zn, Cd, Pb, Ni, Fe, Mn, Cr, Cu, Mg, Al and P in the supernatant have been similar in comparison to water treated controls. Therefore it is concluded that AGRAGEL has no relevant potential to interact and mobilize heavy metal ions from compost or soil.
Conclusions regarding Environmental Fate
Taking into account all investigations with respect to biodegradability research has shown that AGRAGEL does not constitute a persistent polymer, but is susceptible to natural, fungal degradative processes known to occur in the environment. After solubilization, degradation and mineralisation the constituents of the polymer are integrated into the natural carbon and nitrogen cycle. AGRAGEL is safe and compatible to the environment without any negative effect on the natural soil compartment.
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