Phytotoxicity Assessment of Wastewater from Industrial Pulp Production ^†

Abstract

During the production of pulp from natural raw materials, such as wood, large amounts of waste are generated. When they are reused, e.g., in agriculture, ecological requirements towards the environment must be met, and the associated risks must be known. In the framework of this study, the phytotoxicity of liquid waste generated during the industrial processing of pulp using the sulfite process was investigated. The results of semichronic toxicity tests on Lepidium sativum L. and Sinapis alba L. showed a direct effect on the growth and development of plants and proved that the undiluted samples were phytotoxic. However, the phytotoxicity of the investigated waste samples was influenced by their dilution ratio. Thus, finding the optimal solution ratio is crucial for the reuse of liquid waste. A nonphytotoxic effect on the roots of the tested plants was proven with a solution in a ratio of 1:100. In addition, the control samples diluted with a medium manifested phytotoxicity due to the activity of internal microorganisms.

1. Introduction

The paper industry is one of the largest global polluters of the environment. Large amounts of gaseous, liquid, and solid waste are released into the environment, especially during the sulfite process of pulp production. The biggest problems in terms of environmental pollution include wastewater from production and emissions into the air [1]. Wastewater from pulp production mainly contains organic compounds (resins and organic acids), chlorinated compounds, nitrates, and phosphates, while toxic metals and metalloids are found in low quantities [2].

Due to the relatively high number of organic substances in waste, it can be assumed that the use of pulp production waste may be suitable for agricultural purposes and the production of horticultural substrates. However, during the sulfite process, large amounts of chemicals are used, and some of them can be toxic to the environment. Therefore, the application of pulp waste in agriculture can represent a serious environmental risk. In particular, it can inhibit or stimulate plant growth. Ultimately, it can also affect the quality and species diversity of soil microorganisms [3,4].

A suitable alternative could be the introduction of technology that uses inorganic matter to reduce the content of contaminants without causing phytotoxicity. Such methods include, for example, biosorption, where waste plant biomass is used as a biosorbent, the basic building blocks of which are cellulose, hemicellulose, and lignin. This technology does not depend on the metabolism of microorganism cells; rather, it is based on physicochemical interactions between metal and functional groups of plant cell walls. The process is characterized by relatively high speed and the possibility of recovering adsorbed metal from the biosorbent using reverse desorption processes [5,6].

The chemical composition of waste generated during the sulfite process may vary, and it is thus advisable to regularly assess the waste from the point of view of ecotoxicity [7]. Through acute toxicity tests, it is possible to assess its direct effect on the biotic component of the environment [8]. From the point of view of practical use of the waste in agriculture, it is essential to carry out regular monitoring of its phytotoxic effect, which is assessed using tests on selected plants [9].

The aim of this research was to assess the phytotoxicity of liquid waste (wastewater) generated during the production of pulp and to search for optimal dilution of wastewater that reduces the inhibitory effect on plant growth.

2. Materials and Methods

2.1. Tested Samples and Their Composition

The tested waste samples from the sulfite pulping process were a mixture of waste and water containing mixtures of waste fibers (FB), sludge from mechanical processing and sorting (SL), and industrial wastewater produced during the pulping process (Lenzing Biocel Paskov a.s., Paskov, Czech Republic). The composition of individual samples is shown in

Table 1. Composition of the basic (undiluted) tested samples for evaluating the phytotoxicity of liquid waste.

Sample Identification	FB [g]	SL [g]	WW [mL]	TSB [mL]
A	50	50	300	-
B	50	100	450	-
C	100	50	450	-
D	50	50	200	100

Notes: FB: waste fibers, SL: sludge, WW: wastewater, TSB: tryptone–soybean culture medium.

; the control samples contained only distilled water. Regarding the composition of the samples, an increased activity of microorganisms (primarily cellulolytic microorganisms) was assumed in them. To stimulate their activity, a tryptone–soybean culture medium, TSB (M011, Soyabean Casein Digest Medium, HiMedia Laboratories, Mumbai India), was added only to sample D.

Samples in Table 1 were filtered through filter paper (KA-2, Papírna Perštějn spol. s.r.o., Czech Republic) before testing. These samples were further diluted with sterile distilled water in ratios of 1:10, 1:50, and 1:100. The samples prepared in this way were then subjected to semichronic toxicity testing according to the current Czech legislation [10,11] on white mustard (Sinapis alba L.) and cress (Lepidium sativum L.).

2.2. Phytotoxicity Tests

To test the phytotoxicity of industrial wastewater, tests were carried out on mustard (Sinapis alba L.), and cress (Lepidium sativum L) plants according to standard ČSN EN ISO 18763 (836447) [11]. In order to ensure the safe use of wastewater in agriculture in accordance with Decree No. 273/2021 Coll. [11], inhibition or stimulation of root growth higher than 50% compared to the control must be confirmed within a period of 72 h. A total of 20 seeds of the test plants were spread on filter paper in Petri dishes and exposed to the tested wastewater solutions, which consisted of a mixture of waste from pulp production. Root lengths of germinating plants were measured after 72 h seed incubation, and a thermostat set at 20 °C was used, as can be seen from Figure 1. From the results of each observed concentration (undiluted sample and dilution of 1:10, 1:50, and 1:100), the arithmetic means were determined, which were later compared with the arithmetic mean from the control measurement. Based on the arithmetic means of plant root lengths, inhibition was calculated for each concentration.

Inhibition was calculated using the following formula: (arithmetic mean length of roots in control−arithmetic mean length of roots in sample)/(arithmetic mean length of roots in control).

3. Results and Discussion

3.1. Results of the Semichronic Toxicity Test on Mustard (Sinapis alba L.)

Based on the results of the inhibition of growth of the roots of the tested plants for each sample, it can be stated that the undiluted samples of pulp waste mixtures caused inhibition of growth of the roots of the tested plants, as can be seen from Figure 2. In all examined undiluted samples (A–D), there was more than 80% inhibition of Sinapis alba root growth; therefore, the liquid waste was phytotoxic. Inhibition in undiluted samples ranged from 81.63% to 100%. The application of this mixture of waste in agriculture without any subsequent treatment represents an environmental risk.

Better results were obtained with diluted samples A and B in ratios 1:50 and 1:100, where they even showed a stimulating effect on plant development. Compared to the control, stimulation reached values of 19.79–32.72%. Negative effects on growth of Sinapis alba were confirmed for sample D in dilution of 1:10 as the inhibition of plant development and growth was found to be 62.92%, i.e., 12.92% more than the ecotoxicity limit according to Decree No. 273/2021 Coll. [11].

3.2. Results of the Semichronic Toxicity Test on Cress (Lepidium sativum L.)

The results of measurement for Lepidium sativum are shown in Figure 3. The results from the undiluted samples showed inhibition of growth similar to the results for Sinapis alba. The measurement results also showed that in samples A, B, and C, when they were diluted 1:10, the growth of the roots of the tested plants was stimulated. However, phytotoxic results were achieved in sample A at a dilution of 1:10 and sample D at dilutions of 1:10 and 1:50. Sample A in dilution of 1:10 showed stimulation of plant growth and development by 86.63% compared to control samples, which, however, was in accordance with the legislative requirement for evaluation tests according to Decree 273/2021 Coll. [11]. The results must be evaluated as an inhibition phenomenon as the detected stimulation exceeded the standard 50% limit value by 36.63%. Samples B and C diluted in a ratio of 1:10 had stimulating effects on the growth of Lepidium sativum roots. More diluted samples B and C inhibited the growth of the root, with the inhibition reaching a value up to 19.19%. The most toxic was again sample D, which contained a nutrient medium for microorganisms.

3.3. Discusion of the Results

Overall, the results showed that the undiluted samples of the pulp waste mixture had a negative effect on root growth of mustard and cress, and according to the Czech legislation, Decree 273/2021 Coll. [11], we consider these samples phytotoxic due to the above-limit results (more than 50% inhibition or stimulation). The overall results are shown in

Table 2. Evaluation of the phytotoxicity of a mixture of pulp waste according to Decree 273/2021 Coll.

Sample Identification	Dilution	Mustard Results	Cress Results	Complete Results
A	Undiluted	×	×	×
	1:10	✔	×	×
	1:50	✔	✔	✔
	1:100	✔	✔	✔
B	Undiluted	×	×	×
	1:10	✔	✔	✔
	1:50	✔	✔	✔
	1:100	✔	✔	✔
C	Undiluted	×	×	×
	1:10	✔	✔	✔
	1:50	✔	✔	✔
	1:100	✔	✔	✔
D	Undiluted	×	×	×
	1:10	×	×	×
	1:50	✔	×	×
	1:100	✔	✔	✔

Notes: [✔] nonphytotoxic, [×] phytotoxic.

. In sample A with 1:10 dilution, negative effects on the growth of cress were also detected, and we can also consider this sample of waste mixture as phytotoxic. In the other cases of sample A, no phytotoxic effects of waste mixtures on the examined plants were detected, as shown in Table 2.

Samples diluted with tryptone–soybean culture medium, undiluted samples, and samples diluted in ratios of 1:10 and 1:50 were found to be very risky. Because the tryptone-soybean broth supports the growth of indigenous microorganisms, their increased activity can be assumed in all samples in the D series that were diluted with the culture medium. Nutritional support for the growth of microorganisms in the tested samples will be shown by increased production of their secondary metabolites. In accordance with previously published studies [12,13,14,15], it can be assumed that these metabolites exhibit a high level of diversity in their properties, such as structures, phytotoxic activities, and toxicity modes. In particular, secondary metabolites of fungi are poisonous substances to plants produced through naturally occurring biochemical reactions of these microorganisms [16]. Therefore, the phytotoxicity of the mentioned samples of the D series was probably increased by the metabolites of the microorganisms found in this medium. In addition, some expert studies have shown that synergistic effects of the toxic biomolecules produced can occur in different microorganisms, as evidenced, for example, in [16].

4. Conclusions

This research confirms the necessity of monitoring waste from pulp production, especially from the point of view of its reuse in other sectors, e.g., agriculture. The research also showed that in some cases, the application of pulp waste as fertilizer can lead to significant deterioration in the quality and quantity of flora. Undiluted pulp waste was found to slow the growth of mustard (Sinapis alba L.) and cress (Lepidium sativum L.) plants. Choosing the optimal dilution of waste with water can be key to preventing its negative effect. This research confirmed the negative effect of undiluted pulp waste samples. In the case of sample A diluted in a ratio of 1:10, phytotoxic effects on cress were observed. Samples diluted with tryptone–soybean medium also showed phytotoxicity due to the activity of internal microorganisms.