GB/Z 41358-2022Biological test methods for comprehensive characterization of soil health (English PDF)

Foreword

This document was drafted in accordance with the rules given in GB/T 1.1-2020, Directives for standardization - Part 1: Rules for the structure and drafting of standardizing documents.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. The issuing body of this document shall not be held responsible for identifying any or all such patent rights.

This document was proposed by and is under the jurisdiction of the National Technical Committee on Soil Quality of Standardization Administration of China (SAC/TC 404).

The drafting organizations of this document are the Institute of Soil Science, Chinese Academy of Sciences (Nanjing), the Institute of Applied Ecology, Chinese Academy of Sciences (Shenyang), Nanjing Agricultural University, the Nanjing Agricultural Technology Extension Station and the Jiangsu Institute of Quality and Standardization.

The main drafters of this document are Dong Yuanhua, Li Jiangang, Liang Wenju, Hu Feng, Li Qi, Liu Manqiang, Duan Zengqiang, Chen Meijun, Sun Feifei, Gong Xin, Luo Kun, Wang Jiaqian, Li Yingbin, Dang Keke and Du Xiaofang.

Introduction

Soil supports the main life processes on Earth, maintains the balance of ecosystems and provides the food that humanity needs; it plays an irreplaceable role in present-day food security and in the building of the ecological environment. How to coordinate the production function, the environmental protection function, the support function for ecological engineering and the global change mitigation function of soil has become an important task through which modern soil science contributes to the sustainable development of human society.

Soil health is an intrinsic property of soil that cannot be obtained directly by human observation, and there is as yet no unified international standard for soil health assessment methods. Soil hosts rich and diverse groups of organisms, which play an important role in soil ecological functions such as the formation and improvement of soil structure, the accumulation and turnover of organic matter, the fixation and transformation of nutrients, the fixation and degradation of pollutants, and the transmission and control of soil-borne diseases.

The soil food web is the complex network of relationships formed among the different groups of soil organisms through feeding and being fed upon; it is the main pathway reflecting material cycling and energy flow in the ecosystem and an important biological indicator of the degree of soil health. This document selects the main biological functional groups occupying different trophic levels of the soil food web - plant roots, bacteria, nematodes and earthworms - and characterizes the degree of soil health from different aspects through, respectively, the kidney bean root morphology test method, the luminescent bacteria bioassay, the nematode community bioassay and the earthworm bioassay. This document can provide simple and practicable guidance for the health assessment of different types of soil such as grain fields, vegetable fields, orchards and grassland.

1 Scope

This document gives the principles, sampling, testing and test report requirements of the biological test methods for the comprehensive characterization of soil health, namely the kidney bean root morphology test method, the luminescent bacteria bioassay, the nematode community bioassay and the earthworm bioassay.

This document is applicable to the biological testing of the degree of soil health of cultivated land, garden land and grassland.

2 Normative references

The following documents are referred to in the text in such a way that some or all of their content constitutes requirements of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.

GB/T 15441-1995 Water quality - Determination of the acute toxicity - Luminescent bacteria test

GB/T 36197-2018 Soil quality - Guidance on soil sampling techniques

HJ/T 166-2004 The technical specification for soil environmental monitoring

NY/T 52-1987 Method for determination of soil water content

3 Terms and definitions

For the purposes of this document, the following terms and definitions apply.

3.1 soil health: The capacity of soil, as a living system, to sustainably maintain the productivity, biodiversity and environmental services of the soil system. Note: a healthy soil has no potential risk of soil-borne disease outbreaks and no soil heavy metals exceeding the limits, has a diversified soil biological community, is conducive to increasing soil productivity and ultimately keeps the soil from degrading.

3.2 soil health assessment: The overall qualitative and quantitative evaluation of the degree of soil health using biological factors such as kidney beans, luminescent bacteria, nematodes and earthworms.

3.3 bioassay: A method that systematically uses biological responses to test the effects or hazards caused by one or more pollutants or environmental factors present alone or in combination.

3.4 community: The assemblage of the populations of the various species gathered in the same area at the same time. Note: in this document it mainly refers to the community structure of nematodes and earthworms.

3.5 luminescent bacteria: Bacteria containing the luciferase gene that are able to emit visible light under normal physiological conditions.

4 General

This document includes four bioassays: the kidney bean root morphology test method is mainly aimed at soil health testing related to soil-borne diseases; the luminescent bacteria bioassay is mainly aimed at health testing of polluted soils; the nematode community bioassay is mainly aimed at health testing of degraded grassland soils; and the earthworm bioassay is mainly aimed at testing the comprehensive degree of health of orchard, vegetable field and dryland soils.

In practice, where several of the bioassays in this document are used at the same time, the luminescent bacteria bioassay is applied first to evaluate the degree of pollution of the soil under test, and the health grade of the soil is determined by the result of that method. If the soil tested by that method is healthy, the health grade of the soil is determined by the lowest grade obtained with the other methods.

5 Bioassays

5.1 Kidney bean root morphology test method.

5.1.1 Principle. In a greenhouse pot experiment, kidney beans are planted in the soil under test and grown for four weeks under suitable conditions; the kidney bean root systems are then collected intact, the health status of the roots grown in the soil is observed, and the soil health grade is determined against the root health grading established in this document.

5.1.2 Sampling equipment. The following shall be prepared: a) tools: iron spade, iron shovel, cylindrical soil auger, mud sampler, spiral soil auger, bamboo strips and tools suited to special sampling requirements; b) equipment: global positioning system (GPS), camera, tape measure, sample bags, sample boxes, polyethylene buckets, etc.

5.1.3 Sampling procedure. a) Select representative sampling points and remove the litter, above-ground vegetation, debris and other cover from the soil surface. b) With a clean iron spade, dig vertically in the field a rectangular strip about 20 cm long, 14 cm wide and 5 cm high; the size of each soil sample shall be kept consistent. c) Pick out dead branches and leaves, plant roots, stubble, insect bodies and stones, and put the collected soil into a clean polyethylene bucket. d) Determine the number of sampling points according to the actual conditions of the test plot, in accordance with 6.2.3.2 of HJ/T 166-2004. e) Thoroughly mix the soil from all points into one composite sample by the method of 5.3 of GB/T 36197-2018, put it into a sample bag and, after returning, pass the soil through an 8 mm sieve for later use. f) Fill in two soil sample labels, one placed inside the sample bag and one tied to its mouth, stating sample number, sampling location, characteristics, sampling depth, sampling date and sampling personnel. Note: representative sampling points avoid locally very wet or very dry areas, raised spots in the field, field borders, fertilization points or strips, and areas near roads.

5.1.4 Indicator plant. Kidney beans with plump seeds of uniform size and of the same variety.

5.1.5 Test tools. The pots used are cylindrical polyvinyl chloride (PVC) tubes 30 cm high and 10 cm in diameter with holes at the bottom; a cotton ball, not packed too tightly so that water can drain, is placed at the bottom of each tube.

5.1.6 Test procedure. a) Place the PVC tubes on a perforated tube rack, leaving one empty hole between every two tubes (see A.1 in Annex A for the apparatus); label the front of each tube with the treatment and arrange them in a row; each soil sample is replicated 10 times. b) Fill each tube with the soil sample using a plastic funnel to 2.0 cm from the top of the tube, avoiding cross-contamination between samples. c) Place one kidney bean seed in the centre of the soil surface layer of each tube (at a depth of 1.0 cm), with the hilum side horizontal, simulating field conditions as far as possible. d) Cover the top of the tube with a 1.0 cm layer of quartz sand to prevent soil crusting during watering. e) Place the racks in a greenhouse for cultivation with a daily light period of 12 h to 14 h, a temperature of 30 °C +/- 3 °C and a relative humidity of 60 % to 70 %, under routine management, keeping the soil water content at 20 % to 30 %. f) After four weeks, carefully remove the plants from the tubes, avoiding damage to the roots. g) Wash the roots and evaluate and record root morphology and growth status.

5.1.7 Expression of test results. 5.1.7.1 Results are expressed mainly according to the coverage rate of root lesions, in 4 grades: a) grade 1 (healthy): roots white, lesion coverage <= 10 %; b) grade 2 (slight disease): roots turning from white to slightly yellow, lesion coverage > 10 % to 30 %; c) grade 3 (moderate disease): moderate destruction of root tissue, lesion coverage of the hypocotyl > 30 % to 50 %; d) grade 4 (severe disease): main root brown, with > 50 % of the hypocotyl and roots showing disease symptoms.

5.1.7.2 The root morphology of grades 1 to 4 is shown in Figure 1, which presents photographs of washed kidney bean roots for each grade, from dense pale healthy roots (grade 1) to a browned hypocotyl and darkened, sparse roots (grade 4). 5.1.7.3 The degree of health of diseased soil is determined from the root grade of the indicator plant; the kidney bean root grade is the health grade of the continuously cropped soil. 5.1.7.4 When evaluating kidney bean root health, if the root morphology of kidney beans grown in different replicates of the same soil sample differs markedly (health grade inconsistent with the other replicates of the group), the deviating samples are removed before statistics are compiled; however, at least 7 replicates of consistent phenotype shall be retained, otherwise the test shall be repeated.

5.2 Luminescent bacteria bioassay.

5.2.1 Principle. Luminescent bacteria are added to an extract of the test soil to measure the effect of the soil on the luminous intensity of the bacteria, so as to determine the strength of soil toxicity, characterize the pollution in the test system and carry out the soil health assessment. 5.2.2 Sampling procedure. Soil samples are collected in accordance with 5.1.3 and air-dried.

5.2.3 Soil extraction. 5.2.3.1 Weigh 2.0 g of air-dried soil into a conical flask and mix with 20 mL of 2.0 % sodium chloride solution (NaCl, analytical grade); 20 mL of 2.0 % NaCl solution without soil sample is used as the control. 5.2.3.2 Shake on a shaker for 16 h at 200 r/min. 5.2.3.3 Filter the shaken samples for testing. Note: if natural (moist) soil is used, the sample mass is converted according to the soil water content so that it equals 2.0 g of air-dried soil. 5.2.4 Test procedure. Take 10 mL to 15 mL of filtrate and carry out the toxicity test and result calculation in accordance with GB/T 15441-1995.

5.2.5 Calculation of the index. The relative luminosity (%) of the sample is calculated with formula (1), RL = LT / LCK, and the mean of three measurements is taken, where RL is the relative luminosity, LT is the light output of the mercuric chloride tube or of the sample tube, and LCK is the light output of the control tube.

5.2.6 Expression of test results. 5.2.6.1 According to the ratio of the luminosity of the sample to that of the blank control, 4 grades are defined: a) grade 1 (healthy): relative luminosity of the sample above 90 % of the blank control; b) grade 2 (slight pollution): 70 % to 90 % of the blank control; c) grade 3 (moderate pollution): 50 % to below 70 % of the blank control; d) grade 4 (severe pollution): less than 50 % of the blank control. 5.2.6.2 The degree of soil health is determined according to 5.2.6.1, and the soil health grade is assigned and the result expressed.

5.3 Nematode community bioassay.

5.3.1 Principle. The Baermann funnel extraction method is used to investigate the composition of the nematode community in a given soil, and the health status of the soil is evaluated from the main ecological indices of the nematode community.

5.3.2 Reagents and instruments. a) Reagent: formalin (formaldehyde volume fraction 6 %). b) Tools: plastic measuring cup (1 L), beakers (250 mL), qualitative filter paper (mean pore size 0.85 mm), watch glasses (diameter 3 cm to 5 cm), funnels (diameter 10 cm to 15 cm), test tubes (50 mL), specimen bottles (5 mL to 10 mL), glass Petri dishes (diameter larger than the funnel), counting dishes (Petri dishes with a grid drawn on the bottom) and counters. c) Instruments: balance (capacity greater than 100 g), vacuum pump (displacement greater than 20 L/min), water bath with temperature control from room temperature to 100 °C, dissecting microscope (magnification 10x to 40x) and optical microscope (magnification 10x to 100x).

5.3.3 Selection of the reference plot. A plot whose plant community is close to the natural state or little disturbed by human activity is selected as the reference plot. In the reference plot and in the typical area, 3 or more measurement plots are set at random to collect soil samples, and the total number of soil nematodes and the numbers of nematodes of the different trophic groups are determined. Soil samples of the evaluated object and of the reference plot are collected in accordance with 5.1.3. 5.3.4 Determination of soil water content. The soil water content shall be determined by the method of NY/T 52-1987.

5.3.5 Nematode extraction. Nematodes are extracted by the Baermann funnel method (apparatus in A.2): a) fix the funnel on a stand, connect a 10 cm rubber tube to its lower end and fit a spring clip on the tube; b) gently place 50 g or 100 g of weighed fresh soil on the funnel lined with facial tissue, add a small amount of water to submerge the soil, open the spring clip after about 24 h and collect about 5 mL of liquid in a test tube, let it stand for 1 h and draw off the excess supernatant; c) count the collected nematode suspension under a dissecting microscope, then heat the counted sample in a water bath at 60 °C for 3 min to kill the nematodes; d) add formalin at a ratio of 1:1, pour into a specimen bottle, label, seal and store for subsequent identification. Note: take appropriate precautions when handling formalin to avoid inhalation or skin contact; samples can be handled in a fume hood wearing gloves and a mask.

5.3.6 Nematode identification. 100 nematodes are drawn at random from each sample and identified under the optical microscope; if the total is less than 100, all are identified. The numbers in the 4 main trophic groups - bacterivorous nematodes, fungivorous nematodes, plant-parasitic nematodes and predatory/omnivorous nematodes - are counted separately (see Figure A.4).

5.3.7 Calculation of indices. 5.3.7.1 The total number of soil nematodes (per 100 g dry soil) is calculated with formula (2), QT = QF / (1 + W), where QT is the total number of nematodes per 100 g dry soil, QF the total number per 100 g fresh soil and W the soil water content. 5.3.7.2 The number of predatory/omnivorous nematodes per 100 g dry soil is calculated with formula (3), Qp/o = QT x (QIp/o / QI), where QIp/o is the number of predatory/omnivorous nematodes among those identified and QI is the total number of nematodes identified. 5.3.7.3 The number of fungivorous nematodes per 100 g dry soil is calculated with formula (4), QF = QT x (QFI / QI), where QFI is the number of fungivorous nematodes among those identified.

5.3.8 Expression of test results. 5.3.8.1 The total number of nematodes, the number of predatory/omnivorous nematodes and the number of fungivorous nematodes of the typical area are compared with the corresponding indices of the reference plot; the soil degradation grades are given in Table 1. If no corresponding reference plot can be obtained, a total of 700 to 1 000 nematodes per 100 g dry soil shall be used as the reference. 5.3.8.2 The comparison result of each grading index is matched against the grading characteristics of Table 1; where they agree or are close, the index is assigned to that grade, giving the degree of soil health for that index. Because of differences in management and soil type, suitable local soil health indices should be selected according to local conditions. 5.3.8.3 When the 3 grading indices fall in the same grade, that grade is the soil degradation grade; if not, the grade with the most indices is the grade of the degree of soil health; if two grades have the same number of indices, the more severe grade is taken as the grade of the degree of soil health.

Table 1 Grading of the degree of soil health: grade 1 (slight degradation) - total nematodes increased by 30 % to 40 % or decreased by less than 30 %, predatory/omnivorous nematodes decreased by less than 30 %, fungivorous nematodes decreased by less than 30 %; grade 2 (moderate degradation) - each of the three indices decreased by 30 % to 50 %; grade 3 (severe degradation) - each of the three indices decreased by more than 50 %.

5.4 Earthworm bioassay.

5.4.1 Principle. Earthworms in the soil at the sampling point are collected by electrical shocking, the number of earthworms living in the soil is surveyed, and the soil health grade is determined according to a set grading to carry out the soil health assessment. 5.4.2 Test tools. A 12 V lithium battery, a transformer (8 high capacitors; instantaneous discharge voltage 9 000 V) and 50 cm metal probes; the apparatus is shown in A.3.

5.4.3 Test procedure. a) Choose a spot with a moist surface, with vegetation or organic material cover, or where traces of earthworm activity are clearly visible; in paddy fields sampling takes place after the rice harvest. Mark out a 100 cm x 100 cm quadrat on the surface of the soil under test and remove excess surface organic matter; depending on the size of the sampling area the quadrat is replicated 5 to 10 times. b) Wearing rubber gloves, insert the two metal probes vertically into the soil to a depth of about 30 cm with the two electrodes 50 cm to 100 cm apart; connect the probes to the output of the transformer with wires, connect the 12 V DC power supply to the transformer input and switch on the transformer after ensuring there is no short circuit. c) Observe the area within a radius of 50 cm centred on the two probes; as earthworms emerge, pick them up and put them into a sampling bag, touching mainly the head of the earthworm and wearing rubber gloves throughout. After the transformer has worked for 15 min, cut off the power, wait 1 min and pull the electrodes out of the soil. d) Count the earthworms in each quadrat, photograph and record, and then return the earthworms to the original collection site.

5.4.4 Expression of test results. 5.4.4.1 Results are expressed mainly according to the number of earthworms per square metre, in 4 grades: a) grade 1 (healthy): more than 50 earthworms; b) grade 2 (moderate degradation): > 20 to 50 earthworms; c) grade 3 (severe degradation): > 4 to 20 earthworms; d) grade 4 (unhealthy): 4 earthworms or fewer. 5.4.4.2 The degree of soil health is determined from the earthworm grading index; the earthworm number grade is the soil health grade. 5.4.4.3 When grading earthworm numbers, if the numbers in different replicates of the same plot differ markedly (health grade inconsistent with the other replicates of the group), the deviating samples shall be removed.

6 Test report

Where several bioassays of this document are used at the same time, it is recommended that the luminescent bacteria bioassay be applied first to evaluate the degree of pollution of the soil under test for the soil health assessment, the health grade of the soil being determined by the result of that method. If the soil tested by that method is healthy, the final test report is issued with the soil health grade taken as the lowest grade obtained with the other methods.

After the test a test report shall be issued, including: a) the test method, test date, end date and environmental conditions under which the test was carried out; b) the background of the plot, the sampling method, the number of sampling points, the number of replicates of the soil samples and the characteristics of the soil under test; c) the original records and test results; d) any operational details or events that may have affected the test results.

Annex A (informative) Test apparatus and selection of sampling points and samples

A.1 Kidney bean pot test apparatus. The apparatus is shown in Figure A.1, a schematic of a single PVC tube 30 cm high and 10 cm in diameter with holes at the bottom, and of the tubes arranged in staggered rows on a perforated rack. Kidney beans are planted in the tubes filled with test soil; a cotton ball, not packed too tightly so that water can drain, is placed at the bottom of each tube. After four weeks of growth under suitable conditions, the kidney bean roots are collected intact, their growth is observed and the roots are graded in accordance with 5.1.7.1 for the soil health assessment.

A.2 Baermann funnel filtration apparatus. Figure A.2 shows a funnel (top width 12 cm) lined with filter paper, whose stem is connected to a rubber tube fitted with a stop valve and placed in a beaker; key: 1 filter paper, 2 funnel, 3 stop valve, 4 rubber tube. The funnel is fixed on a stand with a 10 cm rubber tube at its lower end fitted with a spring clip; the soil sample is placed gently on the funnel lined with facial tissue and a small amount of water is added to submerge the soil; after about 24 h the spring clip is opened, about 5 mL of liquid is collected in a test tube, left to stand for 1 h and the excess supernatant is drawn off, giving the nematode suspension.

A.3 Earthworm electrical shocking apparatus. Figure A.3 a) is a working schematic of the electrical method: a power supply module (12 V DC battery) feeding a transformer (capacitor bank) connected to metal probes inserted in the ground; Figure A.3 b) is a photograph of the equipment used, with key: 1 transformer, 2 12 V lithium battery, 3 metal probes, 4 wires. Before starting, the metal probes are inserted into the soil of the area to be tested, and insulated wires connect the 3 parts: the power supply module (12 V DC), the transformer unit (earthworm-collecting transformer) and the grounding unit (two metal probes). The power is switched on after checking that everything is correct; to stop, the power is cut off and the probes are pulled out after waiting 1 min.

A.4 Schematic of classification characteristics. Figure A.4 shows the identification key for soil nematode trophic groups, with the anterior body of a nematode labelled stylet, basal knob, median bulb, nerve ring and posterior bulb. Nematodes without a stylet are divided by the presence of lips/setae/a nerve ring structure: if present they are bacterivorous nematodes, if absent predatory/omnivorous nematodes. Nematodes with a stylet are divided by the presence of a posterior oesophageal bulb: if present they are plant-parasitic nematodes, if absent fungivorous nematodes.

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