A METHOD FOR CHARACTERISING NATURAL AND WASTE WATERS USING PLANT SEEDS C H A R L E S A. B I N E Y
Institute of Aquatic Biology, P.O. Box 38, Achimota, Ghana
(Received January 1990) Abstract. Studies were conducted to describe a method for the characterisation of natural and waste waters using plant seed germination as a technique for toxicity testing. Maize seeds were found to be more sensitive to differences in water quality than cowpea seeds and also showed more consistency in results. Trials conducted using natural waters and effluents from gold mining and textile manufacturing operations are also reported.
1. Introduction
Plant organisms can be usefully employed to monitor the state of the environment. For example, mercury contamination near emission sources have been evaluated using plant species (Tamura et al., 1985; Shaw and Panigrahi, 1986) and some studies have investigated accumulation by plants of trace metals (Bargagli et al., 1985; Fayed and Abd-E1-Shafy 1985; Sears et al., 1985). Plants have also been used for toxicity testing although not as extensively as animals. Wong and Bradshaw (1982), studied the comparative toxicity of heavy metals using root elongation of rye grass, Lolium perenne. Wang (1985) also used plant seed germination as a technique for toxicity tests of phenolic compounds. In some industrialised countries such as the United States of America and Great Britain, plants are used by industries and regulatory authorities among the batch tests for new chemicals. Algae, macrophytes and some terrestrial plants are also used in bioassays. Davis and Beckett (1978) reviewed the use of young plants to detect metal pollution. Recently, Burton (1986) in his review of the use of plants in biomonitoring of the environment reported on angiosperms for monitoring metal pollution. Plant seeds have the advantage of being usually dry, dormant and having a long shelf life but with the ability of being activated under suitable conditions. These advantages, coupled with the ready availability of different kinds of seeds, prompted the present study with the aim of describing a simple method for characterising natural and waste waters using plant seed germination. It is based on the premise that since germination is an active and ordered process, certain stages may be inhibited by the presence of some substances. For example, Mallette et al. (1960), have reported that chemical inhibitors of germination include ammonia, hydrocyanic acid, parascorbic acid, essential oils and some alkaloids. Thus, when seeds are exposed to potential inhibitors during germination, the overall rate of the process may be decreased as a function of the concentration of the inhibitors. Where a mixture of such substances occur, additive, synergistic or antagonistic effects may come into play. It is also possible that germination may be enhanced by the presence of certain EnvironmentalMonitoring and Assessment 18: 123-128, 1991. 9 1991 Kluwer Academic Publishers. Printed in the Netherlands.
124
C H A R L E S A. B I N E Y
substances such as low nutrient concentrations. Since it is not possible to conduct a complete or total analysis of a sample of water, this method would be useful for the expression of the total character of a sample. Industrial effluents for instance are very diverse in their characteristics and analysis of even a fraction of their various constitutents is very expensive. Also, conclusions based on the analysis of a few parameters may prove misleading. This method on the other hand provides a cheap and quick means of characterising industrial effluents and may have useful applications especially in developing countries. Although seed germination tests do not provide a substitute for chemical analysis, they can give an indication of toxicity. Thus, it should be possible to quickly screen effluents from different sources and identify those potentially harmful ones whose discharges into the environment may have to be regulated. Extensive chemical analysis may then be conducted on these effluents to determine the causes of their toxicity. The effects of effluent discharges on different sections of a river or stream can also be cheaply monitored by this method. 2. Materials and Methods 2.1. GENERAL Maize seeds, obtained from the Ghana Seed Company, were washed in tap water to remove any surface contamination. To inhibit the growth of yeasts and fungi during germination, seeds were sterilised for 30 min. in 1:10 solution of chlorex after which they were washed ten times in distilled water with stirring, floating seeds being discarded. The seeds were then soaked in test water for a noted period of time, removed and dried on filter paper for about 30 min. For the incubation of seeds, 9 cm petri dishes each lined with filter paper and heated at 105 ~ for 1 hr were used. Prior to incubation the petri dishes were cooled to room temperature and the filter papers moistened with a known volume of test solution. The treated seeds were accurately weighed, transfered into the petri dishes and incubated under normal and uniform laboratory light and temperature conditions. Temperatures ranged from 22 to 28 ~ After a given period of incubation, the germinated seeds were removed from the petri dishes, dried on filter paper and reweighed. All experiments were conducted in duplicate and repeated twice. Distilled water was used as control. 2.2. SELECTIONOF SEED TYPE Initially two kinds of seeds, maize (Zeamays) and a variety of cowpea (Vignaunguiculata), were used to compare the effects on germination of the following test samples; double distilled water, distilled water, tap water, diluted and undiluted effluents from the Ghana Italian Petroleum Company refinery. The seeds were soaked for 5 hr in test water samples of known pH followed by incubation for 96 hr in petri dishes lined with filter papers onto which 5 ml of the respective solutions had been pipetted. The initial weight in each petri dish was 8 g.
NATURAL
AND
WASTE
WATERS
USING
PLANT
SEEDS
125
2.3. DETERMINATIONOF OPTIMUMCONDITIONS For the selected seed type, optimum conditions were determined by varying the length of incubation period, weight of seeds, time of soaking of seeds and volume of incubation water. 2.4. CHARACTERISATIONOF NATURALAND WASTEWATERS After determining the optimum conditions required for sample testing, trials were conducted using both natural and waste waters some chemical parameters of which were also measured. The analytical procedures for these parameters have been previously described (Biney, 1982, 1985, 1986) and are summarised below: pH - measured in the field with a portable Griffin pH meter. Copper, lead, cadmium, zinc, iron - determined by flame atomisation using a VARIAN 1275 atomic absorption spectrophotometer. Mercury - cold vapour atomic absorption spectrophotometry. Orthophosphate - determined colorimetrically using ammonium molybdate and ascorbic acid. Nitrate-nitrogen - determined by hydrazine reduction followed by diazotizing to form an azo dye. Ammonia-nitrogen - measured by the indophenol blue method. Sulphate - determined turbidimetrically using barium chloride. Chloride - estimated by silver nitrate titration using potassium dichromate. Calcium, magnesium - determined by EDTA titration. All samples were filtered through an 0.45 #m filter before analysis with the exception of those to be tested for trace metals which were digested in a teflon bomb. Analysis were carried out in duplicate. 3. Results and Discussions
With the exception of the chemical analysis all results represent the means of six separate readings from three experiments and have been expressed as percentage weight increases or as R values. R was calculated as the ratio of the percentage weight increase of seeds in a test solution to that of a distilled water control. For convenience, this ratio was arbitrarily described as an R value. 3.1. SELECTIONOF SEED TYPE From the results summarised in Table I, maize appeared sensitive to the differences in water quality as it was least inhibited by double distilled water and most inhibited by undiluted refinery effluent. For cowpea seeds, differences in water quality did not affect their germination. Also the low standard deviations of the percentage weight increases for maize showed it to be more consistent in toxicity testing. Further studies towards the elucidation of optimum conditions were therefore limited to maize seeds. It is not likely that these results were greatly influenced by the differences in pH of the
126
CHARLES A. B1NEY TABLE I
Effectsof differentqualitiesof water on germinationof maize and cowpea Sample
Double distilled water Distilled water Tap water Dilute refinery effluent (1:10) Undiluted refinery effluent
% Weight increase
pH
6.6 5.8 6.9 6.3 6.9
_+ 0.3 _+ 0,3 + 0,1 + 0.2 ___0.4
Maize
Cowpea
36.2 35.8 34.4 24.6 9.6
36.8 _-!-12.4 38.3 + 8.0 39.4 + 10.9 36.8 + 13.3 34.4 4- 4.1
_+ 2.1 +_ 1.7 _+ 3.3 _+ 1.8 -i- 3.9
test sample solutions. Wang (1985) showed that pH in the range of 5 to 9 did not have a significant effect on the germination of velvet leaf(Abutilon theophiastOseeds. In this study the pH ranged form 5.8 to 6.9. 3.2. DETERMINATION OF OPTIMUM CONDITIONS
Based on the results of the preliminary experiments during which the test conditions were varied, the following is a description of the procedure for testing water toxicity to maize seed germination. (i) Wash seeds with tap water to remove any surface contamination. (ii) Sterilise seeds in 10% Chlorex solution for 30 min. (iii) Wash ten times with distilled water and discard floating seeds. (iv) Soak equal portions of seeds for 5 hr in test water and in a distilled water control. (v) Dry seeds on filter paper for about 30 min. (vi) Accurately weight about 4 g of seeds into oven-sterilised 9 cm petri dishes each lined with filter paper moistened with 5 ml test water or distilled water. (vii) After 72 hr of incubation under normal laboratory light and temperature (22 to 28 ~ conditions remove seeds, dry and reweigh. (viii) The results may be expressed as an R value which is a ratio of the percentage weight increase of the test sample to that of the distilled water control. 3.3. CHARACTERISATION OF NATURAL AND WASTE WATERS The results of trials conducted under the optimum conditions are presented in Tables II and III. The following test samples collected from different locations in Ghana were used: (i) Waste waters from gold ore precessing operations in Obuasi. (ii) Surface and groundwaters from the Obuasi gold mining area. (iii) Treated water used for domestic purposes in Obuasi. (iv) Untreated waste water from two textile factories in Tema. (v) Water from the Kao Kudi stream, which flows through an urban residential area in Accra. From the ratios (R) of the percentage weight increase of the test samples to that of the controls (Tables II and III) the most toxic of the samples was untreated effluent from gold ore processing with an R value of 0.09. The least toxic was treated potable water with a
127
NATURAL AND WASTE WATERS USING PLANT SEEDS TABLE II R values and mean concentrations (/~g ml-~) of trace metals in natural and waste waters Sample
R
Hg
Cu
Pb
Zn
Fe
Gold mining, Obuasi Untreated effluent Lecheate from tailings River Kwabrafosu River Sibi Water from well Treated potable water
0.09 0.36 0.46 0.77 0.89 1.07
0.06 0.06 0.07 0.05 0.02 0.01
8.91 9.32 8.23
Institute of Aquatic Biology, P.O. Box 38, Achimota, Ghana
(Received January 1990) Abstract. Studies were conducted to describe a method for the characterisation of natural and waste waters using plant seed germination as a technique for toxicity testing. Maize seeds were found to be more sensitive to differences in water quality than cowpea seeds and also showed more consistency in results. Trials conducted using natural waters and effluents from gold mining and textile manufacturing operations are also reported.
1. Introduction
Plant organisms can be usefully employed to monitor the state of the environment. For example, mercury contamination near emission sources have been evaluated using plant species (Tamura et al., 1985; Shaw and Panigrahi, 1986) and some studies have investigated accumulation by plants of trace metals (Bargagli et al., 1985; Fayed and Abd-E1-Shafy 1985; Sears et al., 1985). Plants have also been used for toxicity testing although not as extensively as animals. Wong and Bradshaw (1982), studied the comparative toxicity of heavy metals using root elongation of rye grass, Lolium perenne. Wang (1985) also used plant seed germination as a technique for toxicity tests of phenolic compounds. In some industrialised countries such as the United States of America and Great Britain, plants are used by industries and regulatory authorities among the batch tests for new chemicals. Algae, macrophytes and some terrestrial plants are also used in bioassays. Davis and Beckett (1978) reviewed the use of young plants to detect metal pollution. Recently, Burton (1986) in his review of the use of plants in biomonitoring of the environment reported on angiosperms for monitoring metal pollution. Plant seeds have the advantage of being usually dry, dormant and having a long shelf life but with the ability of being activated under suitable conditions. These advantages, coupled with the ready availability of different kinds of seeds, prompted the present study with the aim of describing a simple method for characterising natural and waste waters using plant seed germination. It is based on the premise that since germination is an active and ordered process, certain stages may be inhibited by the presence of some substances. For example, Mallette et al. (1960), have reported that chemical inhibitors of germination include ammonia, hydrocyanic acid, parascorbic acid, essential oils and some alkaloids. Thus, when seeds are exposed to potential inhibitors during germination, the overall rate of the process may be decreased as a function of the concentration of the inhibitors. Where a mixture of such substances occur, additive, synergistic or antagonistic effects may come into play. It is also possible that germination may be enhanced by the presence of certain EnvironmentalMonitoring and Assessment 18: 123-128, 1991. 9 1991 Kluwer Academic Publishers. Printed in the Netherlands.
124
C H A R L E S A. B I N E Y
substances such as low nutrient concentrations. Since it is not possible to conduct a complete or total analysis of a sample of water, this method would be useful for the expression of the total character of a sample. Industrial effluents for instance are very diverse in their characteristics and analysis of even a fraction of their various constitutents is very expensive. Also, conclusions based on the analysis of a few parameters may prove misleading. This method on the other hand provides a cheap and quick means of characterising industrial effluents and may have useful applications especially in developing countries. Although seed germination tests do not provide a substitute for chemical analysis, they can give an indication of toxicity. Thus, it should be possible to quickly screen effluents from different sources and identify those potentially harmful ones whose discharges into the environment may have to be regulated. Extensive chemical analysis may then be conducted on these effluents to determine the causes of their toxicity. The effects of effluent discharges on different sections of a river or stream can also be cheaply monitored by this method. 2. Materials and Methods 2.1. GENERAL Maize seeds, obtained from the Ghana Seed Company, were washed in tap water to remove any surface contamination. To inhibit the growth of yeasts and fungi during germination, seeds were sterilised for 30 min. in 1:10 solution of chlorex after which they were washed ten times in distilled water with stirring, floating seeds being discarded. The seeds were then soaked in test water for a noted period of time, removed and dried on filter paper for about 30 min. For the incubation of seeds, 9 cm petri dishes each lined with filter paper and heated at 105 ~ for 1 hr were used. Prior to incubation the petri dishes were cooled to room temperature and the filter papers moistened with a known volume of test solution. The treated seeds were accurately weighed, transfered into the petri dishes and incubated under normal and uniform laboratory light and temperature conditions. Temperatures ranged from 22 to 28 ~ After a given period of incubation, the germinated seeds were removed from the petri dishes, dried on filter paper and reweighed. All experiments were conducted in duplicate and repeated twice. Distilled water was used as control. 2.2. SELECTIONOF SEED TYPE Initially two kinds of seeds, maize (Zeamays) and a variety of cowpea (Vignaunguiculata), were used to compare the effects on germination of the following test samples; double distilled water, distilled water, tap water, diluted and undiluted effluents from the Ghana Italian Petroleum Company refinery. The seeds were soaked for 5 hr in test water samples of known pH followed by incubation for 96 hr in petri dishes lined with filter papers onto which 5 ml of the respective solutions had been pipetted. The initial weight in each petri dish was 8 g.
NATURAL
AND
WASTE
WATERS
USING
PLANT
SEEDS
125
2.3. DETERMINATIONOF OPTIMUMCONDITIONS For the selected seed type, optimum conditions were determined by varying the length of incubation period, weight of seeds, time of soaking of seeds and volume of incubation water. 2.4. CHARACTERISATIONOF NATURALAND WASTEWATERS After determining the optimum conditions required for sample testing, trials were conducted using both natural and waste waters some chemical parameters of which were also measured. The analytical procedures for these parameters have been previously described (Biney, 1982, 1985, 1986) and are summarised below: pH - measured in the field with a portable Griffin pH meter. Copper, lead, cadmium, zinc, iron - determined by flame atomisation using a VARIAN 1275 atomic absorption spectrophotometer. Mercury - cold vapour atomic absorption spectrophotometry. Orthophosphate - determined colorimetrically using ammonium molybdate and ascorbic acid. Nitrate-nitrogen - determined by hydrazine reduction followed by diazotizing to form an azo dye. Ammonia-nitrogen - measured by the indophenol blue method. Sulphate - determined turbidimetrically using barium chloride. Chloride - estimated by silver nitrate titration using potassium dichromate. Calcium, magnesium - determined by EDTA titration. All samples were filtered through an 0.45 #m filter before analysis with the exception of those to be tested for trace metals which were digested in a teflon bomb. Analysis were carried out in duplicate. 3. Results and Discussions
With the exception of the chemical analysis all results represent the means of six separate readings from three experiments and have been expressed as percentage weight increases or as R values. R was calculated as the ratio of the percentage weight increase of seeds in a test solution to that of a distilled water control. For convenience, this ratio was arbitrarily described as an R value. 3.1. SELECTIONOF SEED TYPE From the results summarised in Table I, maize appeared sensitive to the differences in water quality as it was least inhibited by double distilled water and most inhibited by undiluted refinery effluent. For cowpea seeds, differences in water quality did not affect their germination. Also the low standard deviations of the percentage weight increases for maize showed it to be more consistent in toxicity testing. Further studies towards the elucidation of optimum conditions were therefore limited to maize seeds. It is not likely that these results were greatly influenced by the differences in pH of the
126
CHARLES A. B1NEY TABLE I
Effectsof differentqualitiesof water on germinationof maize and cowpea Sample
Double distilled water Distilled water Tap water Dilute refinery effluent (1:10) Undiluted refinery effluent
% Weight increase
pH
6.6 5.8 6.9 6.3 6.9
_+ 0.3 _+ 0,3 + 0,1 + 0.2 ___0.4
Maize
Cowpea
36.2 35.8 34.4 24.6 9.6
36.8 _-!-12.4 38.3 + 8.0 39.4 + 10.9 36.8 + 13.3 34.4 4- 4.1
_+ 2.1 +_ 1.7 _+ 3.3 _+ 1.8 -i- 3.9
test sample solutions. Wang (1985) showed that pH in the range of 5 to 9 did not have a significant effect on the germination of velvet leaf(Abutilon theophiastOseeds. In this study the pH ranged form 5.8 to 6.9. 3.2. DETERMINATION OF OPTIMUM CONDITIONS
Based on the results of the preliminary experiments during which the test conditions were varied, the following is a description of the procedure for testing water toxicity to maize seed germination. (i) Wash seeds with tap water to remove any surface contamination. (ii) Sterilise seeds in 10% Chlorex solution for 30 min. (iii) Wash ten times with distilled water and discard floating seeds. (iv) Soak equal portions of seeds for 5 hr in test water and in a distilled water control. (v) Dry seeds on filter paper for about 30 min. (vi) Accurately weight about 4 g of seeds into oven-sterilised 9 cm petri dishes each lined with filter paper moistened with 5 ml test water or distilled water. (vii) After 72 hr of incubation under normal laboratory light and temperature (22 to 28 ~ conditions remove seeds, dry and reweigh. (viii) The results may be expressed as an R value which is a ratio of the percentage weight increase of the test sample to that of the distilled water control. 3.3. CHARACTERISATION OF NATURAL AND WASTE WATERS The results of trials conducted under the optimum conditions are presented in Tables II and III. The following test samples collected from different locations in Ghana were used: (i) Waste waters from gold ore precessing operations in Obuasi. (ii) Surface and groundwaters from the Obuasi gold mining area. (iii) Treated water used for domestic purposes in Obuasi. (iv) Untreated waste water from two textile factories in Tema. (v) Water from the Kao Kudi stream, which flows through an urban residential area in Accra. From the ratios (R) of the percentage weight increase of the test samples to that of the controls (Tables II and III) the most toxic of the samples was untreated effluent from gold ore processing with an R value of 0.09. The least toxic was treated potable water with a
127
NATURAL AND WASTE WATERS USING PLANT SEEDS TABLE II R values and mean concentrations (/~g ml-~) of trace metals in natural and waste waters Sample
R
Hg
Cu
Pb
Zn
Fe
Gold mining, Obuasi Untreated effluent Lecheate from tailings River Kwabrafosu River Sibi Water from well Treated potable water
0.09 0.36 0.46 0.77 0.89 1.07
0.06 0.06 0.07 0.05 0.02 0.01
8.91 9.32 8.23
