Research Articles De-icers

Ecotoxicity of Alternative De-icers

Anneli Joutti*, Eija Schultz, Piia Pessala, Taina Nystfin and Pasi Hellst~n

Finnish Environment Institute (SYKE), Hakuninmaantie 6, 00430 Helsinki, Finland

* Corresponding author (anneli.joutti@ymparisto.fi)

I n t r o d u c t i o n

De-icers for removal of ice from roads are essential to win- tertime road maintenance in Nordic countries (Jones et al. 1992, Blomqvist and Johansson 1999, Viskari 1999). In USA 15 million tons and in Canada 5 million tons of road salts (NaCI, CaCI2, KC1 and small amounts of MgCI2) are ap- plied to roads every year (Buttle and Labadia 1999, Mayer et al. 1999, Robidoux and Delisle 200 t). In Finland 100,000 tons of salt (about 70% NaC1 and 30% CaCI2) is used an- nually and NaCI has polluted groundwater in Finland dur- ing the past forty year (Viskari 1999, Nyst~n and Hellst6n 2002). There has been a growing concern of the environ- mental effects resulting from de-icing. Regulatory bodies have tried to find de-icers which have less harmful impacts on soil and groundwater. Alternative organic de-icers are e.g. potassium formiate (KFo), potassium acetate (KAc), and calcium magnesium acetate (CMA) (Nyst6n and Hellst~n 2002). These substances do not possess the corrosive char- acteristics of NaC1.

At present the environmental hazard of chemicals is evalu- ated primarily by chemical analyses. However, chemical data alone are insufficient to assess the potential environmental hazards of chemicals. Bioassays can help define the ecotoxi- cological effects of environmental contaminants. The bio- assay approach integrates the biological effects and factors such as pH, bioavailability and interactions between com- pounds~ In this respect bioassays representing different tro- phic levels such as plant, microbiological and enzymatic tests could be used.

Experiments with plants are easy technologically and results are welt reproducible. A simple plant test, the Allium cepa test, is based on growth inhibition of the roots of onion bulbs (Fiskesj6 1988, 1997). This plant test has been used for test- ing pesticides, heavy metal salts, industrial chemicals, wastewater and soil samples (Fiskesj6 1997, Kristen 1997)o Duckweed Lemna species have been used to test the toxicity of chemicals, elutriates from sediments and waste leachates (Lewis 1995). In general duckweed species have not been very sensitive when compared to other species, e.g. algae~ fish or daphnids, yet they are widely used to model organ- isms for vascular plants (Lewis t995).

Microbial and enzymatic bioassays are rapid, easy and cost- effective in toxicity testing. The luminescent bacteria Vibrio fischeri test is one of the most widely used microbial tests and has been shown to be sensitive especially to organic

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compounds (Bulich 1979). The mitochondrial reverse elec- tron transport fRET) test is an enzymatic screening test and is based on the phenomena of electron transport and energy coupling (Blondin et al. 1987, Knobeloch et al. 1994, Read et al. 1997) which are ubiquitous in biological systems~ The high sensitivity and relatively good correlation of RET with whole organism tests make it a useful screening tool (Betterman et al. 1996).

While high levels of NaCI have been proven to have adverse effects on the ecosystems (Jones et al. 1992, Blomqvist and Johansson 1999, Buttle and Labadia 1999, Mayer et al. 1999, Viskari 1999), much less is known about the effects of alter- native de-icers. Robidoux and DelMe (2001) studied the ef- fects of NaC1 and two alternative deicers, NaFo and CMA, in artificial and natural soil on growth of macrophytes (cress, barley etc.) and on survival of the earthworm. However, there have been relatively few studies describing the effects of other alternative de-icers.

The goals of our study were

* to characterize sodium chloride NaCI and five alterna- tive de-icers (CaCI2, MgCI2, KFo, KAc and CMA) with biotests described above: two plant tests, Allium cepa and Lemna tests, the enzymatic in vitro test RET test and the luminescent bacteria test BioTox; and

* to compare the sensitivity and applicability of these biotests. We have applied these biotests earlier for waste and soil eluates (Vaajasaari et al. 2000, Joutti et al. 2000, Juvonen et al. 2000, Schultz et al. 2002, Vaajasaari et al. 2002)

1 Materials and Methods

1.1 Sample description

The compounds tested: sodium chloride and calcium chlo- ride (Kemira Chemicals Oy, Finland), magnesium chloride (Kemikaalimiehet Oy, Finland), potassium formiate (Kemira Chemicals Oy, Finland), potassium acetate (Clariant, Swe- den) and calcium magnesium acetate (Kemira Chemicals Oy, Finland) were of technical grade.

1.2 Ecotoxicological tests

The samples were tested with a battery of aquatic bioassays. The results of the bioassays ,were calculated as EC50 values. The EC50 values were also transformed to toxic units TU (=100/EC50).

1.2,1 Plants

The Allium cepa root elongation test was done according to Fiskesj6 (1988, 1993, 1997). Onion Allium cepa bulbs were exposed to eluate dilutions to estimate plant toxicity. The outer scales were removed from fresh, small, equal-sized bulbs of a commercial variety of the AlIium cepa. Each elu- ate was diluted with tap water and tested at concentrations from 100% to 0 39% (dilution factor 2). Controls were grown in tap water. The bulbs were protected from direct sunlight. After 4 days exposure at room temperature, the degree of toxicity was estimated by comparing the length of the root bundles grown in samples to the root length of con-

trols. The mean value of the 3-4 replicates was used for calculations. The percentage inhibition of root lengths com- pared to the control was used for calculation of EC50 val- ues. The EC50 values for the onion test were calculated by graphical interpolation.

Lemna growth inhibition test. A local Lemna species was used as test plant. The sample extracts, at 10 or 50% final concen- tration, were mixed with buffered duckweed nutrient medium (Lemna growth inhibition test, OECD 1996). Ten young plants with two leaves (fronds) were transferred into vessels contain- ing 15 ml of the test solution. They were incubated for 5 days trader the following test conditions: light 4000-5000 lux, light/ dark period 16/8, temperature 20 _* 2 ~ relative humidity 40-60%). The experiments were performed in three replicates for the controls, the sample dilutions and the positive controls (potassium dichromate 5 mg Cr/l). The number of fronds was determined on day 3 and at the end of the test. Fresh weight and the chlorophyll a content were also measured. Chloro- phyll was extracted with ethanol by homogenizing the plant tissue in a Potter glass homogenizer. The growth inhibition was calculated using frond number, fresh weight and chloro- phyll a concentrations.

1.2.2 Microbes

Vibrio fischeri luminescence inhibition test. The sample solu- tions were adjusted to pH 7 (_*0,2) and the tests were per- formed with the BioTox TM test kit (Aboatox Oy, Finland) ac- cording to the standard procedure (SFS-EN ISO 11348-3) and manufacturers instructions. An exception to the standard pro- cedure was the dilution of the samples to suit the osmolarity requirements (equal to 2% NaCI solution) of the bacteria. Osmolality was measured (The Advanced Micro-Osmometer Model 3MO, Advanced Instruments tnc ) and the samples were diluted to test concentrations that equal the osmolatity of 2% NaC1. For example with the KAc solution the highest test concentration was 3 % and the dilutions below that were made with required NaCl additions. The inhibition of the light production of the bacteria was measured with the No-Orbit Sirius 1257-1uminometer (Bio-Orbit, Finland) after 30 minute contact times. All measurements were run as duplicates and EC50 values were obtained by linear regression analysis (BioTox TM Software, Bio-Orbit).

1.2.3 Cell organelles

Reverse electron transport, RET, assay is an in vitro screening test based on the use of sub-mitochondrial particles prepared from isolated beef heart mitochondria (Knobeloch et al. 1994, Read et al. 1997). Electron transfer enzymes are exposed in a reaction mixture containing succinate as substrate, NAD+ as electron acceptor, antimycin A to avoid the loss of electrons to oxygen and ATP as energy supply. The reduction of NAD+ to NADH was measured kinetically at 340 nm in a microplate reader (iEMS, Ascent, Labsystems, Finland). Sample extracts were diluted with water in two-fold serial dilutions to achieve assay concentrations from 66.7% to 0.13% of the eluateso The enzyme activities from the exposed and unexposed as- says were compared and the inhibition percentages were used to calculate the EC50 values.

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Research Articles De-icers

2 Results

The results of all biotests are presented as ECS0 values in Table 1. For a more convenient graphical expression and interpretation of toxicity data, the EC50 values were also transformed to toxic units TU (see Fig. 1-3)o

CMA was unsuitable for RET and BioTox tests because it was not possible to analyze turbid samples with these bioassays. Turbidity, however, did not disturb the plant tests. The toxicities of the de-icers varied both between different de-icers and between different bioassays (Fig. 1). On the whole, the responses varied from no toxicity to 2632 TU (see Fig. 1-3). Most of the responses of the de-icers tested were very toxic or toxic to test organisms.

Hg. 1: Toxicity of six de-icers on Lemna, Atliurn cepa, Vibrio fischeri(BioTox) and RET (results expressed in toxic units TU). It was not possible to test sample CMA with RET and BioTex (see the text)

In the Lemna test, all three parameters (number of fronds, chlorophyll a content and flesh weight) gave the same results (Table 1). The toxicities of de-icers varied considerably: KFo and KAc were substantially more toxic to Lemna than other substances (Fig. 2), although CMA and CaC1 z were also rela- tively toxic to duckweed. The results of the Alliurn cepa root elongation test were very similar to the Lemna test: KFo and KAc were highly toxic, and CMA toxic, to onion root growth. Both plant tests were very sensitive. The BioTox test demon- strated that, of the five samples, only KAc and KFo were slightly toxic, while CaCI2, NaCI, and MgC12 were not toxic to V. fischeri at the tested concentrations. According to the RET test all de-icers except CMA were slightly or relatively toxic.

3 Discussion

3.1 Sensitivity and comparison of the four bioassays

The most sensitive species tested were the plants, duckweed and onion. The sensitivity ranking for tests in decreasing

Fig. 2: Response profiles of the biotests on the de-icers (as TU)

order was Lemna > Allium cepa > RET > Vibrio fischeri (see Fig. 2). The Lemna frond test was 92 times more sensitive and the AIlium test 70 times more sensitive than V. fischeri to KFo. The plant bioassays proved to be sensitive but easy for the biotesting of de-icers It was possible to analyze tur- bid and colorful samples with these tests.

The four bioassays used could be divided into two different groups according to their response to different test organ- isms (see Fig. 2): The two plant tests and BioTox were more sensitive to the influence of organic substances. By contrast, RET was more sensitive to inorganic compounds.

3.2 The toxicity of the de-icers

Most of the responses of the de-icers to the assay organisms were very toxic (TU >100) or toxic (TU 10-100) (toxicity classification according to Bulich, 1982). Fig. 3 clearly shows that the six de-icers differ in their response profiles. On the whole, KFo and KAc were the substantially most toxic sam- pies. The de-icers can be ranked according to the Lemna and Allium tests: KFo > KAc > CMA > CaCI z > NaCt > MgCI z.

The technical grade de-icers used in this study may contain additive chemical compounds whose toxicity may interfere with the toxicities of chemicals of interest. These additive com- pounds can be corrosion inhibitors (phosphorus compounds), and-caking substances (iron(II)cyanide or iron(III)cyanide) etc,

Only a few studies have tested de-icers for ecotoxicity. It is of interest to compare our results to data of Robidoux and Delisle (2001). They studied the effects of three de-icers: NaCI, NaFo and CMA, on survival of the earthworm and on germination and growth of cress and barley. According to this study, the de-icers were also toxic or very toxic to these test organisms and the relative toxicity of deicers was NaCI = NaFo > CMAo

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Fig. 3: The response profiles of different de-icers (as TU)

In the Helsinki area old urban snow contains 6-41 mg/l chloride and wastewater 87 mg/t (H. Pitk/inen, pets. comm.). Considering the concentration of chloride in used snow and the EC50 values of this study (table 1), chloride or NaCI does not appear to affect the growth of onion and duckweed. Onion could be the representative of agri- cultural areas and duckweed the representative of aquatic environments. In Kouvola, southern Finland, old urban snow may contain 3000 mg/l NaCI (P. Wallius, pers. comm.). At this concentration, duckweed growth and RET enzymes would be affected (EC50 3100 and 600 rag/l, re- spectively). Deleterious effects of road salts did not reach far from the road: at distances of 20-30 m, the salt concen- trations and visible injuries in needles were insignificant (Hautata and K/irenlampi 1992).

According to our study KFo and KAc were very toxic to onion and duckweed (EC50 38-80 mg/l). Nysten and Hetlsten (2002), however, showed in an in vitro study that formiate and acetate may be biodegraded completely in 1 m and 3.5 m high soii filters at low temperature (3-6~ Formiate was degraded more efficiently than acetate and after four weeks the concentration of formiate was <5 mg/1 and that of acetate was <20 mg/l. Such low concentrations of formiate and acetate in eluate and the EC50 values of this study indicate that they might not affect growth of onion and duckweed. Formiate and acetate does not affect RET enzyme. However, the degradation of the formiate and ac- etate may be different in the field study scale than in the in vitro study. Thus, we are going to test formiate in the field 2002-2004 in Suomenniemi, southern Finland.

4 Conclusion and Outlook

This study demonstrated that the tested de-icers had signifi- cant effects on plant growth, microbial luminescence and enzymatic activity. Toxicity of the de-icers varied greatly: potassium formiate KFo and potassium acetate KAc were the most toxic compounds. Toxicity could hardly be pre- dicted from the chemical concentrations of these de-icers. However, the decision finding for soil quality assessment is at the moment mainly based on the results of chemical analy- ses without considering ecotoxicological investigations. In summary, these results demonstrate a need for including tox- icity measurements in the evaluation of technologies used in soil treatment and illustrate the potential value of such meas-

urements for ensuring that no or minimal environmental contamination would arise from de-icing process.

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Received: September 16th, 2002 Accepted: December 30th, 2002

OnllneFirst: July 14th, 2003

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